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The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with the myopathy, is also present in LAMP-2-deficient mice. Intellectual dysfunction observed in the human disease suggests a pivotal role of LAMP-2 within brain. LAMP-2A, one specific LAMP-2 isoform, was proposed to be important for the lysosomal degradation of selective proteins involved in neurodegenerative diseases such as Huntington's and Parkinson's disease. To elucidate the neuronal function of LAMP-2 we analyzed knockout mice for neuropathological changes, MA and steady-state levels of CMA substrates. The absence of LAMP-2 in murine brain led to inflammation and abnormal behavior, including motor deficits and impaired learning. The latter abnormality points to hippocampal dysfunction caused by altered lysosomal activity, distinct accumulation of p62-positive aggregates, autophagic vacuoles and lipid storage within hippocampal neurons and their presynaptic terminals. The absence of LAMP-2 did not apparently affect MA or steady-state levels of selected CMA substrates in brain or neuroblastoma cells under physiological and prolonged starvation conditions. Our data contribute to the understanding of intellectual dysfunction observed in Danon disease patients and highlight the role of LAMP-2 within the central nervous system, particularly the hippocampus.

Here we present a male patient with acute myeloid leukemia (AML) initially diagnosed as M5 and with karyotype 46,XY. After induction therapy, he underwent a HLA-matched allogeneic hematopoietic stem cell transplantation, and six years later he relapsed as AML M1 with an abnormal karyotype //47,XX,+10[2]/47,XX,+11[3]/48,XX,+10,+11[2]/46,XX[13]. Based on this, we tested the possibility of donor cell origin by FISH and molecular STR analysis. We found no evidence of Y chromosome presence by FISH and STR analysis consistent with the success of the allogeneic hematopoietic stem cell transplantation from the female donor. FISH studies confirmed trisomies and no evidence of MLL translocation either p53 or ATM deletion. Additionally 28 fusion common leukemia transcripts were evaluated by multiplex reverse transcriptase-polymerase chain reaction assay and were not rearranged. STR analysis showed a complete donor chimerism. Thus, donor cell leukemia (DCL) was concluded, being essential the use of cytological and molecular approaches. Pediatric DCL is uncommon, our patient seems to be the sixth case and additionally it presented a late donor cell leukemia appearance. Different extrinsic and intrinsic mechanisms have been considered to explain this uncommon finding as well as the implications to the patient.

Stem cells are considered as the next generation drug treatment in patients with cardiovascular disease who are resistant to conventional treatment. Among several stem cells used in the clinical setting, cardiac stem cells (CSCs) which reside in the myocardium and epicardium of the heart have been shown to be an effective option for the source of stem cells. In normal circumstances, CSCs primarily function as a cell store to replace the physiologically depleted cardiovascular cells, while under the diseased condition they have been shown to experimentally regenerate the diseased myocardium. In spite of their major functional role, molecular mechanisms regulating the CSCs proliferation and differentiation are still unknown. MicroRNAs (miRs) are small, noncoding RNA molecules that regulate gene expression at the posttranscriptional level. Recent studies have demonstrated the important role of miRs in regulating stem cell proliferation and differentiation, as well as other physiological and pathological processes related to stem cell function. This review summarises the current understanding of the role of miRs in CSCs. A deeper understanding of the mechanisms by which miRs regulate CSCs may lead to advances in the mode of stem cell therapies for the treatment of cardiovascular diseases.

The mature striatum is divided into a labyrinthine system of striosomes embedded in a surrounding matrix compartment. We pulse-labeled striosomal cells (S cells) and matrix cells (M cells) in cats with (3) H-thymidine and followed their distributions during fetal and postnatal development. We identified three maturational phases in S-cell distributions. The early phase (sampled at embryonic day [E]27-E35 following E24-E28 (3) H-thymidine) was characterized by a transient medial accumulation of synchronously generated S cells within the caudate nucleus adjoining the ganglionic eminence, potentially a waiting compartment. Band-like arrangements of synchronously generated S cells then formed beyond this medial band. During the second phase (sampled at E38-E45), the loosely banded S-cell distributions were transformed into clustered arrangements typical of developing striosomes. In the third phase (sampled from E52 into the postnatal period), these developed into the typical mature striosomal architecture. At adulthood, gentle mediolateral birthdate-gradients in S cells were still evident, but M cells, produced over mid to late prenatal ages, became broadly distributed, without apparent gradients or banding arrangements. These findings suggest that the maturational histories of the striosomal and matrix neurons are influenced by their generation times and local environments, and that future S cells have transient, nonstriosomal distributions prior to their aggregation into striosomal clusters, including a putative waiting compartment. Further, the eventual patterning of the striosomal compartment reflects outside-in, band-like gradient patterns of settling of synchronously generated S cells, patterns that could be related both to neural processing in the mature striatum and to patterns of vulnerability of striatal neurons.

<h4>Introduction</h4>Increasing evidence indicates that microRNAs (miRNAs) are important players in oncogenesis. Considering the widespread use of aromatase inhibitors (AIs) in endocrine therapy as a first-line treatment for postmenopausal estrogen receptor α-positive breast cancer patients, identifying deregulated expression levels of miRNAs in association with AI resistance is of utmost importance.<h4>Methods</h4>To gain further insight into the molecular mechanisms underlying the AI resistance, we performed miRNA microarray experiments using a new model of acquired resistance to letrozole (Res-Let cells), obtained by long-term exposure of aromatase-overexpressing MCF-7 cells (MCF-7aro cells) to letrozole, and a model of acquired anastrozole resistance (Res-Ana cells). Three miRNAs (miR-125b, miR-205 and miR-424) similarly deregulated in both AI-resistant cell lines were then investigated in terms of their functional role in AI resistance development and breast cancer cell aggressiveness and their clinical relevance using a cohort of 65 primary breast tumor samples.<h4>Results</h4>We identified the deregulated expression of 33 miRNAs in Res-Let cells and of 18 miRNAs in Res-Ana cells compared with the sensitive MCF-7aro cell line. The top-ranked Kyoto Encyclopedia of Genes and Genomes pathways delineated by both miRNA signatures converged on the AKT/mTOR pathway, which was found to be constitutively activated in both AI-resistant cell lines. We report for the first time, to our knowledge, that ectopic overexpression of either miR-125b or miR-205, or the silencing of miR-424 expression, in the sensitive MCF-7aro cell line was sufficient to confer resistance to letrozole and anastrozole, to target and activate the AKT/mTOR pathway and to increase the formation capacity of stem-like and tumor-initiating cells possessing self-renewing properties. Increasing miR-125b expression levels was also sufficient to confer estrogen-independent growth properties to the sensitive MCF-7aro cell line. We also found that elevated miR-125b expression levels were a novel marker for poor prognosis in breast cancer and that targeting miR-125b in Res-Let cells overcame letrozole resistance.<h4>Conclusion</h4>This study highlights that acquisition of specific deregulated miRNAs is a newly discovered alternative mechanism developed by AI-resistant breast cancer cells to achieve constitutive activation of the AKT/mTOR pathway and to develop AI resistance. It also highlights that miR-125b is a new biomarker of poor prognosis and a candidate therapeutic target in AI-resistant breast cancers.

We have reached a conundrum in assigning cause of death for sudden unexpected infant deaths. We summarize the discordant perspectives and approaches and how they have occurred, and recommend a pathway toward improved consistency. This lack of consistency affects pediatricians and other health care professionals, scientific investigators, medical examiners and coroners, law enforcement agencies, families, and support or advocacy groups. We recommend that an interdisciplinary international committee be organized to review current approaches for assigning cause of death, and to identify a consensus strategy for improving consistency. This effort will need to encompass intrinsic risk factors or infant vulnerability in addition to known environmental risk factors including unsafe sleep settings, and must be sufficiently flexible to accommodate a progressively expanding knowledge base.

To design an estrogen and phenol red free medium for cell culture and check its effectiveness and safety on osteoblast growth it is necessary to maintain the estrogen receptors free for tests. For this purpose, we tested some modifications of the traditional culture media: estrogen depleted fetal bovine serum; estrogen charcoal stripped fetal bovine serum and phenol red free α-MEM. The aim of this work is to examine the effects of its depletion in the proliferation, differentiation, and toxicity of mesenchymal stromal cells differentiated into osteoblasts to obtain an effective interference free culture medium for in vitro studies, focused on non-previously studied estrogen receptors. We performed viability tests using the following techniques: MTT, alkaline phosphatase specific activity, formation of mineralized matrix by Alizarin technique and analysis of SEM/EDX of mineralized nodules. The results showed that the culture media with estrogen free α-MEM + phenol red free α-MEM did not impact viability, alkaline phosphatase activity and mineralization of the osteoblasts culture compared to control. In addition, its nodules possess Ca/P ratio similar to hydroxyapatite nodules on the 14th and 21st day. In conclusion, the modified culture medium with phenol red free α-MEM with estrogen depleted fetal bovine serum can be safely used in experiments where the estrogen receptors need to be free.

Forty-eight Duroc x Landrace x Large White gilts were used to determine the relationship between proteome changes of longissimus muscle and intramuscular fat (IMF) content in arginine-supplemented pigs. Beginning at 60 kg BW, pigs were fed a corn- and soybean meal-based diet supplemented or not with 1% L-arginine until they reached a BW of 100 kg. Supplementation with 1% L-arginine did not affect the growth performance or carcass traits, while it increased IMF content by 32% (P < 0.01), it also decreased the drip loss at 48 h post-mortem and the b* meat color value at 24 h post-mortem; supplementation with 1% dietary L-arginine did not change the proportion of SFA and MUFA in muscle lipids. The proteome changes in longissimus muscle between the control and supplemented pigs showed that L-arginine significantly influenced the abundance of proteins related to energy metabolism, fiber type and structure. The increase in IMF content was positively correlated with the increased abundance of slow twitch troponin I (TNNI1) protein and negatively correlated with myosin heavy chain IIb (MyHC IIb) protein content. It is suggested that the proteome changes in longissimus muscle contributed to the greater IMF content in L-arginine supplemented pigs.

The three butyrophilin BTN3A molecules, BTN3A1, BTN3A2, and BTN3A3, are members of the B7/butyrophilin-like group of Ig superfamily receptors, which modulate the function of T cells. BTN3A1 controls activation of human Vγ9/Vδ2 T cells by direct or indirect presentation of self and nonself phosphoantigens (pAg). We show that the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate binds to the intracellular B30.2 domain of BTN3A1 with an affinity of 1.1 μM, whereas the endogenous pAg isopentenyl pyrophosphate binds with an affinity of 627 μM. Coculture experiments using knockdown cell lines showed that in addition to BTN3A1, BTN3A2 and BTN3A3 transmit activation signals to human γδ T cells in response to (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate and the aminobisphosphonate drug zoledronate that causes intracellular accumulation of isopentenyl pyrophosphate. The plakin family member periplakin, identified in yeast two-hybrid assays, interacted with a membrane-proximal di-leucine motif, located proximal to the B30.2 domain in the BTN3A1 cytoplasmic tail. Periplakin did not interact with BTN3A2 or BTN3A3, which do not contain the di-leucine motif. Re-expression into a BTN3A1 knockdown line of wild-type BTN3A1, but not of a variant lacking the periplakin binding motif, BTN3A1Δexon5, restored γδ T cell responses, demonstrating a functional role for periplakin interaction. These data, together with the widespread expression in epithelial cells, tumor tissues, and macrophages detected using BTN3A antiserum, are consistent with complex functions for BTN3A molecules in tissue immune surveillance and infection, linking the cell cytoskeleton to γδ T cell activation by indirectly presenting pAg to the Vγ9/Vδ2 TCR.

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.

Huntington's disease is an incurable neurodegenerative disease caused by inheritance of an expanded cytosine-adenine-guanine (CAG) trinucleotide repeat within the Huntingtin gene. Extensive volume loss and altered diffusion metrics in the basal ganglia, cortex and white matter are seen when patients with Huntington's disease (HD) undergo structural imaging, suggesting that changes in basal ganglia-cortical structural connectivity occur. The aims of this study were to characterise altered patterns of basal ganglia-cortical structural connectivity with high anatomical precision in premanifest and early manifest HD, and to identify associations between structural connectivity and genetic or clinical markers of HD. 3-Tesla diffusion tensor magnetic resonance images were acquired from 14 early manifest HD subjects, 17 premanifest HD subjects and 18 controls. Voxel-based analyses of probabilistic tractography were used to quantify basal ganglia-cortical structural connections. Canonical variate analysis was used to demonstrate disease-associated patterns of altered connectivity and to test for associations between connectivity and genetic and clinical markers of HD; this is the first study in which such analyses have been used. Widespread changes were seen in basal ganglia-cortical structural connectivity in early manifest HD subjects; this has relevance for development of therapies targeting the striatum. Premanifest HD subjects had a pattern of connectivity more similar to that of controls, suggesting progressive change in connections over time. Associations between structural connectivity patterns and motor and cognitive markers of disease severity were present in early manifest subjects. Our data suggest the clinical phenotype in manifest HD may be at least partly a result of altered connectivity.

Cyclic AMP signaling is critical for activity-dependent refinement of neuronal circuits. Global disruption of adenylyl cyclase 1 (AC1), the major calcium/calmodulin-stimulated adenylyl cyclase in the brain, impairs formation of whisker-related discrete neural modules (the barrels) in cortical layer 4 in mice. Since AC1 is expressed both in the thalamus and the neocortex, the question of whether pre- or postsynaptic (or both) AC1 plays a role in barrel formation has emerged. Previously, we generated cortex-specific AC1 knockout (Cx-AC1KO) mice and found that these animals develop histologically normal barrels, suggesting a potentially more prominent role for thalamic AC1 in barrel formation. To determine this, we generated three new lines of mice: one in which AC1 is disrupted in nearly half of the thalamic ventrobasal nucleus cells in addition to the cortical excitatory neurons (Cx/pTh-AC1KO mouse), and another in which AC1 is disrupted in the thalamus but not in the cortex or brainstem nuclei of the somatosensory system (Th-AC1KO mouse). Cx/pTh-AC1KO mice show severe deficits in barrel formation. Th-AC1KO mice show even more severe disruption in barrel patterning. In these two lines, single thalamocortical (TC) axon labeling revealed a larger lateral extent of TC axons in layer 4 compared to controls. In the third line, all calcium-stimulated adenylyl cyclases (both AC1 and AC8) are deleted in cortical excitatory neurons. These mice have normal barrels. Taken together, these results indicate that thalamic AC1 plays a major role in patterning and refinement of the mouse TC circuitry.

<h4>Background</h4>This study aimed to investigate the bone morphogenetic protein-2 (BMP-2) levels in serum and synovial fluid (SF) of patients with primary knee osteoarthritis (OA) and to exam its correlation with radiographic and symptomatic severity of the disease.<h4>Material/methods</h4>A total of 37 knee OA patients and 20 healthy controls were enrolled in this study. Knee OA radiographic grading was performed according to the Kellgren-Lawrence (KL) grading system by evaluating X-ray changes observed in anteroposterior knee radiography. Symptomatic severity of the disease was evaluated according to the Western Ontario McMaster University Osteoarthritis Index (WOMAC) scores. BMP-2 levels in serum and SF were determined using enzyme-linked immunosorbent assay.<h4>Results</h4>Serum BMP-2 level in patients with knee OA was higher than that in healthy controls. Knee OA patients with KL grade 4 showed significantly elevated BMP-2 levels in the serum and SF compared with those with KL grade 2 and 3. Knee OA patients with KL grade 3 had significant higher SF levels of BMP-2 than those with KL grade 2. BMP-2 levels in the serum and SF of knee OA patients were both positively correlated with KL grades and WOMAC scores.<h4>Conclusions</h4>BMP2 levels in serum and SF were closely related to the radiographic and symptomatic severity of knee OA and may serve as an alternative biochemical parameter to determine disease severity of primary knee OA.

Posttraumatic stress disorder (PTSD) is a heterogeneous disorder that affects individuals exposed to trauma (e.g., combat, interpersonal violence, and natural disasters). Although its diagnostic features have been recently reclassified with the emergence of the Diagnostic and Statistical Manual for Mental Disorders, Fifth Edition, the disorder remains characterized by hyperarousal, intrusive reminders of the trauma, avoidance of trauma-related cues, and negative cognition and mood. This heterogeneity indicates the presence of multiple neurobiological mechanisms underlying the etiology and maintenance of PTSD. Translational research spanning the past few decades has revealed several potential avenues for the identification of diagnostic biomarkers for PTSD. These include, but are not limited to, monoaminergic transmitter systems, the hypothalamic-pituitary-adrenal axis, metabolic hormonal pathways, inflammatory mechanisms, psychophysiological reactivity, and neural circuits. The current review provides an update to the literature with regard to the most promising putative PTSD biomarkers, with specific emphasis on the interaction between neurobiological influences on disease risk and symptom progression. Such biomarkers will most likely be identified by multi-dimensional models derived from comprehensive descriptions of molecular, neurobiological, behavioral, and clinical phenotypes.

<h4>Background</h4>A variety of materials have been used for bone augmentation, distraction osteotomy, and in post-cancer patients following tumor removal. However, a temporary metal implant that would resorb after successful treatment is a new concept. Magnesium was suggested as a suitable material for these purposes because it is biocompatible, has better mechanical properties than titanium, and stimulates new bone formation. This study evaluates histological appearance of magnesium-based implants and the surrounding bone.<h4>Materials and methods</h4>Three magnesium-based biomaterials were tested in a rabbit bone defect model: magnesium-hydroxyapatite (Mg-HA), W4 (96 % magnesium, 4 % yttrium), and pure magnesium (pure Mg). Animals were sacrificed after 6 and 12 weeks and the samples were analyzed histologically and histomorphometrically.<h4>Results</h4>Mg-HA had the highest mean amount of tartrate-resistant acid phosphatase (TRAP) positive cells at the implantation site of all groups. It had shown the fastest degradation rate already at 6 weeks but the least amount of new bone formation. New bone was seen forming in direct contact with pure Mg and W4. The mean gas volume was highest in W4 compared to pure Mg and Mg-HA but this difference was not statistically significant. W4 had the lowest mean number of TRAP-positive cells of all materials.<h4>Conclusion</h4>Pure Mg and W4 were shown to be the most promising materials in this study in respect to the bone response to the implant material. They could be used for screws and plates in bone augmentation procedures.

Marital separation is linked to negative mental and physical health; however, the strength of this link may vary across people. This study examined changes in respiratory sinus arrhythmia (RSA), used to assess cardiac vagal control, in recently separated adults (N = 79; M time since separation = 3.5 months). When reflecting on the separation, self-reported psychological distress following the separation interacted with a polymorphism in the serotonin transporter gene (5-HTTLPR) and a relevant single nucleotide polymorphism (SNP), rs25531, to predict RSA. Among people reporting emotional difficulties after the separation, those who were homozygous for the short allele had lower RSA levels while reflecting on their relationship than other genotypes. The findings, although limited by the relatively small sample size, are discussed in terms of how higher-sensitivity genotypes may interact with psychological responses to stress to alter physiology.

<h4>Purpose</h4>Obesity is a growing public health concern now reaching epidemic status worldwide for children and adults due to multiple problems impacting on energy intake and expenditure with influences on human reproduction and infertility. A positive family history and genetic factors are known to play a role in obesity by influencing eating behavior, weight and level of physical activity and also contributing to human reproduction and infertility. Recent advances in genetic technology have led to discoveries of new susceptibility genes for obesity and causation of infertility. The goal of our study was to provide an update of clinically relevant candidate and known genes for obesity and infertility using high resolution chromosome ideograms with gene symbols and tabular form.<h4>Methods</h4>We used computer-based internet websites including PubMed to search for combinations of key words such as obesity, body mass index, infertility, reproduction, azoospermia, endometriosis, diminished ovarian reserve, estrogen along with genetics, gene mutations or variants to identify evidence for development of a master list of recognized obesity genes in humans and those involved with infertility and reproduction. Gene symbols for known and candidate genes for obesity were plotted on high resolution chromosome ideograms at the 850 band level. Both infertility and obesity genes were listed separately in alphabetical order in tabular form and those highlighted when involved with both conditions.<h4>Results</h4>By searching the medical literature and computer generated websites for key words, we found documented evidence for 370 genes playing a role in obesity and 153 genes for human reproduction or infertility. The obesity genes primarily affected common pathways in lipid metabolism, deposition or transport, eating behavior and food selection, physical activity or energy expenditure. Twenty-one of the obesity genes were also associated with human infertility and reproduction. Gene symbols were plotted on high resolution ideograms and their name, precise chromosome band location and description were summarized in tabular form.<h4>Conclusions</h4>Meaningful correlations in the obesity phenotype and associated human infertility and reproduction are represented with the location of genes on chromosome ideograms along with description of the gene and position in tabular form. These high resolution chromosome ideograms and tables will be useful in genetic awareness and counseling, diagnosis and treatment to improve clinical outcomes.

To examine the impact of common somatic mutations in prostate cancer (PCa) on androgen receptor (AR) signaling, mouse models were designed to perturb sequentially the AR, ETV1, and PTEN pathways. Mice with "humanized" AR (hAR) alleles that modified AR transcriptional strength by varying polyglutamine tract (Q-tract) length were crossed with mice expressing a prostate-specific, AR-responsive ETV1 transgene (ETV1(Tg)). While hAR allele did not grossly affect ETV1-induced neoplasia, ETV1 strongly antagonized global AR regulation and repressed critical androgen-induced differentiation and tumor suppressor genes, such as Nkx3-1 and Hoxb13. When Pten was varied to determine its impact on disease progression, mice lacking one Pten allele (Pten(+/-) ) developed more frequent prostatic intraepithelial neoplasia (PIN). Yet, only those with the ETV1 transgene progressed to invasive adenocarcinoma. Furthermore, progression was more frequent with the short Q-tract (stronger) AR, suggesting that the AR, ETV1, and PTEN pathways cooperate in aggressive disease. On the Pten(+/-) background, ETV1 had markedly less effect on AR target genes. However, a strong inflammatory gene expression signature, notably upregulation of Cxcl16, was induced by ETV1. Comparison of mouse and human patient data stratified by the presence of E26 transformation-specific ETS fusion genes highlighted additional factors, some not previously associated with prostate cancer but for which targeted therapies are in development for other diseases. In sum, concerted use of these mouse models illuminates the complex interplay of AR, ETV1, and PTEN pathways in pre-cancerous neoplasia and early tumorigenesis, disease stages difficult to analyze in man.

Mutations in transferrin receptor 2 (TfR2) cause a rare form of the hereditary hemochromatosis, resulting in iron overload predominantly in the liver. TfR2 is primarily expressed in hepatocytes and is hypothesized to sense iron levels in the blood to positively regulate the expression of hepcidin through activation of the BMP signaling pathway. Hepcidin is a peptide hormone that negatively regulates iron egress from cells and thus limits intestinal iron uptake. In this study, a yeast two-hybrid approach using the cytoplasmic domain of TfR2 identified CD81 as an interacting protein. CD81 is an abundant tetraspanin in the liver. Co-precipitations of CD81 with different TfR2 constructs demonstrated that both the cytoplasmic and ecto-transmembrane domains of TfR2 interact with CD81. Knockdown of CD81 using siRNA significantly increased TfR2 levels by increasing the half-life of TfR2, indicating that CD81 promotes degradation of TfR2. Previous studies showed that CD81 is targeted for degradation by GRAIL, an ubiquitin E3 ligase. Knockdown of GRAIL in Hep3B-TfR2 cells increased TfR2 levels, consistent with inhibition of CD81 ubiquitination. These results suggest that down-regulation of CD81 by GRAIL targets TfR2 for degradation. Surprisingly, knockdown of CD81 decreased hepcidin expression, implying that the TfR2/CD81 complex is involved in the maintenance of hepcidin mRNA. Moreover, knockdown of CD81 did not affect the stimulation of hepcidin expression by BMP6 but increased both the expression of ID1 and SMAD7, direct targets of BMP signaling pathway, and the phosphorylation of ERK1/2, indicating that the CD81 regulates hepcidin expression differently from the BMP and ERK1/2 signaling pathways.

Several single gene disorders share clinical and radiologic characteristics with multiple sclerosis and have the potential to be overlooked in the differential diagnostic evaluation of both adult and paediatric patients with multiple sclerosis. This group includes lysosomal storage disorders, various mitochondrial diseases, other neurometabolic disorders, and several other miscellaneous disorders. Recognition of a single-gene disorder as causal for a patient's 'multiple sclerosis-like' phenotype is critically important for accurate direction of patient management, and evokes broader genetic counselling implications for affected families. Here we review single gene disorders that have the potential to mimic multiple sclerosis, provide an overview of clinical and investigational characteristics of each disorder, and present guidelines for when clinicians should suspect an underlying heritable disorder that requires diagnostic confirmation in a patient with a definite or probable diagnosis of multiple sclerosis.

<h4>Background</h4>Distinguishing between bacterial and viral lower respiratory tract infection (LRTI) remains challenging. Transcriptional profiling is a promising tool for improving diagnosis in LRTI.<h4>Methods</h4>We performed whole blood transcriptional analysis in 118 patients (median age [interquartile range], 61 [50-76] years) hospitalized with LRTI and 40 age-matched healthy controls (median age, 60 [46-70] years). We applied class comparisons, modular analysis, and class prediction algorithms to identify and validate diagnostic biosignatures for bacterial and viral LRTI.<h4>Results</h4>Patients were classified as having bacterial (n = 22), viral (n = 71), or bacterial-viral LRTI (n = 25) based on comprehensive microbiologic testing. Compared with healthy controls, statistical group comparisons (P < .01; multiple-test corrections) identified 3376 differentially expressed genes in patients with bacterial LRTI, 2391 in viral LRTI, and 2628 in bacterial-viral LRTI. Patients with bacterial LRTI showed significant overexpression of inflammation and neutrophil genes (bacterial > bacterial-viral > viral), and those with viral LRTI displayed significantly greater overexpression of interferon genes (viral > bacterial-viral > bacterial). The K-nearest neighbors algorithm identified 10 classifier genes that discriminated between bacterial and viral LRTI with a 95% sensitivity (95% confidence interval, 77%-100%) and 92% specificity (77%-98%), compared with a sensitivity of 38% (18%-62%) and a specificity of 91% (76%-98%) for procalcitonin.<h4>Conclusions</h4>Transcriptional profiling is a helpful tool for diagnosis of LRTI.

Eukaryotic gene expression is tightly regulated transcriptionally and post-transcriptionally by a host of noncoding (nc)RNAs. The best-studied class of short ncRNAs, microRNAs, mainly repress gene expression post-transcriptionally. Long noncoding (lnc)RNAs, which comprise RNAs differing widely in length and function, can regulate gene transcription as well as post-transcriptional mRNA fate. Collectively, ncRNAs affect a broad range of age-related physiologic deteriorations and pathologies, including reduced cardiovascular vigor and age-associated cardiovascular disease. This review presents an update of our understanding of regulatory ncRNAs contributing to cardiovascular health and disease as a function of advancing age. We will discuss (1) regulatory ncRNAs that control aging-associated cardiovascular homeostasis and disease, (2) the concepts, approaches, and methodologies needed to study regulatory ncRNAs in cardiovascular aging and (3) the challenges and opportunities that age-associated regulatory ncRNAs present in cardiovascular physiology and pathology. This article is part of a Special Issue entitled "CV Aging".

Variation in cystic fibrosis (CF) phenotypes, including lung disease severity, age of onset of persistent Pseudomonas aeruginosa (P. aeruginosa) lung infection, and presence of meconium ileus (MI), has been partially explained by genome-wide association studies (GWASs). It is not expected that GWASs alone are sufficiently powered to uncover all heritable traits associated with CF phenotypic diversity. Therefore, we utilized gene expression association from lymphoblastoid cells lines from 754 p.Phe508del CF-affected homozygous individuals to identify genes and pathways. LPAR6, a G protein coupled receptor, associated with lung disease severity (false discovery rate q value = 0.0006). Additional pathway analyses, utilizing a stringent permutation-based approach, identified unique signals for all three phenotypes. Pathways associated with lung disease severity were annotated in three broad categories: (1) endomembrane function, containing p.Phe508del processing genes, providing evidence of the importance of p.Phe508del processing to explain lung phenotype variation; (2) HLA class I genes, extending previous GWAS findings in the HLA region; and (3) endoplasmic reticulum stress response genes. Expression pathways associated with lung disease were concordant for some endosome and HLA pathways, with pathways identified using GWAS associations from 1,978 CF-affected individuals. Pathways associated with age of onset of persistent P. aeruginosa infection were enriched for HLA class II genes, and those associated with MI were related to oxidative phosphorylation. Formal testing demonstrated that genes showing differential expression associated with lung disease severity were enriched for heritable genetic variation and expression quantitative traits. Gene expression provided a powerful tool to identify unrecognized heritable variation, complementing ongoing GWASs in this rare disease.

Mitochondrial disorders are defined as defects that affect the oxidative phosphorylation system (OXPHOS). They are characterized by a heterogeneous array of clinical presentations due in part to a wide variety of factors required for proper function of the components of the OXPHOS system. There is no cure for these disorders owing to our poor knowledge of the pathogenic mechanisms of disease. To understand the mechanisms of human disease numerous mouse models have been developed in recent years. Here we summarize the features of several mouse models of mitochondrial diseases directly related to those factors affecting mtDNA maintenance, replication, transcription, translation as well as other proteins that are involved in mitochondrial dynamics and quality control which affect mitochondrial OXPHOS function without being intrinsic components of the system. We discuss how these models have contributed to our understanding of mitochondrial diseases and their pathogenic mechanisms.

Currently, dental caries is the main reason of patient visits to dentists. A great deal of scientific work is dedicated to the study of enamel caries. The reason for this is the necessity for more detailed study of the pathogenesis of dental caries and other pathological processes occurring in tooth enamel. The application of modern high-technological methods of research has made it possible to study enamel structure in detail. Hard dental tissues are composed of organic and inorganic components and water. The organic substance consists of proteins, lipids, and carbohydrates. At different ages, caries intensity may vary. The carious process often develops during the first months after the tooth eruption but much less in adulthood and old age. These processes are mainly associated with the mechanisms of ionic exchange between the oral cavity and hard dental tissues. Different groups of teeth are differently affected by the carious process. Previous studies have revealed that age is an important factor influencing on the structure and chemical composition of hard dental tissues. Various recent works at the Department of Therapeutic Dentistry of Voronezh N.N. Burdenko State Medical Academy (VSMA) have studied not only the structural-morphological features of the enamel in children and adults but also the level of metabolic processes inside it. As a result of aging, teeth change both the enamel structure and the level of its ionic processes, but unfortunately, these changes have not been well characterized in teeth with conventional age-specific differences.

Huntington's disease (HD) is a neurological genetic disorder caused by the expansion of the CAG trinucleotide repeats (TNR) in the N-terminal region of coding sequence of the Huntingtin's (HTT) gene. This results in the addition of a poly-glutamine tract within the Huntingtin protein, resulting in its pathological form. The mechanism by which TRN expansion takes place is not yet fully understood. We have recently shown that DNA polymerase (Pol) β can promote the microhomology-mediated end joining and triplet expansion of a substrate mimicking a double strand break in the TNR region of the HTT gene. Here we show that TNR expansion is dependent on the structure of the DNA substrate, as well as on the two essential Pol β co-factors: flap endonuclease 1 (Fen1) and DNA ligase 1 (Lig1). We found that Fen1 significantly stimulated TNR expansion by Pol β, but not by the related enzyme Pol λ, and subsequent ligation of the DNA products by Lig1. Interestingly, the deletion of N-terminal domains of Pol λ, resulted in an enzyme which displayed properties more similar to Pol β, suggesting a possible evolutionary mechanism. These results may suggest a novel mechanism for somatic TNR expansion in HD.

A series of diterpenoid derivatives based on podocarpic acid were synthesized and evaluated as anti-influenza A virus agents. Several of the novel podocarpic acid derivatives exhibited nanomolar activities against an H1N1 influenza A virus (A/Puerto Rico/8/34) that was resistant to two anti-influenza drugs, oseltamivir and amantadine. This class of compounds inhibits the influenza virus by targeting the viral hemagglutinin-mediated membrane fusion. These results indicated that podocarpic acid derivatives may serve as potential drug candidates to fight drug-resistant influenza A virus infections.

Neuropsychiatric symptoms of Alzheimer's disease (NPS-AD) are highly prevalent and lead to poor medical and functional outcomes. In spite of the burdensome nature of NPS-AD, we are continuing to refine the nosology and only beginning to understand the underlying pathophysiology. Cluster analyses have frequently identified three to five subsyndromes of NPS-AD: behavioral dysfunction (for example, agitation/aggressiveness), psychosis (for example, delusions and hallucinations), and mood disturbance (for example, depression or apathy). Recent neurobiological studies have used new neuroimaging techniques to elucidate behaviorally relevant circuits and networks associated with these subsyndromes. Several fronto-subcortical circuits, cortico-cortical networks, and neurotransmitter systems have been proposed as regions and mechanisms underlying NPS-AD. Common to most of these subsyndromes is the broad overlap of regions associated with the salience network (anterior cingulate and insula), mood regulation (amygdala), and motivated behavior (frontal cortex). Treatment strategies for dysregulated mood syndromes (depression and apathy) have primarily targeted serotonergic mechanisms with antidepressants or dopaminergic mechanisms with psychostimulants. Psychotic symptoms have largely been targeted with anti-psychotic medications despite controversial risk/benefit tradeoffs. Management of behavioral dyscontrol, including agitation and aggression in AD, has encompassed a wide range of psychoactive medications as well as non-pharmacological approaches. Developing rational therapeutic approaches for NPS-AD will require a firmer understanding of the underlying etiology in order to improve nosology as well as provide the empirical evidence necessary to overcome regulatory and funding challenges to further study these debilitating symptoms.

A challenge lies ahead in ensuring that consultant workforce planning within Intensive Care Medicine meets changing and expanding health care needs at a time of financial constraint. During the development of an enlarging consultant intensivist workforce, it is important to explore existing practices to ensure subsequent contracts provide optimal work intensity and on-call frequency as well as appropriate SPA time for clinical governance and professional development. We conducted a survey across 14 deaneries, 43 ICUs and 398 consultant job plans to compare current working practices and set these against new guidelines and standards. It demonstrated that 93% of consultants work 10 PAs or more, with an average of 5.14 Direct Clinical Care PAs in Intensive Care and an average of 2.14 SPAs. Seven of 43 ICUs had consultant-to-patient ratios greater than 1:8 and 33 ICUs had insufficient resident cover overnight, highlighting challenges with trainee staffing and anticipated service reconfiguration.

<h4>Background</h4>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motor neuron degeneration in motor cortex, brainstem and spinal cord. microRNAs (miRNAs) are small non-coding RNAs that bind complementary target sequences and modulate gene expression; they are key molecules for establishing a neuronal phenotype, and in neurodegeneration. Here we investigated neural miR-9, miR-124a, miR-125b, miR-219, miR-134, and cell cycle-related miR-19a and -19b, in G93A-SOD1 mouse brain in pre-symptomatic and late stage disease.<h4>Results</h4>Expression of miR-9, miR-124a, miR-19a and -19b was significantly increased in G93A-SOD1 whole brain at late stage disease compared to B6.SJL and Wt-SOD1 control brains. These miRNAs were then analyzed in manually dissected SVZ, hippocampus, primary motor cortex and brainstem motor nuclei in 18-week-old ALS mice compared to same age controls. In SVZ and hippocampus miR-124a was up-regulated, miR-219 was down-regulated, and numbers of neural stem progenitor cells (NSPCs) were significantly increased. In G93A-SOD1 brainstem motor nuclei and primary motor cortex, miR-9 and miR-124a were significantly up-regulated, miR-125b expression was also increased. miR-19a and -19b were up-regulated in primary motor cortex and hippocampus, respectively. Expression analysis of predicted miRNA targets identified miRNA/target gene pairs differentially expressed in G93A-SOD1 brain regions compared to controls.<h4>Conclusions</h4>Hierarchical clustering analysis, identifying two clusters of miRNA/target genes, one characterizing brainstem motor nuclei and primary motor cortex, the other hippocampus and SVZ, suggests that altered expression of neural and cell cycle-related miRNAs in these brain regions might contribute to ALS pathogenesis in G93A-SOD1 mice. Re-establishing their expression to normal levels could be a new therapeutic approach to ALS.

With several therapeutic approaches in development for Huntington's disease, there is a need for easily accessible biomarkers to monitor disease progression and therapy response. We performed next-generation sequencing-based transcriptome analysis of total RNA from peripheral blood of 91 mutation carriers (27 presymptomatic and, 64 symptomatic) and 33 controls. Transcriptome analysis by DeepSAGE identified 167 genes significantly associated with clinical total motor score in Huntington's disease patients. Relative to previous studies, this yielded novel genes and confirmed previously identified genes, such as H2AFY, an overlap in results that has proven difficult in the past. Pathway analysis showed enrichment of genes of the immune system and target genes of miRNAs, which are downregulated in Huntington's disease models. Using a highly parallelized microfluidics array chip (Fluidigm), we validated 12 of the top 20 significant genes in our discovery cohort and 7 in a second independent cohort. The five genes (PROK2, ZNF238, AQP9, CYSTM1 and ANXA3) that were validated independently in both cohorts present a candidate biomarker panel for stage determination and therapeutic readout in Huntington's disease. Finally we suggest a first empiric formula predicting total motor score from the expression levels of our biomarker panel. Our data support the view that peripheral blood is a useful source to identify biomarkers for Huntington's disease and monitor disease progression in future clinical trials.

Mutations in more than 500 genes have been associated with intellectual disability (ID) and related disorders of cognitive function, such as autism and schizophrenia. Here we aimed to unravel the molecular epidemiology of non-specific ID in a genetic isolate using a combination of population and molecular genetic approaches. A large multigenerational pedigree was ascertained within a Dagestan Genetic Heritage research program in a genetic isolate of indigenous ethnics. Clinical characteristics of the affected members were based on combining diagnoses from regional psychiatric hospitals with our own clinical assessment, using a Russian translation of the structured psychiatric interviews, the Diagnostic Interview for Genetic Studies and the Family Interview for Genetic Studies, based on DSM-IV criteria. Weber/CHLC 9.0 STRs set was used for multipoint parametric linkage analyses (Simwalk2.91). Next, we checked CNVs and LOH (based on Affymetrix SNP 5.0 data) in the linked with ID genomic regions with the aim to identify candidate genes associated with mutations in linked regions. The number of statistically significant (p ≤ 0.05) suggestive linkage peaks with 1.3 < LOD < 3.0 we detected in a total of 10 genomic regions: 1q41, 2p25.3-p24.2, 3p13-p12.1, 4q13.3, 10p11, 11q23, 12q24.22-q24.31, 17q24.2-q25.1, 21q22.13 and 22q12.3-q13.1. Three significant linkage signals with LOD >3 were obtained at 2p25.3-p24.2 under the dominant model, with a peak at 21 cM flanked by loci D2S2976 and D2S2952; at 12q24.22-q24.31 under the recessive model, with a peak at -120 cM flanked by marker D12S2070 and D12S395 and at 22q12.3 under the dominant model, with a peak at 32 cM flanked by marker D22S683 and D22S445. After a set of genes had been designated as possible candidates in these specific chromosomal regions,we conducted an exploratory search for LOH and CNV based on microarray data to detect structural genomic variants within five ID-linked regions with LOD scores between 2.0 and 3.9. In these selected regions we obtained 173 ROH segments and 98 CN segments. Further analysis of region 2p25.3-p24.2 revealed deletions within genes encoding MYTL, SNTG2 and TPO among five of 21 affected cases at 2p25.3-p24.2. In the ID-linked region at 12q24.22-12q24.31 19 out of 21 ID cases carried segmental CNV and 20 of 21 them displayed ROH segments with mean size lengths for ID cases 2512 kb (500-6,472 kb) and for healthy control 682 kb (531-986 kb), including the genes MED13L, HRK, FBXW8, TESC, CDK2AP1 and SBNO1. Seven of 21 affected pedigree members displayed segmental deletions at 22q12.3 that includes the gene LARGE. Eight affected pedigree members carried ROH segments and 6 CN segments at 10p11.23-p11.21 containing the genes ZEB1, c10orf68 and EPC1. Our linkage and structural genomic variation analyses in a remote highland genetic isolate with aggregation of ID demonstrated that even highly isolated single kindred ID has oligo/polygenic pathogenesis. The results obtained implicate 10 genomic regions linked with ID that contain some of previously reported candidate genes, including HRK, FBXW8, TESC, CDK2AP1 and SBNO1 at 12q24 that were shown in recent studies as associated with brain measures derived from MRI scans.

Microbial systems often exhibit staggering diversity, making the study of rare, interesting species challenging. For example, metagenomic analyses of mixed-cell populations are often dominated by the sequences of the most abundant organisms, while those of rare microbes are detected only at low levels, if at all. To overcome this, selective cultivation or fluorescence-activated cell sorting (FACS) can be used to enrich for the target species prior to sequence analysis; however, since most microbes cannot be grown in the lab, cultivation strategies often fail, while cell sorting requires techniques to uniquely label the cell type of interest, which is often not possible with uncultivable microbes. Here, we introduce a culture-independent strategy for sorting microbial cells based on genomic content, which we term PCR-activated cell sorting (PACS). This technology, which utilizes the power of droplet-based microfluidics, is similar to FACS in that it uses a fluorescent signal to uniquely identify and sort target species. However, PACS differs importantly from FACS in that the signal is generated by performing PCR assays on the cells in microfluidic droplets, allowing target cells to be identified with high specificity with suitable design of PCR primers and TaqMan probes. The PACS assay is general, requires minimal optimization and, unlike antibody methods, can be developed without access to microbial antigens. Compared to non-specific methods in which cells are sorted based on size, granularity, or the ability to take up dye, PACS enables genetic sequence-specific sorting and recovery of the cell genomes. In addition to sorting microbes, PACS can be applied to eukaryotic cells, viruses, and naked nucleic acids.

In this work we have investigated the effects of strontium (Sr) dopant on in vitro protein release kinetics and in vivo osteogenic properties of plasma sprayed hydroxyapatite (HA) coatings, along with their dissolution behavior. Plasma sprayed HA coatings are widely used in load-bearing implants. Apart from osseointegration, the new generation of HA coating is expected to deliver biomolecules and/or drugs that can induce osteoinduction. This paper reports the preparation of crystalline and amorphous HA coatings on commercially pure titanium (Cp-Ti) using inductively coupled radio frequency (RF) plasma spray, and their stability at different solution pH. Coatings prepared at 110 mm working distance from the nozzle showed an average Ca ion release of 18 and 90 ppm in neutral and acidic environments, respectively. Decreasing the working distance to 90 mm resulted in the formation of a coating with less crystalline HA and phases with higher solubility products, and consequently higher dissolution over 32 days. A 92% release of a model protein bovine serum albumin (BSA) in phosphate buffer with pH of 7.4 was measured for Sr-doped HA (Sr-HA) coating, while only a 72% release could be measured for pure HA coating. Distortion of BSA during adsorption on coatings revealed a strong interaction between the protein and the coating, with an increase in α-helix content. Osteoid formation was found on Sr-HA implants as early as 7 weeks post implantation compared to HA coated and uncoated Ti implants. After 12 weeks post implantation, osteoid new bone was formed on HA implants; whereas, bone mineralization started on Sr-HA samples. While no osteoid was formed on bare Ti surfaces, bone was completely mineralized on HA and Sr-HA coatings after 16 weeks post implantation. Our results show that both phase stability and chemistry can have a significant influence toward in vitro and in vivo response of HA coatings on Ti implants.

Protein conformation diseases, including polyglutamine (polyQ) diseases, result from the accumulation and aggregation of misfolded proteins. Huntington's disease (HD) is one of nine diseases caused by an expanded polyQ repeat within the affected protein and is hallmarked by intracellular inclusion bodies composed of aggregated N-terminal huntingtin (Htt) fragments and other sequestered proteins. Fluorescence microscopy and filter trap assay are conventional methods to study protein aggregates, but cannot be used to analyze the presence and levels of post-translational modifications of aggregated Htt such as ubiquitination. Ubiquitination of proteins can be a signal for degradation and intracellular localization, but also affects protein activity and protein-protein interactions. The function of ubiquitination relies on its mono- and polymeric isoforms attached to protein substrates. Studying the ubiquitination pattern of aggregated Htt fragments offers an important possibility to understand Htt degradation and aggregation processes within the cell. For the identification of aggregated Htt and its ubiquitinated species, solubilization of the cellular aggregates is mandatory. Here we describe methods to identify post-translational modifications such as ubiquitination of aggregated mutant Htt. This approach is specifically described for use with mammalian cell culture and is suitable to study other disease-related proteins prone to aggregate.

It is suggested that genetic variations explain a significant portion of the variability in pain perception; therefore, increased understanding of pain-related genetic influences may identify new targets for therapies and treatments. The relative contribution of the different genes to the variance in clinical and experimental pain responses remains unknown. It is suggested that the genetic contributions to pain perception vary across pain modalities. For example, it has been suggested that more than 60% of the variance in cold pressor responses can be explained by genetic factors; in comparison, only 26% of the variance in heat pain responses is explained by these variations. Thus, the selection of pain model might markedly influence the magnitude of the association between the pain phenotype and genetic variability. Thermal pain sensation is complex with multiple molecular and cellular mechanisms operating alone and in combination within the peripheral and central nervous system. It is thus highly probable that the thermal pain experience is affected by genetic variants in one or more of the pathways involved in the thermal pain signaling. This review aims to present and discuss some of the genetic variations that have previously been associated with different experimental thermal pain models.

Peroxiredoxin 6 (Prdx6), an important antioxidant enzyme that can eliminate reactive oxygen species (ROS) to maintain homeostasis, is a bifunctional protein that possesses the activities of both glutathione peroxidase and phospholipase A2. In this study, a novel full-length Prdx6 cDNA (OaPrdx6) was cloned from Sheep (Ovis aries) using rapid amplification of cDNA ends (RACE). The full-length cDNA of OaPrdx6 was 1753bp containing a 5'-untranslated region (UTR) of 93bp, a 3'-UTR of 985bp with a poly(A) tail, and an open reading frame (ORF) of 675bp encoding a protein of 224 amino acid residues with a predicted molecular weight of 25.07kDa. The recombinant protein OaPrdx6 was expressed and purified, and its DNA protection activity was identified. In order to analyze the Prdx6 protein expression in tissues from O. aries, monoclonal antibodies against OaPrdx6 were prepared. Western blotting results indicated that OaPrdx6 protein could be detected in heart, liver, spleen, lung, kidney, stomach, intestine, muscle, lymph node and white blood cells, and the highest expression was found in lung while the lowest expression in muscle. Compared to the normal sheep group, the mRNA transcription level of Prdx6 in buffy coat was up-regulated in the group infected with a virulent field strain of Brucella melitensis, and down-regulated in the group inoculated with a vaccine strain S2 of brucellosis. The results indicated that Prdx6 was likely to be involved in the host immune responses against Brucella infection, and probably regarded as a molecular biomarker for distinguishing between animals infected with virulent Brucella infection and those inoculated with vaccine against brucellosis.

<h4>Purpose</h4>In this study we hypothesized that the mRNA vector Staufen mediates RNA relocalization during meiotic maturation, and by virtue of its interactions with endoplasmic reticulum, provides a possible mechanism by which protein synthesis is regulated.<h4>Methods</h4>We assessed the expression of staufen (STAU) and calreticulin (CALR), the latter adopted as a marker of the endoplasmic reticulum, in human oocytes at different stages of maturation: GV, metaphase MI and MII. Oocytes were subjected to polymerase chain reaction in order to investigate the expression of STAU and CALR. The corresponding protein products were identified by immunofluorescence and confocal laser scanning microscopy.<h4>Results</h4>STAU and CALR were constantly expressed and selectively localized during oocyte maturation. At the GV stage the both proteins displayed a dispersed distribution localization throughout the cytoplasm. Progressing to the MII stage, STAU tended to compartmentalize towards the cortical area of the oocyte clustering in granules of larger sizes. At the MII stage, CALR assumed a pattern reminiscent and possibly coincident with the position of the meiotic spindle.<h4>Conclusions</h4>The changing pattern of STAU distribution during meiotic maturation of human oocytes implicates a novel mechanism for the regulation of protein synthesis based on mRNA localization. Moreover, the unique disposition of CALR at the MII spindle uncovers a physical interaction with endoplasmic reticulum that may mediate cytoskeletal remodelling during oocyte maturation.

<h4>Background</h4>Despite the easy accessibility and diagnostic utility of PBMCs and their potential to show distinct expression patterns associated with the accelerated disease progression in HIV/HCV co-infection, there has not been a systematic study focusing on the global dysregulations of the biological pathways in PBMCs from HIV, HCV mono- and co-infected individuals. This study aimed at identifying the transcriptome distinctions of PBMCs between these patient groups.<h4>Methods</h4>Genome-wide transcriptomes of PBMCs from 10 HIV/HCV co-infected patients, 7 HIV+ patients, 5 HCV+ patients, and 5 HIV/HCV sero-negative healthy controls were analyzed using Illumina microarray. Pairwise comparisons were performed to identify differentially expressed genes (DEGs), followed by gene set enrichment analysis (GSEA) to detect the global dysregulations of the biological pathways between HIV, HCV mono- and co-infection.<h4>Results</h4>Forty-one, 262, and 44 DEGs with fold change > 1.5 and FDR (false discovery rate) <0.05 for the comparisons of HCV versus co-infection, HIV versus co-infection, and HIV versus HCV were identified, respectively. Significantly altered pathways (FDR < 0.05), featured by those involved in immune system, signaling transduction, and cell cycle, were detected. Notably, the differential regulation of cytotoxicity pathway discriminated between HIV, HCV mono- and co-infection (up-regulated in the former versus the latter group: co-infection versus HIV or HCV, HIV versus HCV; FDR <0.001 ~ 0.019). Conversely, the cytokine-cytokine receptor interaction pathway was down-regulated in co-infection versus either HCV (FDR = 0.003) or HIV (FDR = 0.028). For the comparison of HIV versus HCV, the cell cycle (FDR = 0.016) and WNT signaling (FDR = 0.006) pathways were up- and down-regulated in HIV, respectively.<h4>Conclusions</h4>Our study is the first to identify the differential regulation of cytotoxicity pathway discriminating between HIV, HCV mono- and co-infection, which may reflect the distinct patterns of virus-host cell interactions underlying disease progression. Further inspection of cytotoxicity pathway has pinned down to the expression of the KIR genes to be associated with specific patterns of particular virus-host interactions. Between HIV and HCV, the altered cell cycle and WNT signaling pathways may suggest the different impact of HIV and HCV on cell proliferation and differentiation.

<h4>Background</h4>Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.<h4>Methods</h4>The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey's post-hoc test were used in the analysis of data.<h4>Results</h4>MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers.<h4>Conclusion</h4>Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.

<h4>Background</h4>Ubiquitination is a highly dynamic and reversible process with a central role in cell homeostasis. Deregulation of several deubiquitinating enzymes has been linked to tumor development but their specific role in prostate cancer progression remains unexplored.<h4>Methods</h4>RNAi screening was used to investigate the role of the ovarian tumor proteases (OTU) family of deubiquitinating enzymes on the proliferation and invasion capacity of prostate cancer cells. RhoA activity was measured in relation with OTUB1 effects on prostate cancer cell invasion. Tumor xenograft mouse model with stable OTUB1 knockdown was used to investigate OTUB1 influence in tumor growth.<h4>Results</h4>Our RNAi screening identified OTUB1 as an important regulator of prostate cancer cell invasion through the modulation of RhoA activation. The effect of OTUB1 on RhoA activation is important for androgen-induced repression of p53 expression in prostate cancer cells. In localized prostate cancer tumors OTUB1 was found overexpressed as compared to normal prostatic epithelial cells. Prostate cancer xenografts expressing reduced levels of OTUB1 exhibit reduced tumor growth and reduced metastatic dissemination in vivo.<h4>Conclusions</h4>OTUB1 mediates prostate cancer cell invasion through RhoA activation and promotes tumorigenesis in vivo. Our results suggest that drugs targeting the catalytic activity of OTUB1 could potentially be used as therapeutics for metastatic prostate cancer.

Atresia, or absence, of the inferior vena cava (AIVC) is a rare, usually fortuitous finding on advanced imaging that predisposes patients to deep venous thrombosis (DVT). In young adults who lack predisposing risk factors but nonetheless develop extensive or bilateral DVTs, AIVC should be considered. We describe a case of a previously healthy 17-year-old male patient who developed an extensive renal vein thrombus due to the absence of the superior portion of his IVC compounded with previously undiagnosed antithrombin III deficiency. We discuss the diagnosis and management of this complicated condition.

The major cholesterol metabolite in brain, 24(S)-hydroxycholesterol (24S-HC), serves as a vehicle for cholesterol removal. Its effects on neuronal function, however, have only recently begun to be investigated. Here, we review that nascent work. Our own studies have demonstrated that 24S-HC has potent positive modulatory effects on N-methyl-d-aspartate (NMDA) receptor (NMDAR) function. This could have implications not only for brain plasticity but also for pathological NMDAR overuse. Other work has demonstrated effects of 24S-HC on neuronal survival and as a possible biomarker of neurodegenerative disease. Depending on circumstances, both upregulation/mimicry of 24S-HC signaling and down-regulation/antagonism may have therapeutic potential. We are interested in the possibility that synthetic analogues of 24S-HC with positive effects at NMDARs may hold neurotherapeutic promise, given the role of NMDA receptor hypofunction in certain neuropsychiatric disorders.

Hoyeraal-Hreidarsson syndrome (HHS) is a severe form of Dyskeratosis congenita characterized by developmental defects, bone marrow failure and immunodeficiency and has been associated with telomere dysfunction. Recently, mutations in Regulator of Telomere ELongation helicase 1 (RTEL1), a helicase first identified in Mus musculus as being responsible for the maintenance of long telomeres, have been identified in several HHS patients. Here we show that RTEL1 is required for the export and the correct cytoplasmic trafficking of the small nuclear (sn) RNA pre-U2, a component of the major spliceosome complex. RTEL1-HHS cells show abnormal subcellular partitioning of pre-U2, defects in the recycling of ribonucleotide proteins (RNP) in the cytoplasm and splicing defects. While most of these phenotypes can be suppressed by re-expressing the wild-type protein in RTEL1-HHS cells, expression of RTEL1 mutated variants in immortalized cells provokes cytoplasmic mislocalizations of pre-U2 and other RNP components, as well as splicing defects, thus phenocopying RTEL1-HHS cellular defects. Strikingly, expression of a cytoplasmic form of RTEL1 is sufficient to correct RNP mislocalizations both in RTEL1-HHS cells and in cells expressing nuclear mutated forms of RTEL1. This work unravels completely unanticipated roles for RTEL1 in RNP trafficking and strongly suggests that defects in RNP biogenesis pathways contribute to the pathology of HHS.

Nucleotides are required for a wide variety of biological processes and are constantly synthesized de novo in all cells. When cells proliferate, increased nucleotide synthesis is necessary for DNA replication and for RNA production to support protein synthesis at different stages of the cell cycle, during which these events are regulated at multiple levels. Therefore the synthesis of the precursor nucleotides is also strongly regulated at multiple levels. Nucleotide synthesis is an energy intensive process that uses multiple metabolic pathways across different cell compartments and several sources of carbon and nitrogen. The processes are regulated at the transcription level by a set of master transcription factors but also at the enzyme level by allosteric regulation and feedback inhibition. Here we review the cellular demands of nucleotide biosynthesis, their metabolic pathways and mechanisms of regulation during the cell cycle. The use of stable isotope tracers for delineating the biosynthetic routes of the multiple intersecting pathways and how these are quantitatively controlled under different conditions is also highlighted. Moreover, the importance of nucleotide synthesis for cell viability is discussed and how this may lead to potential new approaches to drug development in diseases such as cancer.

Mutations in the mtDNA genome have long been suspected to play an important role in cancer. Although most cancer cells harbor mtDNA mutations, the question of whether such mutations are associated with clinical prognosis of lung cancer remains unclear. We resequenced the entire mitochondrial genomes of tumor tissue from a population of 250 Korean patients with non-small cell lung cancer (NSCLC). Our analysis revealed that the haplogroup (D/D4) was associated with worse overall survival (OS) of early-stage NSCLC [adjusted hazard ratio (AHR), 1.95; 95% CI, 1.14-3.33; P trend = 0.03]. By comparing the mtDNA variations between NSCLC tissues and matched blood samples, we found that haplogroups M/N and/or D/D4 were hotspots for somatic mutations, suggesting a more complicated mechanism of mtDNA somatic mutations other than the commonly accepted mechanism of sequential accumulation of mtDNA mutations.

<h4>Background</h4>The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification.<h4>Methods</h4>The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres.<h4>Results</h4>Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine.<h4>Conclusions</h4>The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation in the zebrafish, a role similar to that ascribed to its murine ortholog.

A rapid increase in utility of engineered nanomaterials, including carbon nanotubes (CNTs), has raised a concern over their safety. Based on recent evidence from animal studies, pulmonary exposure of CNTs may lead to nanoparticle accumulation in the deep lung without effective clearance which could interact with local lung cells for a long period of time. Physicochemical similarities of CNTs to asbestos fibers may contribute to their asbestos-like carcinogenic potential after long-term exposure, which has not been well addressed. More studies are needed to identify and predict the carcinogenic potential and mechanisms for promoting their safe use. Our previous study reported a long-term in vitro exposure model for CNT carcinogenicity and showed that 6-month sub-chronic exposure of single-walled carbon nanotubes (SWCNT) causes malignant transformation of human lung epithelial cells. In addition, the transformed cells induced tumor formation in mice and exhibited an apoptosis resistant phenotype, a key characteristic of cancer cells. Although the potential role of p53 in the transformation process was identified, the underlying mechanisms of oncogenesis remain largely undefined. Here, we further examined the gene expression profile by using genome microarrays to profile molecular mechanisms of SWCNT oncogenesis. Based on differentially expressed genes, possible mechanisms of SWCNT-associated apoptosis resistance and oncogenesis were identified, which included activation of pAkt/p53/Bcl-2 signaling axis, increased gene expression of Ras family for cell cycle control, Dsh-mediated Notch 1, and downregulation of apoptotic genes BAX and Noxa. Activated immune responses were among the major changes of biological function. Our findings shed light on potential molecular mechanisms and signaling pathways involved in SWCNT oncogenic potential.

Identification of molecular-based signatures is one of the critical steps toward finding therapeutic targets in cancer. In this paper, we propose methods to discover prognostic gene signatures under a causal structure learning framework across the whole genome. The causal structures are represented by directed acyclic graphs (DAGs), wherein we construct gene-specific network modules that constitute a gene and its corresponding regulators. The modules are then subsequently used to correlate with survival times, thus, allowing for a network-oriented approach to gene selection to adjust for potential confounders, as opposed to univariate (gene-by-gene) approaches. Our methods are motivated by and applied to a clear cell renal cell carcinoma (ccRCC) study from The Cancer Genome Atlas (TCGA) where we find several prognostic genes associated with cancer progression - some of which are novel while others confirm existing findings.

<h4>Introduction</h4>There is paucity of data on tuberculosis and antituberculous therapy (ATT) induced hepatotoxicity in patients with chronic liver disease.<h4>Aim</h4>To study demographic, clinical characteristics of tuberculosis, pattern of drug induced liver injury and treatment responses to ATT in patients with liver cirrhosis.<h4>Material and method</h4>All cases of liver cirrhosis diagnosed with tuberculosis (TB) between January 2010 and June 2013 were enrolled. Drug induced liver injury (DILI) was defined as follows (1) an aspartate aminotransferase (AST)/alanine aminotransferase (ALT) value exceeding 3 times the normal upper limit if the baseline level was normal (<40 IU/L), or an AST/ALT exceeding 2 times the baseline level if the baseline level was abnormal and an absolute increase in serum bilirubin >2 mg/dl from baseline.<h4>Results</h4>Sixty seven patients had confirmed TB with underlying cirrhosis and formed the study group. The mean age was 52 ± 12 years and M:F ratio was 57:10. Mean Child Turcotte Pugh (CTP) score 8.5 ± 1.5 (CTP A:B:C:7:44:16). The sites of TB included: pulmonary (25, 37%); pleural effusion (10, 16%) peritoneal (19, 29%); chest lymph nodes (3, 4%); liver (1, 1.5%); intestines (3, 4%), vertebra (3, 4%), brain (1, 1.5%) and disseminated (2, 3%). Thus, extrapulmonary TB was more common in the cirrhotic patients as compared to pulmonary TB. Patients with Child's status A (n = 7) received 4 drugs (R: rifampicin, H: Isoniazid, E: ethambutol, Z: pyrizinamide) and could tolerate well even during follow up without any drug induced toxicity. In rest of patients commonest regimen followed was combination of drugs (RHEO, n = 32) followed by RHE (n = 11). DILI occurred in 35% started with either RHEO, HEO and REO. Median time of onset of DILI was 12 days (4-34) days. There was no DILI related death during hospital stay or follow up.<h4>Conclusions</h4>Extrapulmonary TB is common in patients with cirrhosis and DILI is common in Child B and C with combination of rifampicin and isoniazid regimen.

Cullin 3 (Cul3)-family ubiquitin ligases use the BTB-domain-containing proteins for the recruitment of substrates, but the regulation of this family of ubiquitin ligases has not been completely understood. KLHL20 is a BTB-family protein and targets tumor suppressor promyelocytic leukemia protein (PML) and death-associated protein kinase (DAPK) to its kelch-repeat domain for ubiquitination and degradation. Here, we show that another BTB-kelch protein KLHL39 is recruited to the substrate-binding domain of KLHL20 but is not a substrate of Cul3-KLHL20 complex. Interestingly, KLHL39 does not bind Cul3 because of the absence of certain conserved residues in the BTB domain. Instead, KLHL39 blocks KLHL20-mediated ubiquitination of PML and DAPK by disrupting the binding of these substrates to KLHL20 as well as the binding of KLHL20 to Cul3. Through the two mechanisms, KLHL39 increases the stability of PML and DAPK. In human colon cancers, downregulations of KLHL39, PML and DAPK are associated with metastatic progression. Furthermore, preclinical data indicate that KLHL39 promotes colon cancer migration, invasion and survival in vitro and metastasis in vivo through a PML- and DAPK-dependent mechanism. Our study identifies KLHL39 as a negative regulator of Cul3-KLHL20 ubiquitin ligase and reveals a role of KLHL39-mediated PML and DAPK stabilization in colon cancer metastasis.

Leukocyte common antigen-related receptor protein tyrosine phosphatases--comprising LAR, PTPδ, and PTPσ--are synaptic adhesion molecules that organize synapse development. Here, we identify glypican 4 (GPC-4) as a ligand for PTPσ. GPC-4 showed strong (nanomolar) affinity and heparan sulfate (HS)-dependent interaction with the Ig domains of PTPσ. PTPσ bound only to proteolytically cleaved GPC-4 and formed additional complex with leucine-rich repeat transmembrane protein 4 (LRRTM4) in rat brains. Moreover, single knockdown (KD) of PTPσ, but not LAR, in cultured neurons significantly reduced the synaptogenic activity of LRRTM4, a postsynaptic ligand of GPC-4, in heterologous synapse-formation assays. Finally, PTPσ KD dramatically decreased both the frequency and amplitude of excitatory synaptic transmission. This effect was reversed by wild-type PTPσ, but not by a HS-binding-defective PTPσ mutant. Our results collectively suggest that presynaptic PTPσ, together with GPC-4, acts in a HS-dependent manner to maintain excitatory synapse development and function.

BRAF (v-raf murine sarcoma viral oncogene homolog B) inhibitors elicit a transient anti-tumor response in ∼ 80% of BRAF(V600)-mutant melanoma patients that almost uniformly precedes the emergence of resistance. Here we used a mouse model of melanoma in which melanocyte-specific expression of Braf(V618E) (analogous to the human BRAF(V600E) mutation) led to the development of skin hyperpigmentation and nevi, as well as melanoma formation with incomplete penetrance. Sleeping Beauty insertional mutagenesis in this model led to accelerated and fully penetrant melanomagenesis and synchronous tumor formation. Treatment of Braf(V618E) transposon mice with the BRAF inhibitor PLX4720 resulted in tumor regression followed by relapse. Analysis of transposon insertions identified eight genes including Braf, Mitf, and ERas (ES-cell expressed Ras) as candidate resistance genes. Expression of ERAS in human melanoma cell lines conferred resistance to PLX4720 and induced hyperphosphorylation of AKT (v-akt murine thymoma viral oncogene homolog 1), a phenotype reverted by combinatorial treatment with PLX4720 and the AKT inhibitor MK2206. We show that ERAS expression elicits a prosurvival signal associated with phosphorylation/inactivation of BAD, and that the resistance of hepatocyte growth factor-treated human melanoma cells to PLX4720 can be reverted by treatment with the BAD-like BH3 mimetic ABT-737. Thus, we define a role for the AKT/BAD pathway in resistance to BRAF inhibition and illustrate an in vivo approach for finding drug resistance genes.

<h4>Background</h4>Gastric cancer (GC) is an aggressive malignancy whose mechanisms of development and progression are poorly understood. The identification of prognosis-related genomic loci and genes may suffer from the relatively small case numbers and a lack of systematic validation in previous studies.<h4>Methods</h4>Array-based comparative genomic hybridization (aCGH) coupled with patient clinical information was applied to identify prognosis-related loci and genes with high-frequency recurrent gains in 129 GC patients. The candidate loci and genes were then validated using an independent cohort of 384 patients through branched DNA signal amplification analysis (QuantiGene assays).<h4>Results</h4>In the 129 patients, a copy number gain of three chromosome regions-namely, 8q22 (including ESRP1 and CCNE2), 8q24 (including MYC and TNFRSF11B), and 20q11-q13 (including SRC, MMP9, and CSE1L)--conferred poor survival for patients. In addition, the correlation between the branched DNA signal amplification analysis results and the aCGH results was analyzed in 73 of these 129 patients, and MYC, TNFRSF11B, ESRP1, CSE1L, and MMP9 were found to be well correlated. Further validation using an independent cohort (n = 384) verified that only MYC and TNFRSF11B within 8q24 are related to survival. Patients with gains in both MYC and TNFRSF11B had poorer survival than those with no gains, particularly those with noncardia GC. Gains in both of these genes were also a significant independent prognostic indicator.<h4>Conclusions</h4>Our results revealed that copy number gains in MYC and TNFRSF11B located at 8q24 are associated with survival in GC, particularly noncardia GC.

Over the past years, certain "vulnerability genes" have been identified that play a key role in the development of mood and anxiety disorders. In particular, a low-expressing variant of the human serotonin transporter (5-HTT) gene has been described that renders individuals more susceptible to adverse experience and hence to the development of psychiatric diseases. However, some authors have recently argued that lower 5-HTT expression not only increases vulnerability to adverse experiences, but also enhances susceptibility to beneficial experiences, thus promoting phenotypic plasticity. The aim of the present study was to assess the effects of 5-HTT expression on susceptibility to beneficial experience in a hypothesis-driven experimental approach. Using a well-established rodent model for the human polymorphism, male heterozygous 5-HTT knockout (HET) and 5-HTT wildtype (WT) mice were either provided with the beneficial experience of cohabitation with a female (mating experience) or kept as naïve controls in single-housing conditions. Following the experimental treatment, they were tested for their anxiety-like behaviour and exploratory locomotion in three widely used behavioural tests. Interestingly, while cohabitation reduced anxiety-like behaviour and increased exploratory locomotion in the open field test in HET mice, it did not affect WT mice, pointing to a genotype-dependent susceptibility to the beneficial experience. Thus, our results might support the view of the low expressing version of the 5-HTT gene as a "plasticity" rather than a "vulnerability" variant.

Transcriptional mechanisms mediated by the binding of transcription factors (TFs) to cis-acting regulatory elements (CREs) in DNA play crucial roles in directing gene expression. While TFs have been extensively studied, less effort has gone towards the identification and functional characterization of CREs and associated epigenetic modulation. However, owing to methodological and analytical advances, more comprehensive studies of regulatory elements and mechanisms are now possible. We summarize recent progress in integrative analyses of these regulatory components in the development of the cerebral neocortex, the part of the brain involved in cognition and complex behavior. These studies are uncovering not only the underlying transcriptional regulatory networks, but also how these networks are altered across species and in neurological and psychiatric disorders.

<h4>Background</h4>Longitudinal research is critical for understanding the biological mechanisms underlying the development of depression. Researchers recruit high-risk cohorts to understand how risk is transmitted from one generation to the next. Biological measurements have been incorporated into these longitudinal high-risk (LHR) studies in order to illuminate mechanistic pathways.<h4>Methods</h4>To frame our review, we first present heritability estimates along the gene-by-environment continuum as a foundation. We then offer a Biomarkers of Intergenerational Risk for Depression (BIRD) model to describe the multiple hits individuals at risk receive and to allow for greater focus on the interactive effects of markers. BIRD allows for the known multifinality of pathways towards depression and considers the context (i.e., environment) in which these mechanisms emerge. Next, we review the extant LHR cohort studies that have assessed central nervous system (electroencephalography (EEG), neuroimaging), endocrine (hypothalamic-pituitary-adrenal axis (HPA)/cortisol), autonomic (startle, heart rate), genetic, sleep, and birth characteristics.<h4>Results</h4>Results to date, in conjunction with the proposed model, point towards several pathways of discovery in understanding mechanisms, providing clear direction for future research examining potential endophenotypes.<h4>Limitations</h4>Our review is based on relatively narrow inclusion and exclusion criteria. As such, many interesting studies were excluded, but this weakness is offset by strengths such as the increased reliability of findings.<h4>Conclusions</h4>Blanket prevention programs are inefficient and plagued by low effect sizes due to low rates of actual conversion to disorder. The inclusion of biomarkers of risk may lead to enhanced program efficiency by targeting those at greatest risk.

<h4>Unlabelled</h4>Functional inactivation of HFE or hemojuvelin (HJV) is causatively linked to adult or juvenile hereditary hemochromatosis, respectively. Systemic iron overload results from inadequate expression of hepcidin, the iron regulatory hormone. While HJV regulates hepcidin by amplifying bone morphogenetic protein (BMP) signaling, the role of HFE in the hepcidin pathway remains incompletely understood. We investigated the pathophysiological implications of combined Hfe and Hjv ablation in mice. Isogenic Hfe (-)/(-) and Hjv (-)/(-) mice were crossed to generate double Hfe (-)/(-) Hjv (-)/(-) progeny. Wild-type control and mutant mice of all genotypes were analyzed for serum, hepatic, and splenic iron content, expression of iron metabolism proteins, and expression of hepcidin and Smad signaling in the liver, in response to a standard or an iron-enriched diet. As expected, Hfe (-)/(-) and Hjv (-)/(-) mice developed relatively mild or severe iron overload, respectively, which corresponded to the degree of hepcidin inhibition. The double Hfe (-)/(-) Hjv (-)/(-) mice exhibited an indistinguishable phenotype to single Hjv (-)/(-) counterparts with regard to suppression of hepcidin, serum and hepatic iron overload, splenic iron deficiency, tissue iron metabolism, and Smad signaling, under both dietary regimens. We conclude that the hemochromatotic phenotype caused by disruption of Hjv is not further aggravated by combined Hfe/Hjv deficiency. Our results provide genetic evidence that Hfe and Hjv operate in the same pathway for the regulation of hepcidin expression and iron metabolism.<h4>Key messages</h4>Combined disruption of Hfe and Hjv phenocopies single Hjv deficiency. Single Hjv(-)/(-) and double Hfe(-)/(-)Hjv(-)/(-) mice exhibit comparable iron overload. Hfe and Hjv regulate hepcidin via the same pathway.

A small set of gastric adenocarcinomas (9%) harbor Epstein-Barr virus (EBV) DNA within malignant cells, and the virus is not an innocent bystander but rather is intimately linked to pathogenesis and tumor maintenance. Evidence comes from unique genomic features of host DNA, mRNA, microRNA and CpG methylation profiles as revealed by recent comprehensive genomic analysis by The Cancer Genome Atlas Network. Their data show that gastric cancer is not one disease but rather comprises four major classes: EBV-positive, microsatellite instability (MSI), genomically stable and chromosome instability. The EBV-positive class has even more marked CpG methylation than does the MSI class, and viral cancers have a unique pattern of methylation linked to the downregulation of CDKN2A (p16) but not MLH1. EBV-positive cancers often have mutated PIK3CA and ARID1A and an amplified 9p24.1 locus linked to overexpression of JAK2, CD274 (PD-L1) and PDCD1LG2 (PD-L2). Multiple noncoding viral RNAs are highly expressed. Patients who fail standard therapy may qualify for enrollment in clinical trials targeting cancer-related human gene pathways or promoting destruction of infected cells through lytic induction of EBV genes. Genomic tests such as the GastroGenus Gastric Cancer Classifier are available to identify actionable variants in formalin-fixed cancer tissue of affected patients.

<h4>Background</h4>The analgesic efficacy of opioids is well known to vary widely among individuals, and various factors related to individual differences in opioid sensitivity have been identified. However, a prediction model to calculate appropriate opioid analgesic requirements has not yet been established. The present study sought to construct prediction formulas for individual opioid analgesic requirements based on genetic polymorphisms and clinical data from patients who underwent cosmetic orthognathic surgery and validate the utility of the prediction formulas in patients who underwent major open abdominal surgery.<h4>Methods</h4>To construct the prediction formulas, we performed multiple linear regression analyses using data from subjects who underwent cosmetic orthognathic surgery. The dependent variable was 24-h postoperative or perioperative fentanyl use, and the independent variables were age, gender, height, weight, pain perception latencies (PPL), and genotype data of five single-nucleotide polymorphisms (SNPs). To examine the utility of the prediction formulas, we performed simple linear regression analyses using subjects who underwent major open abdominal surgery. Actual 24-h postoperative or perioperative analgesic use and the predicted values that were calculated using the multiple regression equations were incorporated as dependent and independent variables, respectively.<h4>Results</h4>Multiple linear regression analyses showed that the four SNPs, PPL, and weight were retained as independent predictors of 24-h postoperative fentanyl use (R² = 0.145, P = 5.66 × 10⁻¹⁰) and the two SNPs and weight were retained as independent predictors of perioperative fentanyl use (R² = 0.185, P = 1.99 × 10⁻¹⁵). Simple linear regression analyses showed that the predicted values were retained as an independent predictor of actual 24-h postoperative analgesic use (R² = 0.033, P = 0.030) and perioperative analgesic use (R² = 0.100, P = 1.09 × 10⁻⁴), respectively.<h4>Conclusions</h4>We constructed prediction formulas, and the possible utility of these prediction formulas was found in another type of surgery.

<h4>Objective</h4>Biological risk factors underlying psychosis are poorly understood. Biological underpinnings of the dimension of psychosis can be derived using genetic associations with intermediate phenotypes such as subcomponents of auditory event-related potentials (ERPs). Various ERP subcomponent abnormalities in schizophrenia and psychotic bipolar disorder are heritable and are expressed in unaffected relatives, although studies investigating genetic contributions to ERP abnormalities are limited. The authors used a novel parallel independent component analysis (para-ICA) to determine which empirically derived gene clusters are associated with data-driven ERP subcomponents, assuming a complex etiology underlying psychosis.<h4>Method</h4>The authors examined the multivariate polygenic association of ERP subcomponents from 64-channel auditory oddball data in 144 individuals with schizophrenia, 210 psychotic bipolar disorder probands, and 95 healthy individuals from the multisite Bipolar-Schizophrenia Network on Intermediate Phenotypes study. Data were reduced by principal components analysis to two target and one standard ERP waveforms. Multivariate association of compressed ERP waveforms with a set of 20,329 single-nucleotide polymorphisms (SNPs) (reduced from a 1-million-SNP array) was examined using para-ICA. Genes associated with SNPs were further examined using pathway analysis tools.<h4>Results</h4>Para-ICA identified four ERP components that were significantly correlated with three genetic components. Enrichment analysis revealed complement immune response pathway and multiple processes that significantly mediate ERP abnormalities in psychosis, including synaptic cell adhesion, axon guidance, and neurogenesis.<h4>Conclusions</h4>This study identified three genetic components comprising multiple genes mediating ERP subcomponent abnormalities in schizophrenia and psychotic bipolar disorder. The data suggest a possible polygenic structure comprising genes influencing key neurodevelopmental processes, neural circuitry, and brain function mediating biological pathways plausibly associated with psychosis.

Neural circuits in the medial entorhinal cortex (MEC) encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations.

Intermediate beta-thalassemia has a broad spectrum of sequelae and affected subjects may require occasional blood transfusions over their lifetime to correct anemia. Iron overload in intermediate beta-thalassemia results from a paradoxical intestinal absorption, iron release from macrophages and hepatocytes, and sporadic transfusions. Pathological iron accumulation in parenchyma is caused by chronic exposure to non-transferrin bound iron in plasma. The iron scavenger and transport protein transferrin is a potential treatment being studied for correction of anemia. However, transferrin may also function to prevent or reduce iron loading of tissues when exposure to non-transferrin bound iron increases. Here we evaluate the effects of apotransferrin administration on tissue iron loading and early tissue pathology in non-transfused and transfused Hbb(th3/+) mice. Mice with the Hbb(th3/+) phenotype have mild to moderate anemia and consistent tissue iron accumulation in the spleen, liver, kidneys and myocardium. Chronic apotransferrin administration resulted in normalization of the anemia. Furthermore, it normalized tissue iron content in the liver, kidney and heart and attenuated early tissue changes in non-transfused Hbb(th3/+) mice. Apotransferrin treatment was also found to attenuate transfusion-mediated increases in plasma non-transferrin bound iron and associated excess tissue iron loading. These therapeutic effects were associated with normalization of transferrin saturation and suppressed plasma non-transferrin bound iron. Apotransferrin treatment modulated a fundamental iron regulatory pathway, as evidenced by decreased erythroid Fam132b gene (erythroferrone) expression, increased liver hepcidin gene expression and plasma hepcidin-25 levels and consequently reduced intestinal ferroportin-1 in apotransferrin-treated thalassemic mice.

Over the past decade, increasing evidence has implied a significant connection between caspase-6 activity and the pathogenesis of Huntington's disease (HD). Consequently, inhibiting caspase-6 activity was suggested as a promising therapeutic strategy to reduce mutant Huntingtin toxicity, and to provide protection from mutant Huntingtin-induced motor and behavioral deficits. Here, we describe a novel caspase-6 inhibitor peptide based on the huntingtin caspase-6 cleavage site, fused with a cell-penetrating sequence. The peptide reduces mutant Huntingtin proteolysis by caspase-6, and protects cells from mutant Huntingtin toxicity. Continuous subcutaneous administration of the peptide protected pre-symptomatic BACHD mice from motor deficits and behavioral abnormalities. Moreover, administration of the peptide in an advanced disease state resulted in the partial recovery of motor performance, and an alleviation of depression-related behavior and cognitive deficits. Our findings reveal the potential of substrate-based caspase inhibition as a therapeutic strategy, and present a promising agent for the treatment of HD.

Mesenchymal-epithelial transitions (METs) are important in both development and the growth of secondary tumours. Although the molecular basis for epithelial polarity is well studied, less is known about the cues that induce MET. Here we show that Netrins, well known as chemotropic guidance factors, provide a basal polarising cue during the Drosophila midgut MET. Both netrinA and netrinB are expressed in the visceral mesoderm, the substrate upon which midgut cells migrate, while their receptor frazzled (fra) is expressed in midgut cells. Netrins are required to polarise Fra to the basal surface, and Netrins and Fra undergo mutually-dependent endocytosis, with Fra subsequently trafficking to late endosomes. Mutations to fra and netrins affect both migration and MET but to different degrees. Loss of fra strongly delays migration, midgut cells fail to extend protrusions, and apico-basal polarisation of proteins and epithelium formation is inhibited. In netrin mutants, the migration phenotype is weaker and cells still extend protrusions. However, apico-basal polarisation of proteins, including Fra, and FActin is greatly disrupted and a monolayer fails to form. Delocalised accumulations of FActin are prevalent in netrin mutants but not fra mutants suggesting delocalised Fra may disrupt the MET. βPS localisation is also affected in netrin mutants in that a basal gradient is reduced while localisation to the midgut/VM interface is increased. Since a similar effect is seen when endocytosis is inhibited, Netrin and Fra may regulate Integrin turnover. The results suggest Netrin-dependent basal polarisation of Fra is critical for the formation of an epithelium.

Prostacyclin synthase (CYP8A1) is an enzyme responsible for the biosynthesis of prostacyclin (PGI2) which inhibits platelet activation and exhibits anti-inflammatory effect. The objectives of this study were to identify CYP8A1 genetic variants and characterize functional consequences of CYP8A1 variants. In total, 27 variants including four previously unidentified single-nucleotide polymorphisms (SNPs) were identified by direct DNA sequencing in Koreans (n=48). Among them, CYP8A1 A447T and E314Stop were newly assigned as CYP8A1(∗)5 and CYP8A1(∗)6 by the Human Cytochrome P450 Allele Nomenclature Committee, respectively. CYP8A1(∗)5 was found in the heme binding area in three individuals as a heterozygous mutation. To investigate the functional change of CYP8A1(∗)5, CYP8A1(∗)5 and wild-type CYP8A1 protein were overexpressed in an Escherichia coli expression system and purified. Metabolism of PGH2 by the CYP8A1(∗)5 protein exhibited significantly decreased activity, resulting in a 45% decrease in Vmax and a 1.8-fold decrease in intrinsic clearance compared to the wild-type. Based on the predicted crystal structure of CYP8A1(∗)5 using the Molecular Operating Environment platform, the distance from CYP8A1 Cys441 to the heme was altered with a significantly changed binding free energy for the mutant protein. Further studies would be needed to determine the effect of CYP8A1(∗)5 on PGI2 levels in humans.

Recent studies have reported a widespread distribution of extended-spectrum β-lactamase (ESBL)-producing bacteria, not only in the nosocomial setting, but also in the community; some local communities in Southeast Asia have been reported to show a high prevalence of ESBL-producing bacteria. However, the details regarding the quantitative/qualitative state of ESBL-producing bacterial spread in Southeast Asia are currently unclear. Thus, the aim of this study was to assess the state of ESBL-producing bacterial spread in community residents from the Indochinese peninsula, as a representative region of Southeast Asia. In order to achieve this aim, local community residents in Laos and Vietnam were examined for fecal carriage of ESBL-producing Enterobacteriaceae, and the findings were compared with data from a previous study in Thailand which was conducted in the same manner as this study. Between 47.0%-70.2% of the Laotian and Vietnamese residents carried ESBL-producing CTX-M genotype Enterobacteriaceae. The most common sub-genotypes of CTX-M were CTX-M-1 (33.0%-47.5%) and CTX-M-9 (47.5%-64.1%), and these rates were similar among all three countries. Taken together, these results confirmed that ESBL-producing Enterobacteriaceae are widely disseminated in Indochinese countries, such as Thailand, Laos, and Vietnam.

<h4>Background</h4>Although feeding behavior and food habit are ecologically and economically important properties, little is known about formation and evolution of herbivory. Grass carp (Ctenopharyngodon idella) is an ecologically appealing model of vertebrate herbivore, widely cultivated in the world as edible fish or as biological control agents for aquatic weeds. Grass carp exhibits food habit transition from carnivory to herbivory during development. However, currently little is known about the genes regulating the unique food habit transition and the formation of herbivory, and how they could achieve higher growth rates on plant materials, which have a relatively poor nutritional quality.<h4>Results</h4>We showed that grass carp fed with duckweed (modeling fish after food habit transition) had significantly higher relative length of gut than fish before food habit transition or those fed with chironomid larvae (fish without transition). Using transcriptome sequencing, we identified 10,184 differentially expressed genes between grass carp before and after transition in brain, liver and gut. By eliminating genes potentially involved in development (via comparing fish with or without food habit transition), we identified changes in expression of genes involved in cell proliferation and differentiation, appetite control, circadian rhythm, and digestion and metabolism between fish before and after food habit transition. Up-regulation of GHRb, Egfr, Fgf, Fgfbp1, Insra, Irs2, Jak, STAT, PKC, PI3K expression in fish fed with duckweed, consistent with faster gut growth, could promote the food habit transition. Grass carp after food habit transition had increased appetite signal in brain. Altered expressions of Per, Cry, Clock, Bmal2, Pdp, Dec and Fbxl3 might reset circadian phase of fish after food habit transition. Expression of genes involved in digestion and metabolism were significantly different between fish before and after the transition.<h4>Conclusions</h4>We suggest that the food habit transition from carnivory to herbivory in grass carp might be due to enhanced gut growth, increased appetite, resetting of circadian phase and enhanced digestion and metabolism. We also found extensive alternative splicing and novel transcript accompanying food habit transition. These differences together might account for the food habit transition and the formation of herbivory in grass carp.

The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them.

Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT.

<h4>Background</h4>Pediatric acute myeloid leukemia (AML) comprises up to 20% of all childhood leukemia. Recent research shows that aberrant DNA methylation patterning may play a role in leukemogenesis. The epigenetic silencing of the EBF3 locus is very frequent in glioblastoma. However, the expression profiles and molecular function of EBF3 in pediatric AML is still unclear.<h4>Methods</h4>Twelve human acute leukemia cell lines, 105 pediatric AML samples and 30 normal bone marrow/idiopathic thrombocytopenic purpura (NBM/ITP) control samples were analyzed. Transcriptional level of EBF3 was evaluated by semi-quantitative and real-time PCR. EBF3 methylation status was determined by methylation specific PCR (MSP) and bisulfite genomic sequencing (BGS). The molecular mechanism of EBF3 was investigated by apoptosis assays and PCR array analysis.<h4>Results</h4>EBF3 promoter was hypermethylated in 10/12 leukemia cell lines. Aberrant EBF3 methylation was observed in 42.9% (45/105) of the pediatric AML samples using MSP analysis, and the BGS results confirmed promoter methylation. EBF3 expression was decreased in the AML samples compared with control. Methylated samples revealed similar survival outcomes by Kaplan-Meier survival analysis. EBF3 overexpression significantly inhibited cell proliferation and increased apoptosis. Real-time PCR array analysis revealed 93 dysregulated genes possibly implicated in the apoptosis of EBF3-induced AML cells.<h4>Conclusion</h4>In this study, we firstly identified epigenetic inactivation of EBF3 in both AML cell lines and pediatric AML samples for the first time. Our findings also showed for the first time that transcriptional overexpression of EBF3 could inhibit proliferation and induce apoptosis in AML cells. We identified 93 dysregulated apoptosis-related genes in EBF3-overexpressing, including DCC, AIFM2 and DAPK1. Most of these genes have never been related with EBF3 over expression. These results may provide new insights into the molecular mechanism of EBF3-induced apoptosis; however, further research will be required to determine the underlying details. Our findings suggest that EBF3 may act as a putative tumor suppressor gene in pediatric AML.

In human, Lymphoblastoid cell lines (LCLs) from the CEPH/CEU (Centre d'Etude du Polymorphisme Humain - Utah) family resource have been extensively used for examining the genetics of gene expression levels. However, we noted that CEU/CEPH cell lines were collected and transformed approximately thirty years ago, much earlier than the other cell lines from the pertaining individuals, which we suspected could potentially affect gene expression, data analysis and results interpretation. In this study, by analyzing RNA sequencing data of CEU and the other three European populations as well as an African population, we systematically examined and evaluated the potential confounding effect of LCL age on gene expression levels and patterns. Our results indicated that gene expression profiles of CEU samples have been biased by the older age of CEU cell lines. Interestingly, most of CEU-specific expressions are associated with functions related to cell proliferation, which are more likely due to older age of cell lines than intrinsic characters of the population. We suggested the results be carefully explained when CEU LCLs are used for transcriptomic data analysis in future studies.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by aberrant expansion of CAG repeat in the huntingtin gene. Mutant Huntingtin (mHtt) alters multiple cellular processes, leading to neuronal dysfunction and death. Among those alterations, impaired mitochondrial metabolism seems to have a major role in HD pathogenesis. In this study, we used the Drosophila model system to further investigate the role of mitochondrial damages in HD. We first analyzed the impact of mHtt on mitochondrial morphology, and surprisingly, we revealed the formation of abnormal ring-shaped mitochondria in photoreceptor neurons. Because such mitochondrial spheroids were previously detected in cells where mitophagy is blocked, we analyzed the effect of PTEN-induced putative kinase 1 (PINK1), which controls Parkin-mediated mitophagy. Consistently, we found that PINK1 overexpression alleviated mitochondrial spheroid formation in HD flies. More importantly, PINK1 ameliorated ATP levels, neuronal integrity and adult fly survival, demonstrating that PINK1 counteracts the neurotoxicity of mHtt. This neuroprotection was Parkin-dependent and required mitochondrial outer membrane proteins, mitofusin and the voltage-dependent anion channel. Consistent with our observations in flies, we demonstrated that the removal of defective mitochondria was impaired in HD striatal cells derived from HdhQ111 knock-in mice, and that overexpressing PINK1 in these cells partially restored mitophagy. The presence of mHtt did not affect Parkin-mediated mitochondrial ubiquitination but decreased the targeting of mitochondria to autophagosomes. Altogether, our findings suggest that mitophagy is altered in the presence of mHtt and that increasing PINK1/Parkin mitochondrial quality control pathway may improve mitochondrial integrity and neuroprotection in HD.

Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-linked β-N-acetylglucosamine (O-GlcNAc) is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines and secreted proteins whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. We have validated the relative levels of several of these factors using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating secreted protein expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple secreted proteins reflect the modulation observed at the protein level. We validate the transcript levels in male mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet-induced mouse model, we have confirmed that the secreted proteins regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. By comparing the promoters of similarly regulated genes, we determine that Sp1 is a common cis-acting element. Furthermore, we show that the LPL and SPARC promoters are enriched for Sp1 and O-GlcNAc modified proteins during insulin resistance in adipocytes. Thus, the O-GlcNAc modification of proteins bound to promoters, including Sp1, is linked to adipocytokine transcription during insulin resistance.

The predominant population of γδ T cells in human blood express a T cell receptor (TCR) composed of a Vγ9 (Vγ2 in an alternate nomenclature) and Vδ2 domains. These cells came into the limelight when it was discovered they can respond to certain microbial infections and tumorigenic cells through the detection of small, pyrophosphate containing organic molecules collectively called "phosphoantigens" or "pAgs." These molecules are intermediates in both eukaryotic and prokaryotic metabolic pathways. Chemical variants of these intermediates have been used in the clinic to treat a range of different cancers, however, directed optimization of these molecules requires a full understanding of their mechanism of action on target cells. We and others have identified a subclass of butyrophilin-related molecules (BTN3A1-3) that are directly involved in pAg sensing in the target cell, leading to engagement and activation of the T cell through the TCR. Our data and that of others support the pAg binding site to be the intracellular B30.2 domain of BTN3A1, which is the only isoform capable of mediating pAg-dependent stimulation of Vγ9Vδ2 T cells. Here, we review the data demonstrating pAg binding to the B30.2 domain and our studies of the structural conformations of the BTN3A extracellular domains. Finally, we synthesize a model linking binding of pAg to the intracellular domain with T cell detection via the extracellular domains in an "inside-out" signaling mechanism of the type characterized first for integrin molecule signaling. We also explore the role of Vγ9Vδ2 TCR variability in the CDR3 γ and δ loops and how this may modulate Vγ9Vδ2 cells as a population in surveillance of human health and disease.

<h4>Background and aim</h4>Currently, non-alcoholic steatohepatitis (NASH) can only be diagnosed histopathologically. Our objective was to establish a new scoring system for the fibrotic stage of NASH.<h4>Methods</h4>We enrolled 139 patients with histologically proven NASH and divided them into two groups to construct (n = 90) and validate (n = 49) a fibrotic score for NASH (FSN). We used 17 variables and their natural logarithmic transformations in the multivariate analysis. To assess the accuracy of the FSN in determining NASH advanced fibrosis (stages 3-4), we compared various fibrotic scores for NASH.<h4>Results</h4>In the construct group, multivariate regression analysis ultimately obtained the following function: z = 1.022 × ln (type IV collagen 7S) (ng/mL) - 0.00680 × (platelet count) (×10(9)/L) + 1.925 × ln (AST) (IU/L) - 1.239 × ln (ALT) (IU/L) + 0.249. Median values of the FSN for stages 1, 2, 3 and 4 were 1.87, 2.14, 3.26 and 3.89, respectively. The multiple regression coefficient and coefficient of determination were 0.70 and 0.46, respectively. In the validation group, the median value was 2.00, 2.83, 3.08 and 4.37 in each stage. With regard to the utility of the FSN for predicting advanced fibrosis of NASH (stage ≥3), the area under the receiver operating characteristic curves (AUROC), 0.909 (95% CI 0.847-0.970, p < 0.001), was higher than that for the other fibrotic scores (APRI, NAFLD fibrosis score, FIB-4 index, BARD score, NIKEI) in the construct group.<h4>Conclusions</h4>This simple scoring system accurately predicts fibrotic stage and discriminates patients with advanced fibrosis of NASH.

The highly complex structure of the human brain is strongly shaped by genetic influences. Subcortical brain regions form circuits with cortical areas to coordinate movement, learning, memory and motivation, and altered circuits can lead to abnormal behaviour and disease. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume and intracranial volume. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10(-33); 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability in human brain development, and may help to determine mechanisms of neuropsychiatric dysfunction.

<h4>Background</h4>Cardiovascular disease including vascular calcification (VC) remains the leading cause of death in patients suffering from chronic kidney disease (CKD). The process of VC seems likely to be a tightly regulated process where vascular smooth muscle cells are playing a key role rather than just a mere passive precipitation of calcium phosphate. Characterisation of the chemical and crystalline structure of VC was mainly led in patients or animal models with CKD. Likewise, Mg2+ was found to be protective in living cells although a potential role for Mg2+ could not be excluded on crystal formation and precipitation. In this study, the crystal formation and the role of Mg2+ were investigated in an in vitro model of primary human aortic vascular smooth muscle cells (HAVSMC) with physical techniques.<h4>Methodology/principal findings</h4>In HAVSMC incubated with increased Ca x Pi medium, only calcium phosphate apatite crystals (CPA) were detected by Micro-Fourier Transform InfraRed spectroscopy (µFTIR) and Field Effect Scanning Electron Microscope (FE-SEM) and Energy Dispersive X-ray spectrometry (EDX) at the cell layer level. Supplementation with Mg2+ did not alter the crystal composition or structure. The crystal deposition was preferentially positioned near or directly on cells as pictured by FE-SEM observations and EDX measurements. Large µFTIR maps revealed spots of CPA crystals that were associated to the cellular layout. This qualitative analysis suggests a potential beneficial effect of Mg2+ at 5 mM in noticeably reducing the number and intensities of CPA µFTIR spots.<h4>Conclusions/significance</h4>For the first time in a model of HAVSMC, induced calcification led to the formation of the sole CPA crystals. Our data seems to exclude a physicochemical role of Mg2+ in altering the CPA crystal growth, composition or structure. Furthermore, Mg2+ beneficial role in attenuating VC should be linked to an active cellular role.

White matter abnormalities have been reported in premanifest Huntington's disease (HD) subjects before overt striatal neuronal loss, but whether the white matter changes represent a necessary step towards further pathology and the underlying mechanism of these changes remains unknown. Here, we characterized a novel knock-in mouse model that expresses mouse HD gene homolog (Hdh) with extended CAG repeat- HdhQ250, which was derived from the selective breeding of HdhQ150 mice. HdhQ250 mice manifest an accelerated and robust phenotype compared with its parent line. HdhQ250 mice exhibit progressive motor deficits, reduction in striatal and cortical volume, accumulation of mutant huntingtin aggregation, decreased levels of DARPP32 and BDNF and altered striatal metabolites. The abnormalities detected in this mouse model are reminiscent of several aspects of human HD. In addition, disturbed myelination was evident in postnatal Day 14 HdhQ250 mouse brain, including reduced levels of myelin regulatory factor and myelin basic protein, and decreased numbers of myelinated axons in the corpus callosum. Thinner myelin sheaths, indicated by increased G-ratio of myelin, were also detected in the corpus callosum of adult HdhQ250 mice. Moreover, proliferation of oligodendrocyte precursor cells is altered by mutant huntingtin both in vitro and in vivo. Our data indicate that this model is suitable for understanding comprehensive pathogenesis of HD in white matter and gray matter as well as developing therapeutics for HD.

Aging and carcinogenesis coincide with the accumulation of DNA damage and mutations in stem and progenitor cells. Molecular mechanisms that influence responses of stem and progenitor cells to DNA damage remain to be delineated. Here, we show that niche positioning and Wnt signaling activity modulate the sensitivity of intestinal stem and progenitor cells (ISPCs) to DNA damage. ISPCs at the crypt bottom with high Wnt/β-catenin activity are more sensitive to DNA damage compared to ISPCs in position 4 with low Wnt activity. These differences are not induced by differences in cell cycle activity but relate to DNA damage-dependent activation of Wnt signaling, which in turn amplifies DNA damage checkpoint activation. The study shows that instructed enhancement of Wnt signaling increases radio-sensitivity of ISPCs, while inhibition of Wnt signaling decreases it. These results provide a proof of concept that cell intrinsic levels of Wnt signaling modulate the sensitivity of ISPCs to DNA damage and heterogeneity in Wnt activation in the stem cell niche contributes to the selection of ISPCs in the context of DNA damage.

<h4>Background</h4>Identification of genes with ascending or descending monotonic expression patterns over time or stages of stem cells is an important issue in time-series microarray data analysis. We propose a method named Monotonic Feature Selector (MFSelector) based on a concept of total discriminating error (DEtotal) to identify monotonic genes. MFSelector considers various time stages in stage order (i.e., Stage One vs. other stages, Stages One and Two vs. remaining stages and so on) and computes DEtotal of each gene. MFSelector can successfully identify genes with monotonic characteristics.<h4>Results</h4>We have demonstrated the effectiveness of MFSelector on two synthetic data sets and two stem cell differentiation data sets: embryonic stem cell neurogenesis (ESCN) and embryonic stem cell vasculogenesis (ESCV) data sets. We have also performed extensive quantitative comparisons of the three monotonic gene selection approaches. Some of the monotonic marker genes such as OCT4, NANOG, BLBP, discovered from the ESCN dataset exhibit consistent behavior with that reported in other studies. The role of monotonic genes found by MFSelector in either stemness or differentiation is validated using information obtained from Gene Ontology analysis and other literature. We justify and demonstrate that descending genes are involved in the proliferation or self-renewal activity of stem cells, while ascending genes are involved in differentiation of stem cells into variant cell lineages.<h4>Conclusions</h4>We have developed a novel system, easy to use even with no pre-existing knowledge, to identify gene sets with monotonic expression patterns in multi-stage as well as in time-series genomics matrices. The case studies on ESCN and ESCV have helped to get a better understanding of stemness and differentiation. The novel monotonic marker genes discovered from a data set are found to exhibit consistent behavior in another independent data set, demonstrating the utility of the proposed method. The MFSelector R function and data sets can be downloaded from: http://microarray.ym.edu.tw/tools/MFSelector/.

<h4>Background</h4>Single-nucleotide polymorphisms (SNPs) selection and identification are the most important tasks in Genome-wide association data analysis. The problem is difficult because genome-wide association data is very high dimensional and a large portion of SNPs in the data is irrelevant to the disease. Advanced machine learning methods have been successfully used in Genome-wide association studies (GWAS) for identification of genetic variants that have relatively big effects in some common, complex diseases. Among them, the most successful one is Random Forests (RF). Despite of performing well in terms of prediction accuracy in some data sets with moderate size, RF still suffers from working in GWAS for selecting informative SNPs and building accurate prediction models. In this paper, we propose to use a new two-stage quality-based sampling method in random forests, named ts-RF, for SNP subspace selection for GWAS. The method first applies p-value assessment to find a cut-off point that separates informative and irrelevant SNPs in two groups. The informative SNPs group is further divided into two sub-groups: highly informative and weak informative SNPs. When sampling the SNP subspace for building trees for the forest, only those SNPs from the two sub-groups are taken into account. The feature subspaces always contain highly informative SNPs when used to split a node at a tree.<h4>Results</h4>This approach enables one to generate more accurate trees with a lower prediction error, meanwhile possibly avoiding overfitting. It allows one to detect interactions of multiple SNPs with the diseases, and to reduce the dimensionality and the amount of Genome-wide association data needed for learning the RF model. Extensive experiments on two genome-wide SNP data sets (Parkinson case-control data comprised of 408,803 SNPs and Alzheimer case-control data comprised of 380,157 SNPs) and 10 gene data sets have demonstrated that the proposed model significantly reduced prediction errors and outperformed most existing the-state-of-the-art random forests. The top 25 SNPs in Parkinson data set were identified by the proposed model including four interesting genes associated with neurological disorders.<h4>Conclusion</h4>The presented approach has shown to be effective in selecting informative sub-groups of SNPs potentially associated with diseases that traditional statistical approaches might fail. The new RF works well for the data where the number of case-control objects is much smaller than the number of SNPs, which is a typical problem in gene data and GWAS. Experiment results demonstrated the effectiveness of the proposed RF model that outperformed the state-of-the-art RFs, including Breiman's RF, GRRF and wsRF methods.

As a biomarker of beta-amyloid, positron emission tomography (PET) amyloid imaging offers a unique opportunity to detect the presence of this protein in the human body during life. Besides Alzheimer's disease (AD), deposits of beta-amyloid in the brain are also present in other neurodegenerative diseases associated to dementia, such as Parkinson's disease and dementia with Lewy bodies, as well as in other processes affecting brain function, such as cerebral amyloid angiopathy, brain trauma, Down's syndrome and meningiomas, as shown by post-mortem pathology studies. Furthermore, in systemic amyloidosis other organs besides the brain are affected, and amyloid PET imaging may be suitable for the identification of these extra-cerebral amyloid depositions. Finally, the potential use of amyloid PET tracer accumulation in cerebral white matter (WM) as a marker of myelin is being investigated, leading to some promising results in patients with WM lesions and multiple sclerosis. In this article, a review of the ongoing research pointing to a broader application of amyloid PET imaging in clinical practice beyond AD is provided.

The objective of this work was to verify whether, as in the case of the plasma membrane of human vascular smooth muscle cells (hVSMCs), cytosolic ET-1-induced increase of nuclear calcium is mediated via the activation of calcium influx through the steady-state R-type calcium channel. Pharmacological tools to identify the R-type calcium channels, as well as real 3-D confocal microscopy imaging techniques coupled to calcium fluorescent probes, were used to study the effect of cytosolic ET-1 on nuclear calcium in isolated nuclei of human hepatocytes and plasma membrane perforated hVSMCs. Our results showed that pre-treatment with pertussis toxin (PTX) or cholera toxin (CTX) prevented cytosolic ET-1 (10(-9) mol/L) from inducing a sustained increase in nuclear calcium. Furthermore, the L-type calcium channel blocker nifedipine did not prevent cytosolic ET-1 from inducing an increase in nuclear calcium, as opposed to the dual L- and R-type calcium channel blocker isradipine (PN200-110) (in the presence of nifedipine). In conclusion, the preventative effect with PTX and CTX, and the absence of an effect with nifedipine, as well as the blockade by isradipine on cytosolic ET-1-induced increase in nuclear calcium, suggest that this nuclear calcium influx in hVSMCs is due to activation of the steady-state R-type calcium channel. The sarcolemmal and nuclear membrane R-type calcium channels in hVSMCs are involved in ET-1 modulation of vascular tone in physiology and pathology.

HOX genes are highly expressed in many acute myeloid leukemia (AML) samples, but the patterns of expression and associated regulatory mechanisms are not clearly understood. We analyzed RNA sequencing data from 179 primary AML samples and normal hematopoietic cells to understand the range of expression patterns in normal versus leukemic cells. HOX expression in AML was restricted to specific genes in the HOXA or HOXB loci, and was highly correlated with recurrent cytogenetic abnormalities. However, the majority of samples expressed a canonical set of HOXA and HOXB genes that was nearly identical to the expression signature of normal hematopoietic stem/progenitor cells. Transcriptional profiles at the HOX loci were similar between normal cells and AML samples, and involved bidirectional transcription at the center of each gene cluster. Epigenetic analysis of a subset of AML samples also identified common regions of chromatin accessibility in AML samples and normal CD34(+) cells that displayed differences in methylation depending on HOX expression patterns. These data provide an integrated epigenetic view of the HOX gene loci in primary AML samples, and suggest that HOX expression in most AML samples represents a normal stem cell program that is controlled by epigenetic mechanisms at specific regulatory elements.

Somatostatin (SST) deficits are common pathological features in depression and other neurological disorders with mood disturbances, but little is known about the contribution of SST deficits to mood symptoms or causes of these deficits. Here we show that mice lacking SST (Sst(KO)) exhibit elevated behavioral emotionality, high basal plasma corticosterone and reduced gene expression of Bdnf, Cortistatin and Gad67, together recapitulating behavioral, neuroendocrine and molecular features of human depression. Studies in Sst(KO) and heterozygous (Sst(HZ)) mice show that elevated corticosterone is not sufficient to reproduce the behavioral phenotype, suggesting a putative role for Sst cell-specific molecular changes. Using laser capture microdissection, we show that cortical SST-positive interneurons display significantly greater transcriptome deregulations after chronic stress compared with pyramidal neurons. Protein translation through eukaryotic initiation factor 2 (EIF2) signaling, a pathway previously implicated in neurodegenerative diseases, was most affected and suppressed in stress-exposed SST neurons. We then show that activating EIF2 signaling through EIF2 kinase inhibition mitigated stress-induced behavioral emotionality in mice. Taken together, our data suggest that (1) low SST has a causal role in mood-related phenotypes, (2) deregulated EIF2-mediated protein translation may represent a mechanism for vulnerability of SST neurons and (3) that global EIF2 signaling has antidepressant/anxiolytic potential.

<h4>Background</h4>With the ubiquitous Mini-Mental State Exam now under copyright, attention is turning to alternative cognitive screening tests. The aim of the present study was to investigate three common cognitive screening tools: the Montreal Cognitive Assessment (MoCA), the Rowland Universal Dementia Assessment Scale (RUDAS), and the recently revised Addenbrooke's Cognitive Assessment Version III (ACE-III).<h4>Methods</h4>The ACE-III, MoCA and RUDAS were administered in random order to a sample of 37 participants with diagnosed mild dementia and 47 comparison participants without dementia. The diagnostic accuracy of the three tests was assessed.<h4>Results</h4>All the tests showed good overall accuracy as assessed by area under the ROC Curve, 0.89 (95% CI = 0.80-0.95) for the ACE-III, 0.84 (0.75-0.91) for the MoCA, and 0.86 (0.77-0.93) for RUDAS. The three tests were strongly correlated: r(84) = 0.85 (0.78-0.90) between the ACE-III and MoCA, 0.70 (0.57-0.80) between the ACE-III and RUDAS; and 0.65 (0.50-0.76) between the MoCA and RUDAS. The data derived optimal cut-off points for were lower than the published recommendations for the ACE-III (optimal cut-point ≤76, sensitivity = 81.1%, specificity = 85.1%) and the MoCA (≤20, sensitivity = 78.4%, specificity = 83.0%), but similar for the RUDAS (≤22, sensitivity = 78.4%, specificity = 85.1%).<h4>Conclusions</h4>All three tools discriminated well overall between cases of mild dementia and controls. To inform interpretation of these tests in clinical settings, it would be useful for future research to address more inclusive and potentially age-stratified local norms.

With the goal to remove the roots of cancer, eliminate metastatic seeds, and overcome therapy resistance, the 2014 inaugural International Cancer Stem Cell (CSC) Conference at Cleveland, OH, convened together over 320 investigators, including 55 invited world-class speakers, 25 short oral presenters, and 100 poster presenters, to gain an in-depth understanding of CSCs and explore therapeutic opportunities targeting CSCs. The meeting enabled intriguing discussions on several topics including: genetics and epigenetics; cancer origin and evolution; microenvironment and exosomes; metabolism and inflammation; metastasis and therapy resistance; single cell and heterogeneity; plasticity and reprogramming; as well as other new concepts. Reports of clinical trials targeting CSCs emphasized the urgent need for strategically designing combinational CSC-targeting therapies against cancer.

Myotonic dystrophy type 1 is caused by abnormal expansion of a CTG-trinucleotide repeat in the gene encoding Dystrophia Myotonica Protein Kinase (DMPK), which in turn leads to global deregulation of gene expression in affected individuals. The transcribed mRNA contains a massive CUG-expansion in the 3' untranslated region (3'UTR) facilitating nucleation of several regulatory RNA-binding proteins, which are thus unable to perform their normal cellular function. These CUG-expanded mRNA-protein aggregates form distinct, primarily nuclear foci. In differentiated muscle cells, most of the CUG-expanded RNA remains in the nuclear compartment, while in dividing cells such as fibroblasts a considerable fraction of the mutant RNA reaches the cytoplasm, consistent with findings that both nuclear and cytoplasmic events are mis-regulated in DM1. Recent evidence suggests that the nuclear aggregates, or ribonuclear foci, are more dynamic than previously anticipated and regulated by several proteins, including RNA helicases. In this review, we focus on the homeostasis of DMPK mRNA foci and discuss how their dynamic regulation may affect disease-causing mechanisms in DM1.

"Pinhead sperm," or "acephalic sperm," a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

In vitro evolution methodologies are powerful approaches to identify RNA with new functionalities. While Systematic Evolution of Ligands by Exponential enrichment (SELEX) is an efficient approach to generate new RNA aptamers, it is less suited for the isolation of efficient ribozymes as it does not select directly for the catalysis. In vitro compartmentalization (IVC) in aqueous droplets in emulsions allows catalytic RNAs to be selected under multiple-turnover conditions but suffers severe limitations that can be overcome using the droplet-based microfluidics workflow described in this paper. Using microfluidics, millions of genes in a library can be individually compartmentalized in highly monodisperse aqueous droplets and serial operations performed on them. This allows the different steps of the evolution process (gene amplification, transcription, and phenotypic assay) to be uncoupled, making the method highly flexible, applicable to the selection and evolution of a variety of RNAs, and easily adaptable for evolution of DNA or proteins. To demonstrate the method, we performed cycles of random mutagenesis and selection to evolve the X-motif, a ribozyme which, like many ribozymes selected using SELEX, has limited multiple-turnover activity. This led to the selection of variants, likely to be the optimal ribozymes that can be generated using point mutagenesis alone, with a turnover number under multiple-turnover conditions, k(ss) cat, ∼ 28-fold higher than the original X-motif, primarily due to an increase in the rate of product release, the rate-limiting step in the multiple-turnover reaction.

<h4>Background</h4>DNA methylation is an epigenetic modification that plays an important role during mammalian development. Around birth in humans, the main site of red blood cell production moves from the fetal liver to the bone marrow. DNA methylation changes at the β-globin locus and a switch from fetal to adult hemoglobin production characterize this transition. Understanding this globin switch may improve the treatment of patients with sickle cell disease and β-thalassemia, two of the most common Mendelian diseases in the world. The goal of our study was to describe and compare the genome-wide patterns of DNA methylation in fetal and adult human erythroblasts.<h4>Methods</h4>We used the Illumina HumanMethylation 450 k BeadChip to measure DNA methylation at 402,819 CpGs in ex vivo-differentiated erythroblasts from 12 fetal liver and 12 bone marrow CD34+ donors.<h4>Results</h4>We identified 5,937 differentially methylated CpGs that overlap with erythroid enhancers and binding sites for erythropoiesis-related transcription factors. Combining this information with genome-wide association study results, we show that erythroid enhancers define particularly promising genomic regions to identify new genetic variants associated with fetal hemoglobin (HbF) levels in humans. Many differentially methylated CpGs are located near genes with unanticipated roles in red blood cell differentiation and proliferation. For some of these new candidate genes, we confirm the correlation between DNA methylation and gene expression levels in red blood cell progenitors. We also provide evidence that DNA methylation and genetic variation at the β-globin locus independently control globin gene expression in adult erythroblasts.<h4>Conclusions</h4>Our DNA methylome maps confirm the widespread dynamic changes in DNA methylation that occur during human erythropoiesis. These changes tend to happen near erythroid enhancers, further highlighting their importance in erythroid regulation and HbF production. Finally, DNA methylation may act independently of the transcription factor BCL11A to repress fetal hemoglobin production. This provides cues on strategies to more efficiently re-activate HbF production in sickle cell disease and β-thalassemia patients.

Cyclin-dependent kinase 5 regulatory subunit associated protein 1 (CDK5RAP1) is an enzyme which post-synthetically converts the RNA modification N6-iso-pentenyladenosine (i6A) into 2-methylthio-N6-isopentenyladenosine (ms2i6A). However, the interaction between CDK5RAP1 deficiency and cell apoptosis has not been studied. Breast cancer has long been a leading cause of mortality in the world. Therefore, in the present study, CDK5RAP1 deficiency in a human breast cancer cell line was investigated. CDK5RAP1 small interfering RNA (siRNA) and negative control siRNA were transfected into MCF-7 cells, and the cells were further incubated for 48 h. CDK5RAP1 deficiency suppressed tumor growth in MCF-7 cells and arrested the cells at G2/M phase. CDK5RAP1 deficiency also induced cell apoptosis and reactive oxygen species (ROS) generation. Furthermore, western blot analysis showed that the expression of phospho-c-Jun N-terminal kinase (p-JNK), p53, caspase-9 and caspase-3 were upregulated in CDK5RAP1-deficient MCF-7 cells. Pretreatment with N-acetyl-cysteine (NAC), the inhibitor of ROS, or with SP600125, the inhibitor of JNK, prevented the apoptosis and the high expression of p-JNK, p53, caspase-9 and caspase-3 in CDK5RAP1-deficient MCF-7 cells. Taken together, these data indicated that CDK5RAP1 deficiency induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells by the ROS/JNK signaling pathway. Our findings indicated a novel therapeutic strategy for cancer.

Spinobulbar muscular atrophy (SBMA) is an X-linked disease characterized by degeneration of motor neurons, muscle atrophy, and progressive weakness. It is caused by a polyglutamine (polyQ) expansion in the androgen receptor (AR), a transcription factor that is activated upon hormone binding. The polyQ expansion in AR causes it to form intracellular aggregates and impairs transcriptional activity. Intriguingly, SUMOylation (where SUMO indicates small ubiquitin-like modifier) of AR inhibits its transcriptional activity and reduces aggregation of the polyQ form of this protein, but it is unclear whether SUMOylation plays a pathogenic or protective role in SBMA. In this issue of the JCI, Chua et al. address this question by generating knockin mice in which the native AR is replaced by either a polyQ AR or a polyQ AR lacking the two lysine residues that are SUMOylated. The results from this study demonstrate that inhibiting SUMOylation of polyQ AR restores much of its transcriptional activity and prevents many (but not all) SBMA-associated symptoms in this mouse model.

Working memory deficit is the core neurocognitive disorder in schizophrenia patients. To identify the factors underlying working memory deficit in schizophrenia patients and to explore the implication of possible genes in the working memory using genome-wide association study (GWAS) of schizophrenia, computerized delay-matching-to-sample (DMS) and whole genome genotyping data were obtained from 100 first-episode, treatment-naïve patients with schizophrenia and 140 healthy controls from the Mental Health Centre of the West China Hospital, Sichuan University. A composite score, delay-matching-to-sample total correct numbers (DMS-TC), was found to be significantly different between the patients and control. On associating quantitative DMS-TC with interactive variables of groups × genotype, one SNP (rs1411832), located downstream of YWHAZP5 in chromosome 10, was found to be associated with the working memory deficit in schizophrenia patients with lowest p-value (p = 2.02 × 10(-7)). ConsensusPathDB identified that genes with SNPs for which p values below the threshold of 5 × 10(-5) were significantly enriched in GO:0007155 (cell adhesion, p < 0.001). This study indicates that working memory, as an endophenotype of schizophrenia, could improve the efficacy of GWAS in schizophrenia. However, further study is required to replicate the results from our study.

Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13, also known as Unc13) and Ca(2+)-dependent activator proteins for secretion (CAPS) families, which prime vesicles for exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms. Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein that is crucial for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of the presynaptic active zone. Electron tomography of otoferlin-deficient IHC synapses revealed a reduction of short tethers holding vesicles at the active zone, which might be a structural correlate of impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an unconventional priming machinery that involves otoferlin.

Alzheimer's disease (AD), the most prevalent dementia in the elderly, is characterized by progressive synaptic and neuronal loss. Mitochondrial dysfunctions have been consistently reported as an early event in AD and appear before Aβ deposition and memory decline. In order to define a new neuroprotectant strategy in AD targeting mitochondrial alterations, we develop tetrahydro-N,N-dimethyl-2,2-diphenyl-3-furanmethanamine (ANAVEX2-73, AE37), a mixed muscarinic receptor ligand and a sigma-1 receptor (σ1R) agonist. We previously reported that ANAVEX2-73 shows anti-amnesic and neuroprotective activities in mice injected intracerebroventricular (ICV) with oligomeric amyloid-β25-35 peptide (Aβ25-35). The σ1R is present at mitochondria-associated endoplasmic reticulum (ER) membranes, where it acts as a sensor/modulator of ER stress responses and local Ca(2+) exchanges with the mitochondria. We therefore evaluated the effect of ANAVEX2-73 and PRE-084, a reference σ1R agonist, on preservation of mitochondrial integrity in Aβ25-35-injected mice. In isolated mitochondria from hippocampus preparations of Aβ25-35 injected animals, we measured respiration rates, complex activities, lipid peroxidation, Bax/Bcl-2 ratios and cytochrome c release into the cytosol. Five days after Aβ25-35 injection, mitochondrial respiration in mouse hippocampus was altered. ANAVEX2-73 (0.01-1 mg/kg IP) restored normal respiration and PRE-084 (0.5-1 mg/kg IP) increased respiration rates. Both compounds prevented Aβ25-35-induced increases in lipid peroxidation levels, Bax/Bcl-2 ratio and cytochrome c release into the cytosol, all indicators of increased toxicity. ANAVEX2-73 and PRE-084 efficiently prevented the mitochondrial respiratory dysfunction and resulting oxidative stress and apoptosis. The σ1R, targeted selectively or non-selectively, therefore appears as a valuable target for protection against mitochondrial damages in AD.

In addition to the disruption of neural function below spinal cord injuries (SCI), there also can be changes in neuronal properties above and below the lesion site. The relevance of these changes is generally unclear, but they must be understood if we are to provide rational interventions. Pharmacological approaches to improving locomotor function have been studied extensively, but it is still unclear what constitutes an optimal approach. Here, we have used the lamprey to compare the modulatory effects of 5-HT and lesion-induced changes in cellular and synaptic properties in unlesioned and lesioned animals. While analyses typically focus on the sub-lesion spinal cord, we have also examined effects above the lesion to see if there are changes here that could potentially contribute to the functional recovery. Cellular and synaptic properties differed in unlesioned and lesioned spinal cords and above and below the lesion site. The cellular and synaptic modulatory effects of 5-HT also differed in lesioned and unlesioned animals, again in region-specific ways above and below the lesion site. A role for 5-HT in promoting recovery was suggested by the potential for improvement in locomotor activity when 5-HT was applied to poorly recovered animals, and by the consistent failure of animals to recover when they were incubated in PCPA to deplete 5-HT. However, PCPA did not affect swimming in animals that had already recovered, suggesting a difference in 5-HT effects after lesioning. These results show changes in 5-HT modulation and cellular and synaptic properties after recovery from a spinal cord transection. Importantly, effects are not confined to the sub-lesion spinal cord but also occur above the lesion site. This suggests that the changes may not simply reflect compensatory responses to the loss of descending inputs, but reflect the need for co-ordinated changes above and below the lesion site. The changes in modulatory effects should be considered in pharmacological approaches to functional recovery, as assumptions based on effects in the unlesioned spinal cord may not be justified.

<h4>Purpose</h4>Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3-5 (P3-5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors.<h4>Methods</h4>Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR).<h4>Results</h4>Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes' promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random.<h4>Conclusions</h4>This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).

<h4>Aims</h4>It is well established that the serotonergic system contributes to the regulation of glucose homeostasis and feeding and therefore it has been presumed to contribute to the biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI). 5-HTTLPR is a serotonin transporter (5-HTT) gene-linked polymorphic region that regulates the transcriptional activity of 5-HTT. Our aim was to investigate the possible association of 5-HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter gene with T2DM and/or higher BMI in Pakistani population.<h4>Methods</h4>In this study, 574 subjects diagnosed with T2DM and 402 unrelated normal controls from the general Pakistani population were genotyped for 5-HTTLPR polymorphism by PCR amplification and agarose gel electrophoresis. The genotyping data (S/S, S/L and L/L) were recorded and analysed statistically using various software and online available tools.<h4>Results</h4>In the total sample, patients with type 2 diabetes and controls without diabetes, genotypes were distributed according to Hardy-Weinberg equilibrium, and S allele frequency was 61.52% (0.61). There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Pakistani population (p=0.12).<h4>Conclusions</h4>No significant statistical association of 5-HTTLPR polymorphism with type 2 diabetes and obesity in Pakistani population shows that 5-HTTLPR polymorphism is not a major factor in determining type 2 diabetes and obesity in Pakistan.

Soft sets have been regarded as a useful mathematical tool to deal with uncertainty. In recent years, many scholars have shown an intense interest in soft sets and extended standard soft sets to intuitionistic fuzzy soft sets, interval-valued fuzzy soft sets, and generalized fuzzy soft sets. In this paper, hesitant fuzzy soft sets are defined by combining fuzzy soft sets with hesitant fuzzy sets. And some operations on hesitant fuzzy soft sets based on Archimedean t-norm and Archimedean t-conorm are defined. Besides, four aggregation operations, such as the HFSWA, HFSWG, GHFSWA, and GHFSWG operators, are given. Based on these operators, a multicriteria group decision making approach with hesitant fuzzy soft sets is also proposed. To demonstrate its accuracy and applicability, this approach is finally employed to calculate a numerical example.

There is a need to identify novel targets in Acute Lymphoblastic Leukemia (ALL), a hematopoietic cancer affecting children, to improve our understanding of disease biology and that can be used for developing new therapeutics. Hence, the aim of our study was to find new genes as targets using in silico studies; for this we retrieved the top 10% overexpressed genes from Oncomine public domain microarray expression database; 530 overexpressed genes were short-listed from Oncomine database. Then, using prioritization tools such as ENDEAVOUR, DIR and TOPPGene online tools, we found fifty-four genes common to the three prioritization tools which formed our candidate leukemogenic genes for this study. As per the protocol we selected thirty training genes from PubMed. The prioritized and training genes were then used to construct STRING functional association network, which was further analyzed using cytoHubba hub analysis tool to investigate new genes which could form drug targets in leukemia. Analysis of the STRING protein network built from these prioritized and training genes led to identification of two hub genes, SMAD2 and CDK9, which were not implicated in leukemogenesis earlier. Filtering out from several hundred genes in the network we also found MEN1, HDAC1 and LCK genes, which re-emphasized the important role of these genes in leukemogenesis. This is the first report on these five additional signature genes in leukemogenesis. We propose these as new targets for developing novel therapeutics and also as biomarkers in leukemogenesis, which could be important for prognosis and diagnosis.

Single-cell genomics and single-cell transcriptomics have emerged as powerful tools to study the biology of single cells at a genome-wide scale. However, a major challenge is to sequence both genomic DNA and mRNA from the same cell, which would allow direct comparison of genomic variation and transcriptome heterogeneity. We describe a quasilinear amplification strategy to quantify genomic DNA and mRNA from the same cell without physically separating the nucleic acids before amplification. We show that the efficiency of our integrated approach is similar to existing methods for single-cell sequencing of either genomic DNA or mRNA. Further, we find that genes with high cell-to-cell variability in transcript numbers generally have lower genomic copy numbers, and vice versa, suggesting that copy number variations may drive variability in gene expression among individual cells. Applications of our integrated sequencing approach could range from gaining insights into cancer evolution and heterogeneity to understanding the transcriptional consequences of copy number variations in healthy and diseased tissues.

Preterm birth is the leading cause of infant morbidity and mortality. Despite extensive research, the genetic contributions to spontaneous preterm birth (SPTB) are not well understood. Term controls were matched with cases by race/ethnicity, maternal age, and parity prior to recruitment. Genotyping was performed using Affymetrix SNP Array 6.0 assays. Statistical analyses utilized PLINK to compare allele occurrence rates between case and control groups, and incorporated quality control and multiple-testing adjustments. We analyzed DNA samples from mother-infant pairs from early SPTB cases (20(0/7)-33(6/7) weeks, 959 women and 979 neonates) and term delivery controls (39(0/7)-41(6/7) weeks, 960 women and 985 neonates). For validation purposes, we included an independent validation cohort consisting of early SPTB cases (293 mothers and 243 infants) and term controls (200 mothers and 149 infants). Clustering analysis revealed no population stratification. Multiple maternal SNPs were identified with association P-values between 10×10(-5) and 10×10(-6). The most significant maternal SNP was rs17053026 on chromosome 3 with an odds ratio (OR) 0.44 with a P-value of 1.0×10(-6). Two neonatal SNPs reached the genome-wide significance threshold, including rs17527054 on chromosome 6p22 with a P-value of 2.7×10(-12) and rs3777722 on chromosome 6q27 with a P-value of 1.4×10(-10). However, we could not replicate these findings after adjusting for multiple comparisons in a validation cohort. This is the first report of a genome-wide case-control study to identify single nucleotide polymorphisms (SNPs) that correlate with SPTB.

<h4>Background</h4>Glycogen storage disease type III is caused by mutations in both alleles of the AGL gene, which leads to reduced activity of glycogen-debranching enzyme. The clinical picture encompasses hypoglycemia, with glycogen accumulation leading to hepatomegaly and muscle involvement (skeletal and cardiac). We sought to identify the genetic cause of this disease within the Inuit community of Nunavik, in whom previous DNA sequencing had not identified such mutations.<h4>Methods</h4>Five Inuit children with a clinical and biochemical diagnosis of glycogen storage disease type IIIa were recruited to undergo genetic testing: 2 underwent whole-exome sequencing and all 5 underwent Sanger sequencing to confirm the identified mutation. Selected DNA regions near the AGL gene were also sequenced to identify a potential founder effect in the community. In addition, control samples from 4 adults of European descent and 7 family members of the affected children were analyzed for the specific mutation by Sanger sequencing.<h4>Results</h4>We identified a homozygous frame-shift deletion, c.4456delT, in exon 33 of the AGL gene in 2 children by whole-exome sequencing. Confirmation by Sanger sequencing showed the same mutation in all 5 patients, and 5 family members were found to be carriers. With the identification of this mutation in 5 probands, the estimated prevalence of genetically confirmed glycogen storage disease type IIIa in this region is among the highest worldwide (1:2500). Despite identical mutations, we saw variations in clinical features of the disease.<h4>Interpretation</h4>Our detection of a homozygous frameshift mutation in 5 Inuit children determines the cause of glycogen storage disease type IIIa and confirms a founder effect.

<h4>Background</h4>Memory T cells (Tms) form a barrier against long-term allograft survival; however, CD4(+)Foxp3(+) regulatory T cells (Tregs) can suppress allograft rejection. The OX40/OX40L pathway is critical to the generation of Tms and turns off Treg suppressor function.<h4>Methods</h4>B6 mice that rejected BALB/c skin grafts after 4 weeks were used as the secondary heart transplant recipients. The skin recipient mice, termed S0, S2 and S3, were treated with the isotype antibodies, anti-CD40L/LFA-1 or anti-OX40L combined with anti-CD40L/LFA-1 mAbs, respectively. The secondary heart recipients, termed H0 and H2, received anti-CD40L/LFA-1 mAbs or not, respectively (Fig. 1).<h4>Results</h4>Four weeks after primary skin transplantation, the Tms in the S3 group that received anti-OX40L with anti-CD40L/LFA-1 mAbs were reduced compared to those in the S2 group (CD4(+) Tm: 32.61 ± 2.20% in S2 vs. 25.36 ± 1.16% in S3; CD8(+) Tm: 27.76 ± 1.96% in S2 vs. 20.95 ± 1.30% in S3; P < 0.01). Meanwhile, the proportions of Tregs in S3 increased compared to those in S2 (P < 0.05). The anti-OX40L with anti-CD40L/LFA-1 mAbs group (S3H2) prolonged the mean survival time (MST) following secondary heart transplantation from 9.5 days to 21 days (P < 0.001). Furthermore, allogeneic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly inhibited in the S3H2 group. Additionally, a higher level of IL-10 was detected in sera and allografts.<h4>Conclusions</h4>Anti-OX40L mAb could prolong secondary heart allograft survival based on CD40/CD40L and LFA-1/ICAM-1 blockade. The mechanism of protecting allografts using anti-OX40L mAb involved impairing the generation of Tm and up-regulating IL-10 producing Tregs, inhibiting the function of T cells.

Lung cancer is the leading cause of cancer death worldwide. Although several genetic variants associated with lung cancer have been identified in the past, stringent selection criteria of genome-wide association studies (GWAS) can lead to missed variants. The objective of this study was to uncover missed variants by using the known association between lung cancer and first-degree family history of lung cancer to enrich the variant prioritization for lung cancer susceptibility regions. In this two-stage GWAS study, we first selected a list of variants associated with both lung cancer and family history of lung cancer in four GWAS (3,953 cases, 4,730 controls), then replicated our findings for 30 variants in a meta-analysis of four additional studies (7,510 cases, 7,476 controls). The top ranked genetic variant rs12415204 in chr10q23.33 encoding FFAR4 in the Discovery set was validated in the Replication set with an overall OR of 1.09 (95% CI=1.04, 1.14, P=1.63×10(-4)). When combining the two stages of the study, the strongest association was found in rs1158970 at Ch4p15.2 encoding KCNIP4 with an OR of 0.89 (95% CI=0.85, 0.94, P=9.64×10(-6)). We performed a stratified analysis of rs12415204 and rs1158970 across all eight studies by age, gender, smoking status, and histology, and found consistent results across strata. Four of the 30 replicated variants act as expression quantitative trait loci (eQTL) sites in 1,111 nontumor lung tissues and meet the genome-wide 10% FDR threshold.

The objective of this study was to investigate the effects of iron dextran on lipid metabolism and to determine the involvement of oxidative stress. Fischer rats were divided into two groups: the standard group (S), which was fed the AIN-93M diet, and the standard plus iron group (SI), which was fed the same diet but also received iron dextran injections. Serum cholesterol and triacylglycerol levels were higher in the SI group than in the S group. Iron dextran was associated with decreased mRNA levels of pparα, and its downstream gene cpt1a, which is involved in lipid oxidation. Iron dextran also increased mRNA levels of apoB-100, MTP, and L-FABP indicating alterations in lipid secretion. Carbonyl protein and TBARS were consistently higher in the liver of the iron-treated rats. Moreover, a significant positive correlation was found between oxidative stress products, lfabp expression, and iron stores. In addition, a negative correlation was found between pparα expression, TBARS, carbonyl protein, and iron stores. In conclusion, our results suggest that the increase observed in the transport of lipids in the bloodstream and the decreased fatty acid oxidation in rats, which was promoted by iron dextran, might be attributed to increased oxidative stress.

Mitchell-Riley syndrome is a genetic disorder characterized by neonatal diabetes, pancreatic hypoplasia, intestinal atresia and/or malrotation, biliary atresia, and gallbladder aplasia or hypoplasia. It was considered a variant of the Martinez-Frias syndrome with similar phenotypic characteristics, except for neonatal diabetes and tracheoesophageal fistula. However, the genetic mutation in (regulatory factor X on chromosome 6) RFX6 was only detected in babies who had diabetes, making it different from the previously known mutations for the disease. This is the first reported case of a classical Mitchell-Riley syndrome in the Arab peninsula along with additional features and novel mutations in the RFX6 gene.

<h4>Background</h4>Iron nanoparticles (FeNPs) have attracted increasing attention over the past two decades owing to their promising application as biomedical agents. However, to ensure safe application, their potential nanotoxicity should be carefully and thoroughly evaluated. Studies on the effects of FeNPs on cells at the transcriptomic level will be helpful for identifying any potential nanotoxicity of FeNPs and providing valuable mechanistic insights into various FeNPs-induced nanotoxicities.<h4>Results</h4>This study investigated the effects of an 11-nm dimercaptosuccinic acid-coated magnetite nanoparticle on the gene expression profiles of two human cell lines, THP-1 and HepG2. It was found that the expression of hundreds of genes was significantly changed by a 24-h treatment with the nanoparticles at two doses, 50 μg/mL and 100 μg/mL, in the two cell types. By identifying the differentially expressed genes and annotating their functions, this study characterized the general and cell-specific effects of the nanoparticles on two cell types at the gene, biological process and pathway levels. At these doses, the overall effects of the nanoparticle on the THP-1 cells were the induction of various responses and repression of protein translation, but in the HepG2 cells, the main effects were the promotion of cell metabolism, growth and mobility. In combination with a previous study, this study also characterized the common genes, biological processes and pathways affected by the nanoparticle in two human and mouse cell lines and identified Id3 as a nanotoxicity biomarker of the nanoparticle.<h4>Conclusion</h4>The studied FeNPs exerted significant effects on the gene expression profiles of human cells. These effects were highly dependent on the innate biological functions of cells, i.e., the cell types. However, cells can also show some cell type-independent effects such as repression of Id3 expression. Id3 can be used as a nanotoxicity biomarker for iron nanoparticles.

Peptide-hormone secretion is partially triggered by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) and gene inactivation of Zn2+-sensitive Cav2.3-type VGCCs is associated with disturbed glucose homeostasis in mice. Zn2+ has been implicated in pancreatic islet cell crosstalk and recent findings indicate that sudden cessation of Zn2+ supply during hypoglycemia triggers glucagon secretion in rodents. Here we show that diethyldithiocarbamate (DEDTC), a chelating agent for Zn2+ and other group IIB metal ions, differentially affects blood glucose and serum peptide hormone level in wild-type mice and mice lacking the Cav2.3-subunit. Fasting glucose and glucagon level were significantly higher in Cav2.3-deficient compared to wild-type mice, while DEDTC Zn2+-chelation produced a significant and correlated increase of blood glucose and serum glucagon concentration in wild-type but not Cav2.3-deficient mice. Glucose tolerance tests revealed severe glucose intolerance in Zn2+-depleted Cav2.3-deficient but not vehicle-treated Cav2.3-deficient or Zn2+-depleted wildtype mice. Collectively, these findings indicate that Cav2.3 channels are critically involved in the Zn2+-mediated suppression of glucagon secretion during hyperglycemia. Especially under conditions of Zn2+ deficiency, ablation or dysfunction of Cav2.3 channels may lead to severe disturbances in glucose homeostasis.

The delivery of chemotherapeutics into tumor cells is a fundamental knot for tumor-target therapy to improve the curative effect and avoid side effects. Here, A54 peptide-functionalized poly(lactic-co-glycolic acid)-grafted dextran (A54-Dex-PLGA) was synthesized. The synthesized A54-Dex-PLGA self-assembled to form micelles with a low critical micelle concentration of 16.79 μg·mL(-1) and diameter of about 50 nm. With doxorubicin (DOX) base as a model antitumor drug, the drug-encapsulation efficiency of DOX-loaded A54-Dex-PLGA micelles (A54-Dex-PLGA/DOX) reached up to 75%. In vitro DOX release from the A54-Dex-PLGA/DOX was prolonged to 72 hours. The A54-Dex-PLGA micelles presented excellent internalization ability into hepatoma cells (BEL-7402 cell line and HepG2 cell line) in vitro, and the cellular uptake of the micelles by the BEL-7402 cell line was specific, which was demonstrated by the blocking experiment. In vitro antitumor activity studies confirmed that A54-Dex-PLGA/DOX micelles suppressed tumor-cell (BEL-7402 cell) growth more effectively than Dex-PLGA micelles. Furthermore, in vivo biodistribution testing demonstrated that the A54-Dex-PLGA micelles had a higher distribution ability to BEL-7402 tumors than that to HepG2 tumors.

<h4>Objectives</h4>Dengue virus (DENV) infection is a significant risk to over a third of the human population that causes a wide spectrum of illness, ranging from sub-clinical disease to intermediate syndrome of vascular complications called dengue fever complicated (DFC) and severe, dengue hemorrhagic fever (DHF). Methods for discriminating outcomes will impact clinical trials and understanding disease pathophysiology.<h4>Study design</h4>We integrated a proteomics discovery pipeline with a heuristics approach to develop a molecular classifier to identify an intermediate phenotype of DENV-3 infectious outcome.<h4>Results</h4>121 differentially expressed proteins were identified in plasma from DHF vs dengue fever (DF), and informative candidates were selected using nonparametric statistics. These were combined with markers that measure complement activation, acute phase response, cellular leak, granulocyte differentiation and viral load. From this, we applied quantitative proteomics to select a 15 member panel of proteins that accurately predicted DF, DHF, and DFC using a random forest classifier. The classifier primarily relied on acute phase (A2M), complement (CFD), platelet counts and cellular leak (TPM4) to produce an 86% accuracy of prediction with an area under the receiver operating curve of >0.9 for DHF and DFC vs DF.<h4>Conclusions</h4>Integrating discovery and heuristic approaches to sample distinct pathophysiological processes is a powerful approach in infectious disease. Early detection of intermediate outcomes of DENV-3 will speed clinical trials evaluating vaccines or drug interventions.

<h4>Purpose</h4>A key step of delivering extracellular agents to its intracellular target is to escape from endosomal/lysosomal compartments, while minimizing the release of digestive enzymes that may compromise cellular functions. In this study, we examined the intracellular distribution of both fluorecent cargoes and enzymes by a particle delivery platform made from the controlled blending of poly(lactic-co-glycolic acid) (PLGA) and a random pH-sensitive copolymer.<h4>Methods</h4>We utilized both microscopic and biochemical methods to semi-quantitatively assess how the composition of blend particles affects the level of endosomal escape of cargos of various sizes and enzymes into the cytosolic space.<h4>Results</h4>We demonstrated that these polymeric particles enabled the controlled delivery of cargos into the cytosolic space that was more dependent on the cargo size and less on the composition of blend particles. Blend particles did not induce the rupture of endosomal/lysosomal compartments and released less than 20% of endosomal/lysosomal enzymes.<h4>Conclusions</h4>This study provides insight into understanding the efficacy and safety of a delivery system for intracellular delivery of biologics and drugs. Blend particles offer a potential platform to target intracellular compartments while potentially minimizing cellular toxicity.

The child day-care centre (DCC) is often considered as one risk factor for gastroenteritis (GE) rather than a complex setting in which the interplay of many factors may influence the epidemiology of GE. This study aimed to identify DCC-level risk factors for GE and major enteropathogen occurrence. A dynamic network of 100 and 43 DCCs participated in a syndromic and microbiological surveillance during 2010-2013. The weekly incidence of GE events and weekly prevalence of five major enteropathogens (rotavirus, norovirus, astrovirus, Giardia lamblia, Cryptosporidium hominis/parvum) were modelled per DCC using mixed-effects negative binomial/Poisson regression models. Sixteen hundred children were surveyed up to 3 years, during which 1829 GE episodes were reported and 5197 faecal samples were analysed. Identified risk factors were: large DCC capacity, crowding, having animals, nappy changing areas, sandpits, paddling pools, cleaning potties in normal sinks, cleaning vomit with paper towels (but without cleaner), mixing of staff between child groups, and staff members with multiple daily duties. Protective factors were: disinfecting fomites with chlorine, cleaning vomit with paper towels (and cleaner), daily cleaning of bed linen/toys, cohorting and exclusion policies for ill children and staff. Targeting these factors may reduce the burden of DCC-related GE.

Proteins belonging to the AP-1 family of transcription factors are known to be involved in the regulation of neuronal viability. While strides have been made to elucidate the mechanisms of how individual members regulate cell death, much remains unknown. We find that the expression of one AP-1 member, c-Fos, is reduced in cerebellar granule neurons (CGNs) induced to die by low potassium (LK) treatment. Restoration and increase of this expression protect CGNs against LK-induced death, whereas knockdown induces death of otherwise healthy neurons. Furthermore, forced expression can protect cortical neurons against homocysteic acid (HCA)-induced toxicity. Taken together, this suggests that c-Fos is necessary for neuronal survival and that elevating c-Fos expression has a neuroprotective effect. Consistent with this idea is the finding that c-Fos expression is reduced selectively in the striatum in two separate mouse models of Huntington's disease and forced expression protects against neuronal death resulting from mutant huntingtin (mut-Htt) expression. Interestingly, neuroprotection by c-Fos does not require its DNA-binding, transcriptional, or heteromerization domains. However, this protective activity can be inhibited by pharmacological inhibition of c-Abl, CK-I, and MEK-ERK signaling. Additionally, expression of point mutant forms of this protein has identified that mutation of a tyrosine residue, Tyr345, can convert c-Fos from neuroprotective to neurotoxic. We show that c-Fos interacts with histone deacetylase-3 (HDAC3), a protein that contributes to mut-Htt neurotoxicity and whose overexpression is sufficient to promote neuronal death. When co-expressed, c-Fos can protect against HDAC3 neurotoxicity. Finally, our study identifies a 21-amino acid region at the C-terminus of c-Fos that is sufficient to protect neurons against death induced by LK, HCA treatment, or mut-Htt expression when expressed via a plasmid transfection or as a cell-permeable peptide. This cell-permeable peptide, designated as Fos-CTF, could have potential as a therapeutic agent for neurodegenerative diseases.

WWOX is a gene that spans an extremely large chromosomal region. It is derived from within chromosomal band 16q23.2 which is a region with frequent deletions and other alterations in a variety of different cancers. This chromosomal band also contains the FRA16D common fragile site (CFS). CFSs are chromosomal regions found in all individuals which are highly unstable. WWOX has also been demonstrated to function as a tumor suppressor that is involved in the development of many cancers. Two other highly unstable CFSs, FRA3B (3p14.2) and FRA6E (6q26), also span extremely large genes, FHIT and PARK2, respectively, and these two genes are also found to be important tumor suppressors. There are a number of interesting similarities between these three large CFS genes. In spite of the fact that they are derived from some of the most unstable chromosomal regions in the genome, they are found to be highly evolutionarily conserved and the chromosomal region spanning the mouse homologs of both WWOX and FHIT are also CFSs in mice. Many of the other CFSs also span extremely large genes and many of these are very attractive tumor suppressor candidates. WWOX is therefore a member of a very interesting family of very large CFS genes.

Macroautophagy is a cellular response that leads to the bulk, nonspecific degradation of cytosolic components, including organelles. In recent years, it has been recognized that autophagy is essential for prevention of neurodegenerative diseases, including Parkinson disease (PD) and Huntington disease (HD). Here, we show that conophylline (CNP), a vinca alkaloid, induces autophagy in an mammalian target of rapamycin-independent manner. Using a cellular model of PD, CNP suppressed protein aggregation and protected cells from cell death caused by treatment with 1-methyl-4-phenylpyridinium, a neurotoxin, by inducing autophagy. Moreover, in the HD model, CNP also eliminated mutant huntingtin aggregates. Our findings demonstrate the possible use of CNP as a therapeutic drug for neurodegenerative disorders, including PD and HD.

Elevated serum ferritin and increased cellular iron concentrations are risk factors for diabetes; however, the etiology of this association is unclear. Metabolic tissues such as pancreas, liver, and adipose tissue (AT), as well as the immune cells resident in these tissues, may be involved. Recent studies demonstrate that the polarization status of macrophages has important relevance to their iron-handling capabilities. Furthermore, a subset of macrophages in AT have elevated iron concentrations and a gene expression profile indicative of iron handling, a capacity diminished in obesity. Because iron overload in adipocytes increases systemic insulin resistance, iron handling by AT macrophages may have relevance not only to adipocyte iron stores but also to local and systemic insulin sensitivity.

Lung injury associated with hyperoxia reflects in part the secondary effects of pulmonary inflammation and the associated production of reactive oxygen species due to activation of NADPH oxidase, type 2 (NOX2). Activation of NOX2 requires the phospholipase A2 (PLA2) activity of peroxiredoxin 6 (Prdx6). Therefore, we evaluated whether blocking Prdx6 PLA2 activity using the inhibitor MJ33 would be protective in a mouse model of acute lung injury resulting from hyperoxic exposure. Mice were treated with an intraperitoneal injection of MJ33 (2.5nmol/g body weight) at the start of exposure (zero time) and at 48h during continuous exposure to 100% O2 for 80h. Treatment with MJ33 reduced the number of neutrophils and the protein content in the fluid obtained by bronchoalveolar lavage, inhibited the increase in lipid peroxidation products in lung tissue, decreased the number of apoptotic cells in the lung, and decreased the perivascular edema associated with the 80h exposure to hyperoxia. Thus, blocking Prdx6 PLA2 activity by MJ33 significantly protected lungs against damage from hyperoxia, presumably by preventing the activation of NOX2 and the amplification of lung injury associated with inflammation. These findings demonstrate that MJ33, a potent inhibitor of Prdx6 PLA2 activity, can protect mouse lungs against the manifestations of acute lung injury due to oxidative stress.

Cullin family proteins function as scaffolds to form numerous E3 ubiquitin ligases with RING proteins, adaptor proteins and substrate recognition receptors. These E3 ligases further recognize numerous substrates to participate in a variety of cellular processes, such as DNA damage and repair, cell death and cell cycle progression. Clinically, cullin-associated E3 ligases have been identified to involve numerous human diseases, especially with regard to multiple cancer types. Over the past few years, our understanding of cullin proteins and their functions in genome stability and tumorigenesis has expanded enormously. Herein, this review briefly provides current perspectives on cullin protein functions, and mainly summarizes and discusses molecular mechanisms of cullin proteins in tumorigenesis.

We developed a new type of molecular rods consisting of two (or more) rigid units linked by a flexible joint. Consequently we called these constructs articulated rods (ARs). The syntheses of ARs were carried out by a flexible and modular approach providing access to a number of compounds with various functionalizations in terminal positions. First applications were presented with pyrene, cinnamoyl and anthracenyl labelled ARs.

The post-translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme-catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme-free system by measuring the rate constants for thiol-disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles.

The African sweetpotato weevil (SPW) Cylas puncticollis Boheman is one of the most important constraints of sweetpotato production in Sub-Saharan Africa and yet is largely an uncharacterized insect pest. Here, we report on the transcriptome analysis of SPW generated using an Illumina platform. More than 213 million sequencing reads were obtained and assembled into 89,599 contigs. This assembly was followed by a gene ontology annotation. Subsequently, a transcriptome search showed that the necessary RNAi components relevant to the three major RNAi pathways, were found to be expressed in SPW. To address the functionality of the RNAi mechanism in this species, dsRNA was injected into second instar larvae targeting laccase2, a gene which encodes an enzyme involved in the sclerotization of insect exoskeleton. The body of treated insects showed inhibition of sclerotization, leading eventually to death. Quantitative Real Time PCR (qPCR) confirmed this phenotype to be the result of gene silencing. Together, our results provide valuable sequence data on this important insect pest and demonstrate that a functional RNAi pathway with a strong and systemic effect is present in SPW and can further be explored as a new strategy for controlling this important pest.

<h4>Background</h4>Adult T-cell leukemia/lymphoma (ATL) is a mature T-cell neoplasia etiologically linked to HTLV-1. Manifestations of ATL are diverse and different clinical types with different tissue involvement and aggressiveness have been described. The mechanisms that lead to the development of ATL clinical types have not yet been clarified. Considering that in ATL patients HTLV-1 infection generally occurs in childhood, a multistep carcinogenesis model has been proposed. Microsatellite alterations are important genetic events in cancer development and these alterations have been reported in the aggressive types of ATL. Little is known about oncogenesis of the less aggressive types.<h4>Methodology/principal findings</h4>In this study we investigated the role of the microsatellite alterations in the pathogenesis mediated by HTLV-1 in the different types of ATL. We examined the presence of microsatellite instability (MSI) and loss of heterozigosity (LOH) in matched pair samples (tumoral and normal) of 24 patients with less aggressive types (smoldering and chronic) and in aggressive types (acute and lymphoma) of ATL. Four microsatellite markers D10S190, D10S191, D1391 and DCC were analyzed. MSI was found in four patients, three smoldering and one chronic, and LOH in four patients, three smoldering and one acute. None of the smoldering patients with microsatellite alterations progressed to aggressive ATL.<h4>Conclusions/significance</h4>To our knowledge, this is the first report describing the presence of MSI and LOH in the less aggressive types of ATL. These results indicate that microsatellite alterations may participate in the development of the less aggressive types of ATL.

In this study, we formulated polyplexes with different compositions for codelivery of DNA and small-interfering RNA (siRNA). Since DNA and siRNA have distinctive and complementary morphological characteristics (DNA is long and winding and siRNA is short and rigid), we hypothesized that their codelivery using polyplex would enhance each other's transfection. To test this hypothesis, cationic polymer branched polyethylenimine (bPEI) as a standard transfecting agent and its derivative arginine-rich oligopeptide-grafted bPEI modified with polyethylene glycol (P(SiDAAr)5P3), synthesized in our laboratory, were used as carriers for transfection. Polyplexes at different nucleic acid to polymer weight ratios were characterized for transfection in breast cancer sensitive (MCF-7) and resistant (MCF-7/Adr) cell lines. Gene silencing effect of polyplexes was determined in MDA-MB-231-luc-D3H2LN cell line. The results demonstrated that the polyplexes formed with derivative P(SiDAAr)5P3 show significantly lower toxicity compared to polyplexes formed using bPEI. Further, codelivery resulted in 20-fold higher DNA transfection and 2-fold higher siRNA transfection as compared to the respective single nucleotide delivery. DNA transfection was ∼100-fold lower in resistant MCF-7/Adr cells than in sensitive MCF-7 cells. Confocal imaging and flow cytometry data demonstrated that enhanced transfection does not solely depend on DNA's cellular uptake, suggesting that other mechanisms contribute to increased transfection. DNA-co-siRNA delivery could be a promising therapeutic approach to achieve synergistic effects because it can simultaneously target and interfere with multiple regulatory levels in a cell to halt and reverse disease progression.

Surfactin originated from genus Bacillus is composed of a heptapeptide moiety bonded to the carboxyl and hydroxyl groups of a β-hydroxy fatty acid and it can be chemically modified to prepare the derivatives with different structures, owing to the existence of two free carboxyl groups in its peptide loop. This article presents the chemical modification of surfactin esterified with three different alcohols, and nine novel surfactin derivatives have been separated from products by the high performance liquid chromatography (HPLC). The novel derivatives, identified with Fourier transform infrared spectroscopy (FT-IR) and electrospray ionization mass spectrometry (ESI-MS), are the mono-hexyl-surfactin C14 ester, mono-hexyl-surfactin C15 ester, mono-2-methoxy-ethyl-surfactin C14 ester, di-hexyl-surfactin C14 ester, di-hexyl-surfactin ester C15, di-2-methoxy-ethyl-surfactin ester C14, di-2-methoxy-ethyl-surfactin ester C15, di-6-hydoxyl-hexyl-surfactin C14 ester and, di-6-hydoxyl-hexyl-surfactin C15 ester. The reaction conditions for esterification were optimized and the dependence of yields on different alcohols and catalysts were discussed. This study shows that esterification is one of the most efficient ways of chemical modification for surfactin and it can be used to prepare more derivatives to meet the needs of study in biological and interfacial activities.

In 1996 a common-source outbreak of severe soft tissue and bloodstream infections erupted among Israeli fish farmers and fish consumers due to changes in fish marketing policies. The causative pathogen was a new strain of Vibrio vulnificus, named biotype 3, which displayed a unique biochemical and genotypic profile. Initial observations suggested that the pathogen erupted as a result of genetic recombination between two distinct populations. We applied a whole genome shotgun sequencing approach using several V. vulnificus strains from Israel in order to study the pan genome of V. vulnificus and determine the phylogenetic relationship of biotype 3 with existing populations. The core genome of V. vulnificus based on 16 draft and complete genomes consisted of 3068 genes, representing between 59 and 78% of the whole genome of 16 strains. The accessory genome varied in size from 781 to 2044 kbp. Phylogenetic analysis based on whole, core, and accessory genomes displayed similar clustering patterns with two main clusters, clinical (C) and environmental (E), all biotype 3 strains formed a distinct group within the E cluster. Annotation of accessory genomic regions found in biotype 3 strains and absent from the core genome yielded 1732 genes, of which the vast majority encoded hypothetical proteins, phage-related proteins, and mobile element proteins. A total of 1916 proteins (including 713 hypothetical proteins) were present in all human pathogenic strains (both biotype 3 and non-biotype 3) and absent from the environmental strains. Clustering analysis of the non-hypothetical proteins revealed 148 protein clusters shared by all human pathogenic strains; these included transcriptional regulators, arylsulfatases, methyl-accepting chemotaxis proteins, acetyltransferases, GGDEF family proteins, transposases, type IV secretory system (T4SS) proteins, and integrases. Our study showed that V. vulnificus biotype 3 evolved from environmental populations and formed a genetically distinct group within the E-cluster. The unique epidemiological circumstances facilitated disease outbreak and brought this genotype to the attention of the scientific community.

The mechanistic requirements of antigen recognition by T cells expressing a γδ TCR has revealed important differences with those of αβ TCR cells and, despite impressive new data generated in the very recent years, they remain poorly understood. Based on the structure of the TCR chains and the tissue distribution, γδ cells are represented in a variety of populations. The major subset of human peripheral blood γδ cells express Vγ9Vδ2 TCR heterodimers and are all stimulated by phosphorylated metabolites (commonly called phosphoantigens). Phosphoantigens are molecules with a very small mass and only stimulate Vγ9Vδ2 cells in the presence of antigen-presenting cells, suggesting a strict requirement for dedicated antigen-presenting molecules. Recent studies have identified butyrophilin (BTN) 3A1 as the molecule necessary to stimulate Vγ9Vδ2 cells. BTN3A1 extracellular, transmembrane, and cytoplasmic domains have different functions, including cognate interaction with the Vγ9Vδ2 TCR, binding of the phosphoantigens, and interaction with cytoplasmic proteins. This review mainly discusses the known molecular mechanisms of BTN3A1-mediated antigen presentation to γδ cells and proposes a model of phosphoantigen presentation, which integrates past and recent studies.

<h4>Background</h4>There is a wealth of evidence detailing gray matter degeneration and loss of cognitive function over time in individuals with Huntington's disease (HD). Efforts to attenuate disease-related brain and cognitive changes have been unsuccessful to date. Multidisciplinary rehabilitation, comprising motor and cognitive intervention, has been shown to positively impact on functional capacity, depression, quality of life and some aspects of cognition in individuals with HD. This exploratory study aimed to evaluate, for the first time, whether multidisciplinary rehabilitation can slow further deterioration of disease-related brain changes and related cognitive deficits in individuals with manifest HD.<h4>Methods</h4>Fifteen participants who manifest HD undertook a multidisciplinary rehabilitation intervention spanning 9 months. The intervention consisted of once-weekly supervised clinical exercise, thrice-weekly self-directed home based exercise and fortnightly occupational therapy. Participants were assessed using MR imaging and validated cognitive measures at baseline and after 9 months.<h4>Results</h4>Participants displayed significantly increased gray matter volume in the right caudate and bilaterally in the dorsolateral prefrontal cortex after 9 months of multidisciplinary rehabilitation. Volumetric increases in gray matter were accompanied by significant improvements in verbal learning and memory (Hopkins Verbal Learning-Test). A significant association was found between gray matter volume increases in the dorsolateral prefrontal cortex and performance on verbal learning and memory.<h4>Conclusions</h4>This study provides preliminary evidence that multidisciplinary rehabilitation positively impacts on gray matter changes and cognitive functions relating to verbal learning and memory in individuals with manifest HD. Larger controlled trials are required to confirm these preliminary findings.

Disturbances in iron metabolism can be genetic or acquired and accordingly manifest as primary or secondary iron overload state. Organ damage may result from iron overload and manifest clinically as cirrhosis, diabetes mellitus, arthritis, endocrine abnormalities and cardiomyopathy. Hemochromatosis inherited as an autosomal recessive disorder is the most common genetic iron overload disorder. Expert societies recommend screening of asymptomatic and symptomatic individuals with hemochromatosis by obtaining transferrin saturation (calculated as serum iron/total iron binding capacity × 100). Further testing for the hemochromatosis gene is recommended if transferrin saturation is >45% with or without hyperferritinemia. However, management of individuals with low or normal transferrin saturation is not clear. In patients with features of iron overload and high serum ferritin levels, low or normal transferrin saturation should alert the physician to other - primary as well as secondary - causes of iron overload besides hemochromatosis. We present here a possible approach to patients with hyperferritinemia but normal transferrin saturation.

<h4>Aims</h4>Early onset of hepatocellular carcinoma (HCC) (males and females under the age of 40 or 50 years old, respectively) has a significant prevalence and poor prognosis; however, few studies have reported the risk factors and development of HCC in such cases.<h4>Methods</h4>In this study, we retrospectively analyzed clinical, laboratory and demographic data from 588 treatment-naïve HCC patients with hepatitis B virus (HBV)-associated liver cirrhosis (LC) and 708 age-matched HBV-associated LC patients as control in Beijing 302 Hospital.<h4>Results</h4>15.1% (89/588) of the HCC patients and 36.7% (181/708) of the LC patients were classified as early onset. Compared with age-matched LC controls, male gender (odds ratio (OR) = 2.09, P < 0.05), family history of HBV infection (OR = 2.45, P < 0.05) and alpha-fetoprotein (AFP) > 200 ng/ml (OR = 30.8, P < 0.05) were independent risk factors for early-onset HCC. Comparing late-onset LC controls, male gender (OR = 1.92, P < 0.05), age (OR = 1.04, P < 0.05), family history of HCC (OR = 2.06, P < 0.05), history of smoking (OR = 1.68, P < 0.05) and AFP > 200 ng/ml (OR = 12.0, P < 0.05) were associated with the development of naturally occurring HCC. Overall, male gender and AFP > 200 ng/ml is associated with HCC development across all ages, whereas a family history of HBV infection may identify younger HBV-associated LC patients at risk for HCC.<h4>Conclusion</h4>Our data suggest that a family history of HBV infection is a unique risk factor for naturally-occurring early-onset HCC patients with HBV-associated LC, who should be considered for intensive screening programs.

Genome-wide expression quantitative trait locus (eQTL) mapping may reveal common genetic variants regulating gene expression. In addition to mapping eQTLs, we systematically evaluated the heritability of the whole blood transcriptome in 5,626 participants from the Framingham Heart Study. Of all gene expression measurements, about 40 % exhibit evidence of being heritable [hgeneExp(2) > 0, (p < 0.05)], the average heritability was estimated to be 0.13, and 10 % display hgeneExp(2) > 0.2. To identify the role of eQTLs in promoting phenotype differences and disease susceptibility, we investigated the proportion of cis/trans eQTLs in different heritability categories and discovered that genes with higher heritability are more likely to have cis eQTLs that explain large proportions of variance in the expression of the corresponding genes. Single cis eQTLs explain 0.33-0.53 of variance in transcripts on average, whereas single trans eQTLs only explain 0.02-0.07. The top cis eQTLs tend to explain more variance in the corresponding gene when its hgeneExp(2) is greater. Taking body mass index (BMI) as a case study, we cross-linked cis/trans eQTLs with both GWAS SNPs and differentially expressed genes for BMI. We discovered that BMI GWAS SNPs in 16p11.2 (e.g., rs7359397) are associated with several BMI differentially expressed genes in a cis manner (e.g. SULT1A1, SPNS1, and TUFM). These BMI signature genes explain a much larger proportion of variance in BMI than do the GWAS SNPs. Our results shed light on the impact of eQTLs on the heritability of the human whole blood transcriptome and its relations to phenotype differences.

<h4>Objectives</h4>To investigate the genetic and acquired thrombophilic risk factors in pregnancy-associated complications and venous thromboembolism (VTE) and evaluate the association between particular thrombophilic risk factors and thromboembolic complications.<h4>Methods</h4>In this study, pregnant women with pregnancy complications and VTE (N = 101) were the study group, and the control group were women with normal pregnancy (N = 102). All women underwent testing for factor V Leiden mutation (FVL), mutation of the coagulation factors II (FII20210), methylenetetrahydrofolate reductase (MTHFR), plasminogen activator inhibitor-1, antithrombin III (ATIII), protein C (PC) and protein S, lupus anticoagulant (LAC) antibodies, anticardiolipin antibodies and anti-beta-2-glycoprotein-1.<h4>Results</h4>In this study group, mutations of the FVL was 15.8% (16/101), FII20210 5.9% (6/101) and the MTHFR at locus 677 was TT in 19.8% (20/101). Deficiency of ATIII and PC were rare: 3.0% and 1.0%, respectively. LAC were significantly higher in the study group than in the control group: 32.7% versus 3.9%; p < 0.0005. Pregnant women with VTE have been more frequent for FVL (41.7%; p < 0.005), PC deficiency (25.0%; p < 0.005) and LAC (33.3%; p < 0.005). Combination of FVL and MTHFR mutation was related to the risk of recurrent fetal death and habitual abortion.<h4>Conclusion</h4>The inherited and the acquired thrombophilic risk factors were found to be up to 10 times more common in the study group than in the control group.

Directed evolution of adeno-associated virus (AAV) through successive rounds of phenotypic selection is a powerful method to isolate variants with improved properties from large libraries of capsid mutants. Importantly, AAV libraries used for directed evolution are based on the "natural" AAV genome organization where the capsid proteins are encoded in cis from replicating genomes. This is necessary to allow the recovery of the capsid DNA after each step of phenotypic selection. For directed evolution to be used successfully, it is essential to minimize the random mixing of capsomers and the encapsidation of nonmatching viral genomes during the production of the viral libraries. Here, we demonstrate that multiple AAV capsid variants expressed from Rep/Cap containing viral genomes result in near-homogeneous capsids that display an unexpectedly high capsid-DNA correlation. Next-generation sequencing of AAV progeny generated by bulk transfection of a semi-random peptide library showed a strong counter-selection of capsid variants encoding premature stop codons, which further supports a strong capsid-genome identity correlation. Overall, our observations demonstrate that production of "natural" AAVs results in low capsid mosaicism and high capsid-genome correlation. These unique properties allow the production of highly diverse AAV libraries in a one-step procedure with a minimal loss in phenotype-genotype correlation.

Elucidating genetic mechanisms involved in Attention-Deficit/Hyperactivity Disorder (ADHD) has been challenging. Relatively unexplored is the fact that genetic mechanisms can differ with age. The current study explored the association between dopaminergic and serotonergic genes, ADHD symptoms, and neurocognitive functioning in relation to age. Associations of three genetic ADHD risk factors, DAT1, DRD4, and 5-HTT with symptoms and six neurocognitive measures were explored in two samples of the NeuroIMAGE study: 756 children, adolescents, and young adults with ADHD, their siblings, and controls (M age 17 years, SD 3.2), and 393 parents with and without ADHD (M age 48 years, SD 4.8). Association analyses were performed in both samples, and effects were compared to address dichotomous age effects. Gene*age interactions were examined to address continuous age effects. Moderating effects of age were found for DRD4-7R carriership and ADHD symptoms in the adult group only; in the adolescents the 5-HTT LL genotype was differentially associated with inhibition and with motor timing at different ages, and to inhibition in adults; DAT1 10-6 haplotype carriership showed differential working memory performance depending on age. None of our effects survived correction for multiple comparisons. Our results are preliminary, but may point to differential genotype-phenotype associations at different ages. This can be seen as a proof of concept for the importance of age in dopaminergic and serotonergic genetic association analyses. Our findings are consistent with the idea that genetic and neurocognitive mechanisms underlying ADHD may change throughout life. © 2015 Wiley Periodicals, Inc.

Vesicular stomatitis virus expressing Zaire Ebola virus (EBOV) glycoprotein (VSVΔG/EBOVgp) could be used as a vaccine to meet the 2014 Ebola virus outbreak. To characterize the host response to this vaccine, we used mRNA sequencing to analyze peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques after VSVΔG/EBOVgp immunization and subsequent EBOV challenge. We found a controlled transcriptional response that transitioned to immune regulation as the EBOV was cleared. This observation supports the safety of the vaccine.

<h4>Purpose</h4>Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer-related death in the United States, and its incidence is on the rise. Advanced disease is nearly uniformly lethal, emphasizing the need to identify PDA at its earliest stages. To discover early biomarkers of PDA, we evaluated the circulating proteome in murine preinvasive and invasive plasma samples and human prediagnostic and diagnostic samples.<h4>Experimental design</h4>Using a customized antibody microarray platform containing >4,000 features, we interrogated plasma samples spanning preinvasive and invasive disease from a highly faithful mouse model of PDA. In parallel, we mined prediagnostic plasma from women in the Women's Health Initiative (WHI) who would later succumb to PDA together with matched, cancer-free control samples. Samples collected after an establishing diagnosis of PDA were also interrogated to further validate markers.<h4>Results</h4>We identified ERBB2 and TNC in our cross-species analyses, and multiple antibodies identified ESR1 in prediagnostic plasma from people that succumb to PDA. This 3-marker panel had an AUC of 0.86 (95% confidence interval [CI], 0.76-0.96) for the diagnostic cohort that increased to 0.97 (95% CI, 0.92-1.0) with CA19-9 included. The 3-marker panel also had an AUC of 0.68 (95% CI, 0.58-0.77) for the prediagnostic cohort.<h4>Conclusions</h4>We identified potential disease detection markers in plasma up to 4 years before death from PDA with superior performance to CA19-9. These markers might be especially useful in high-risk cohorts to diagnose early, resectable disease, particularly in patients that do not produce CA19-9.

Specificity protein 1 (SP1) is a ubiquitous transcription factor that plays an important role in controlling gene expression. Although important in mediating the function of various hormones, the role of SP1 in regulating milk fat formation remains unknown. To investigate the sequence and expression information, as well as its role in modulating lipid metabolism, we cloned SP1 gene from mammary gland of Xinong Saanen dairy goat. The full-length cDNA of the SP1 gene is 4376 bp including 103 bp of 5'UTR, 2358 bp of ORF (HM_236311) and 1915 bp of 3'UTR, which is predicted to encode a 786 amino acids polypeptide. Phylogenetic tree analysis showed that goat SP1 has the closest relationship with sheep, followed by bovines (bos taurus, odobenus and ceratotherium), pig, primates (pongo, gorilla, macaca and papio) and murine (rattus and mus), while the furthest relationship was with canis and otolemur. Expression was predominant in the lungs, small intestine, muscle, spleen, mammary gland and subcutaneous fat. There were no significant expression level differences between the mammary gland tissues collected at lactation and dry-off period. Overexpression of SP1 in goat mammary epithelial cells (GMECs) led to higher mRNA expression level of peroxisome proliferator-activated receptor-γ (PPARγ) and lower liver X receptor α (LXRα) mRNA level, both of which were crucial in regulating fatty acid metabolism, and correspondingly altered the expression of their downstream genes in GMECs. These results were further enhanced by the silencing of SP1. These findings suggest that SP1 may play an important role in fatty acid metabolism.

Huntington disease (HD), an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG trinucleotide repeat in the Huntingtin (HTT) gene, is characterized by extensive neurodegeneration of striatum and cortex and severe diffuse atrophy at MRI. The expression of genes involved in the cholesterol biosynthetic pathway and the amount of cholesterol, lanosterol, lathosterol and 24S-hydroxycholesterol were reduced in murine models of HD. In case of HD-patients, the decrease of plasma 24OHC follows disease progression proportionally to motor and neuropsychiatric dysfunction and MRI brain atrophy, together with lanosterol and lathosterol (markers of cholesterol synthesis), and 27-hydroxycholesterol. A significant reduction of total plasma cholesterol was observed only in advanced stages. It is likely that mutant HTT decreases the maturation of SREBP and the up-regulation LXR and LXR-targeted genes (SREBP, ABCG1 and ABCG4, HMGCoA reductase, ApoE) resulting into a lower synthesis and transport of cholesterol from astrocytes to neurons via ApoE. In primary oligodendrocytes, mutant HTT inhibited the regulatory effect of PGC1α on cholesterol metabolism and on the expression of MBP. HTT seems to play a regulatory role in lipid metabolism. The impairment of the cholesterol metabolism was found to be proportional to the CAG repeat length and to the load of mutant HTT. A dysregulation on PGC1α and mitochondria dysfunction may be involved in an overall reduction of acetyl-CoA and ATP synthesis, contributing to the cerebral and whole body cholesterol impairment. This article is part of a Special Issue entitled Brain Lipids.

Iron abnormalities in chronic liver disease may be the result of genetic diseases or secondary factors. The present study aimed to identify subjects with HFE-HH in order to describe the frequency of clinical manifestations, identify risk factors for iron elevation, and compare the iron profile of HFE-HH to other genotypes in liver disease patients. A total of 108 individuals with hepatic disease, transferrin saturation (TS) > 45%, and serum ferritin (SF) > 350 ng/mL were tested for HFE mutations. Two groups were characterized: C282Y/C282Y or C282Y/H63D genotypes (n = 16) were the HFE hereditary hemochromatosis (HFE-HH) group; and C282Y and H63D single heterozygotes, the H63D/H63D genotype, and wild-type were considered group 2 (n = 92). Nonalcoholic liver disease, alcoholism, and chronic hepatitis C were detected more frequently in group 2, whereas arthropathy, hepatocarcinoma, diabetes, and osteoporosis rates were significantly higher in the HFE-HH group. TS > 82%, SF > 2685 ng/mL, and serum iron > 178 μg/dL were the cutoffs for diagnosis of HFE-HH in patients with liver disease. Thus, in non-Caucasian populations with chronic liver disease, HFE-HH diagnosis is more predictable in those with iron levels higher than those proposed in current guidelines for the general population.

Alterations in glycan structures are early signs of malignancy and have recently been proposed to be in part a driving force behind malignant transformation. Here, we explore whether differences in expression of genes related to the process of glycosylation exist between breast carcinoma subtypes - and look for their association to clinical parameters. Five expression datasets of 454 invasive breast carcinomas, 31 ductal carcinomas in situ (DCIS), and 79 non-malignant breast tissue samples were analysed. Results were validated in 1960 breast carcinomas. 419 genes encoding glycosylation-related proteins were selected. The DCIS samples appeared expression-wise similar to carcinomas, showing altered gene expression related to glycosaminoglycans (GAGs) and N-glycans when compared to non-malignant samples. In-situ lesions with different aggressiveness potentials demonstrated changes in glycosaminoglycan sulfation and adhesion proteins. Subtype-specific expression patterns revealed down-regulation of genes encoding glycan-binding proteins in the luminal A and B subtypes. Clustering basal-like samples using a consensus list of genes differentially expressed across discovery datasets produced two clusters with significantly differing prognosis in the validation dataset. Finally, our analyses suggest that glycolipids may play an important role in carcinogenesis of breast tumors - as demonstrated by association of B3GNT5 and UGCG genes to patient survival. In conclusion, most glycan-specific changes occur early in the carcinogenic process. We have identified glycan-related alterations specific to breast cancer subtypes including a prognostic signature for two basal-like subgroups. Future research in this area may potentially lead to markers for better prognostication and treatment stratification of breast cancer patients.

Soluble cluster of differentiation 40 (sCD40) is proteolytically cleaved from membrane-bound CD40 and binds to CD154, thereby inhibiting CD40-CD154-mediated immune responses. The aim of the present study was to clarify the role of sCD40 in chronic hepatitis B (CHB). The sCD40 levels in sera from 132 patients with CHB and 33 healthy individuals were retrospectively measured. sCD40 concentrations in patients with CHB were higher than those in healthy controls, and sCD40 levels correlated positively with serum levels of the liver dysfunction biomarkers alanine transaminase (ALT) and aspartate transaminase (AST). sCD40 concentrations increased with a rise in the severity of liver necroinflammation and fibrosis. Patients with >75% liver tissue staining positive for hepatitis B virus (HBV) antigen expression showed significantly lower sCD40 levels than those who stained negative for the HBV antigen. The area under the receiver operating characteristic curve of sCD40 was greater than that of ALT and AST; thus, sCD40 levels have a high diagnostic accuracy for detecting severe liver inflammation in patients with CHB, and could serve as an immunological marker of hepatic tissue injury.

<h4>Background</h4>While the influence of testosterone levels on vulnerability to affective disorders is not straightforward, research suggests this hormone may confer some degree of resiliency in men. We recently demonstrated a role for the dentate gyrus in mediating testosterone's protective effects on depressive-like behavior in gonadectomized male rats. Here, testosterone may exert its effects through androgen receptor-mediated mechanisms or via local aromatization to estradiol.<h4>Methods</h4>Gonadectomized male rats were implanted with a placebo, testosterone, or estradiol pellet, and subsequent protective anxiolytic- and antidepressant-like effects of testosterone and its aromatized metabolite, estradiol, were then investigated in the open field and sucrose preference tests, respectively. Moreover, their influence on gene expression in the hippocampus was analyzed by genome-wide complementary DNA microarray analysis. Finally, the contribution of testosterone's aromatization within the dentate gyrus was assessed by local infusion of the aromatase inhibitor fadrozole, whose efficacy was confirmed by liquid chromatography-tandem mass spectrometry.<h4>Results</h4>Both hormones had antidepressant-like effects associated with a substantial overlap in transcriptional regulation, particularly in synaptic plasticity- and mitogen-activated protein kinase pathway-related genes. Further, chronic aromatase inhibition within the dentate gyrus blocked the protective effects of testosterone.<h4>Conclusions</h4>Both testosterone and estradiol exhibit anxiolytic- and antidepressant-like effects in gonadectomized male rats, while similarly regulating critical mediators of these behaviors, suggesting common underlying mechanisms. Accordingly, we demonstrated that testosterone's protective effects are mediated, in part, by its aromatization in the dentate gyrus. These findings thus provide further insight into a role for estradiol in mediating the protective anxiolytic- and antidepressant-like effects of testosterone.

<h4>Background</h4>Although the association between lead and cardiovascular disease is well established, potential mechanisms are still poorly understood. Calcium metabolism plays a role in lead toxicity and thus, vitamin D receptor (VDR) polymorphisms have been suggested to modulate the association between lead and health outcomes. We investigated effect modification by VDR genetic polymorphisms in the association between cumulative lead exposure and pulse pressure, a marker of arterial stiffness.<h4>Methods</h4>We examined 727 participants (3,100 observations from follow-ups from 1991 to 2011) from the Normative Aging Study (NAS), a longitudinal study of aging. Tibia and patella bone lead levels were measured using K-x-ray fluorescence. Four single nucleotide polymorphisms (SNPs) in the VDR gene, Bsm1, Taq1, Apa1, and Fok1, were genotyped. Linear mixed effects models with random intercepts were implemented to take into account repeated measurements.<h4>Results</h4>Adjusting for potential confounders, pulse pressure was 2.5 mmHg (95% CI: 0.4-4.7) and 1.9 mmHg (95% CI: 0.1-3.8) greater per interquartile range (IQR) increase in tibia lead (15 μg/g) and patella lead (20 μg/g), respectively, in those with at least one minor frequency allele in Bsm1 compared with those with major frequency allele homozygotes. The observed interaction effect between bone lead and the Bsm1 genotype persists over time during the follow-up. Similar results were observed in effect modification by Taq1.<h4>Conclusions</h4>This study suggests that subjects with the minor frequency alleles of VDR Bsm1 or Taq1 may be more susceptible to cumulative lead exposure-related elevated pulse pressure.

Recent studies reveal that circular RNAs (circRNAs) are a novel class of abundant, stable and ubiquitous noncoding RNA molecules in animals. Comprehensive detection of circRNAs from high-throughput transcriptome data is an initial and crucial step to study their biogenesis and function. Here, we present a novel chiastic clipping signal-based algorithm, CIRI, to unbiasedly and accurately detect circRNAs from transcriptome data by employing multiple filtration strategies. By applying CIRI to ENCODE RNA-seq data, we for the first time identify and experimentally validate the prevalence of intronic/intergenic circRNAs as well as fragments specific to them in the human transcriptome.

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1(+/-) mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.

Oocytes isolated from female rhesus monkeys following standard ovarian stimulation protocols during the summer months displayed a reduced capacity to mature compared with stimulation during the normal breeding season. Because the gene expression profiles of oocyte-associated cumulus cells and mural granulosa cells (CCs and GCs) are indicative of altered oocyte quality and can provide insight into intrafollicular processes that may be disrupted during oogenesis, we performed array-based transcriptome comparisons of CCs and GCs from summer and normal breeding season stimulation cycles. Summer CCs and GCs both display deficiencies in expression of mRNAs related to cell proliferation, angiogenesis, and endocrine signaling, as well as reduced expression of glycogen phosphorylase. Additionally, CCs display deficiencies in expression of mRNAs related to stress response. These results provide the first insight into the specific molecular pathways and processes that are disrupted in the follicles of rhesus macaque females during the summer season. Some of the changes seen in summer GCs and CCs have been reported in humans and in other model mammalian species. This suggests that the seasonal effects seen in the rhesus monkey may help us to understand better the mechanisms that contribute to reduced oocyte quality and fertility in humans.

<h4>Purpose</h4>Two recent large-scale genome-wide association studies identified significant associations between myopia and single nucleotide polymorphisms (SNPs) near the PRSS56, BMP3, KCNQ5, LAMA2, TOX, TJP2, RDH5, ZIC2, RASGRF1, GJD2, RBFOX1, and SHISA6 genes. Our study is to examine whether rare variants in these genes contribute to high myopia.<h4>Methods</h4>Whole-exome sequencing was performed on samples of 298 probands with early-onset high myopia (eoHM; spherical refraction in each meridian ≤ -6.00 [diopters] D in both eyes; age of onset < 7 years) and 195 controls (different forms of retinal degeneration including Leber congenital amaurosis, cone-rod dystrophy, and familial exudative vitroretinopathy). Potential variations in these genes were selected for further validation and comparison to the controls. Moreover, Sanger sequencing was used to evaluate the coding regions and the upstream 800 bps of GJD2 in 395 additional subjects with late-onset moderate to high myopia (loMHM; spherical refraction in each meridian ≤ -4.00 D; age of onset ≥ 7 years) and 403 healthy controls (-0.50 D ± 1.00 D).<h4>Results</h4>Exome sequencing of the 298 probands with eoHM identified 25 rare variants that were predicted to affect coding residues. The segregation analysis and the distribution of rare variants between patients and controls did not provide evidence to support their involvement in myopia. Sanger sequencing of GJD2 in an additional 395 subjects with loMHM and 403 healthy controls did not identify myopia-associated variants.<h4>Conclusions</h4>We did not find evidence to support the association of myopia with rare variants in these genes, probably due to our limited sample size. Additional studies are expected to validate these results.

The present study was designed to demonstrate the potential of an optimized histology directed protein identification combined with imaging mass spectrometry technology to reveal and identify molecules associated to ectopic calcification in human tissue. As a proof of concept, mineralized and non-mineralized areas were compared within the same dermal tissue obtained from a patient affected by Pseudoxanthoma elasticum, a genetic disorder characterized by calcification only at specific sites of soft connective tissues. Data have been technically validated on a contralateral dermal tissue from the same subject and compared with those from control healthy skin. Results demonstrate that this approach 1) significantly reduces the effects generated by techniques that, disrupting tissue organization, blend data from affected and unaffected areas; 2) demonstrates that, abolishing differences due to inter-individual variability, mineralized and non-mineralized areas within the same sample have a specific protein profile and have a different distribution of molecules; and 3) avoiding the bias of focusing on already known molecules, reveals a number of proteins that have been never related to the disease nor to the calcification process, thus paving the way for the selection of new molecules to be validated as pathogenic or as potential pharmacological targets.

<h4>Objective</h4>Huntington's disease (HD) is a rare neurological disorder, and its current status in Korea is not well investigated. This study aims to determine the prevalence and incidence of HD and to investigate the clinical features of HD patients in Korea.<h4>Methods</h4>We estimated the crude prevalence and annual incidence of HD based on the databases of the Rare Diseases Registry (RDR) and the National Health Insurance (NHI). The clinical data of genetically confirmed HD patients was collected from 10 referral hospitals and analyzed.<h4>Results</h4>The mean calculated annual incidence was 0.06 cases per 100,000 persons, and the mean calculated prevalence was 0.38 based on the NHI database. The estimated crude prevalence based on the RDR was 0.41. Of the sixty-eight HD patients recruited, the mean age of onset was 44.16 ± 14.08 years and chorea was most frequently reported as the initial symptom and chief complaint. The mean CAG repeat number of the expanded allele was 44.7 ± 4.8 and correlated inversely with the age of onset (p < 0.001). About two-thirds of the patients have a positive family history, and HD patients without positive family history showed a delay in onset of initial symptoms, a prolonged interval between initial symptom onset and genetic diagnosis and a delay in the age of genetic diagnosis.<h4>Conclusions</h4>To the best of our knowledge, this is the first study to estimate the prevalence and incidence of HD in Korea and the largest HD series in the Asian population. Our analyses might be useful for further studies and large-scale investigations in HD patients.

<h4>Background</h4>Understanding of human variation in toxicity to environmental chemicals remains limited, so human health risk assessments still largely rely on a generic 10-fold factor (10½ each for toxicokinetics and toxicodynamics) to account for sensitive individuals or subpopulations.<h4>Objectives</h4>We tested a hypothesis that population-wide in vitro cytotoxicity screening can rapidly inform both the magnitude of and molecular causes for interindividual toxicodynamic variability.<h4>Methods</h4>We used 1,086 lymphoblastoid cell lines from the 1000 Genomes Project, representing nine populations from five continents, to assess variation in cytotoxic response to 179 chemicals. Analysis included assessments of population variation and heritability, and genome-wide association mapping, with attention to phenotypic relevance to human exposures.<h4>Results</h4>For about half the tested compounds, cytotoxic response in the 1% most "sensitive" individual occurred at concentrations within a factor of 10½ (i.e., approximately 3) of that in the median individual; however, for some compounds, this factor was > 10. Genetic mapping suggested important roles for variation in membrane and transmembrane genes, with a number of chemicals showing association with SNP rs13120371 in the solute carrier SLC7A11, previously implicated in chemoresistance.<h4>Conclusions</h4>This experimental approach fills critical gaps unaddressed by recent large-scale toxicity testing programs, providing quantitative, experimentally based estimates of human toxicodynamic variability, and also testable hypotheses about mechanisms contributing to interindividual variation.

The effects of antenatal depression and antidepressant treatment during pregnancy on both mother and child are vigorously studied, but the underlying biology for these effects is largely unknown. The placenta plays a crucial role in the growth and development of the fetus. We performed a gene expression study on the fetal side of the placenta to investigate gene expression patterns in mothers with antenatal depression and in mothers using antidepressant treatment during pregnancy. Placental samples from mothers with normal pregnancies, from mothers with antenatal depression, and from mothers using antidepressants were collected. We performed a pilot microarray study to investigate alterations in the gene expression and selected several genes from the microarray for biological validation with qPCR in a larger sample. In mothers with antenatal depression 108 genes were differentially expressed, whereas 109 genes were differentially expressed in those using antidepressants. Validation of the microarray revealed more robust gene expression differences in the seven genes picked for confirmation in antidepressant-treated women than in depressed women. Among the genes that were validated ROCK2 and C12orf39 were differentially expressed in both depressed and antidepressant-treated women, whereas ROCK1, GCC2, KTN1, and DNM1L were only differentially expressed in the antidepressant-treated women. In conclusion, antenatal depression and antidepressant exposure during pregnancy are associated with altered gene expression in the placenta. Findings on those genes picked for validation were more robust among antidepressant-treated women than in depressed women, possibly due to the fact that depression is a multifactorial condition with varying degrees of endocrine disruption. It remains to be established whether the alterations found in the gene expression of the placenta are found in the fetus as well.

In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In Caenorhabditis elegans, dosage compensation is achieved by the dosage compensation complex (DCC) binding to both X chromosomes in hermaphrodites to downregulate gene expression by twofold. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 is increased, whereas acetylation of histone H4 lysine 16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.

Although ribosomes are very stable under most conditions, ribosomal degradation does occur in diverse groups of organisms in response to specific stresses or environmental conditions. While non-functional ribosome decay (NRD) in yeast is well characterized, very little is known of the mechanisms that initiate ribosomal degradation in bacteria. Here we test ribosome degradation in growing Escherichia coli expressing mutant ribosomes. We found that mutations in the 16S rRNA decoding centre (G530U and A1492C) and 23S rRNA active site (A2451G) do not lead to ribosomal degradation. In contrast, 23S rRNA mutation U2585A causes degradation of both the large and small ribosomal subunits in E. coli. We further tested mutations in 23S rRNA, which disrupt ribosomal intersubunit bridges B2a and B3. Deletion of helix 69 of 23S rRNA and the point mutation A1912G in the same helix did not destabilize ribosomes, while expression of mutations A1919G in H69 and A1960G in H71 led to degradation of both mutant and wild-type ribosomes. Our results suggest an actively induced mechanism requiring de novo protein synthesis for ribosomal degradation in E. coli, which degrades both structurally inactive and active ribosomes.

The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper ((65)Cu/(63)Cu) and sulfur ((34)S/(32)S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is (63)Cu-enriched by ∼0.4‰ and sulfur is (32)S-enriched by ∼1.5‰ in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The (32)S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

A series of novel compounds, namely 1-(diethoxyphosphoryl)-3-(4-ones-1H-1,2,3-triazol-1-yl)propan-2-yl carboxylic esters, were designed on the basis of the diazafulvene intermediate of imidazole glycerol phosphate dehydratase (IGPD) and high-activity inhibitors of IGPD, and synthesized as inhibitors targeting IGPD in plants. Their structures were confirmed by 1H-NMR, 13C-NMR, 31P-NMR and HR-MS. The herbicidal evaluation performed by a Petri dish culture method showed that most compounds possessed moderate to good herbicidal activities. Six compounds were chosen for further herbicidal evaluation on barnyard grass by pot experiments. 1-(Diethoxyphosphoryl)-3-(4-phenyl-1H-1,2,3-triazol-1-yl)propan-2-yl 2-(naphthalen-1-yl)acetate (5-A3) and ethyl 1-(2-acetoxy-3-(diethoxyphosphoryl)propyl)-1H-1,2,3-triazole-4-carboxylate (5-B4) showed good herbicidal activities. Compared with the compounds with the best herbicidal activity ever reported, both compounds 5-A3 and 5-B4, which can inhibit the growth of barnyard grass at the concentration of 250g/hm2, efficiently gave rise to a nearly 4-fold increase of the herbicidal potency. However, their herbicidal activities were lower than that of acetochlor (62.5 g/hm2) in the pot experiments.

In order to begin to prepare a novel orthopedic implant that mimics the natural bone environment, the objective of this in vitro study was to synthesize nanocrystalline hydroxyapatite (NHA) and coat it on titanium (Ti) using molecular plasma deposition (MPD). NHA was synthesized through a wet chemical process followed by a hydrothermal treatment. NHA and micron sized hydroxyapatite (MHA) were prepared by processing NHA coatings at 500°C and 900°C, respectively. The coatings were characterized before and after sintering using scanning electron microscopy, atomic force microscopy, and X-ray diffraction. The results revealed that the post-MPD heat treatment of up to 500°C effectively restored the structural and topographical integrity of NHA. In order to determine the in vitro biological responses of the MPD-coated surfaces, the attachment and spreading of osteoblasts (bone-forming cells) on the uncoated, NHA-coated, and MHA-coated anodized Ti were investigated. Most importantly, the NHA-coated substrates supported a larger number of adherent cells than the MHA-coated and uncoated substrates. The morphology of these cells was assessed by scanning electron microscopy and the observed shapes were different for each substrate type. The present results are the first reports using MPD in the framework of hydroxyapatite coatings on Ti to enhance osteoblast responses and encourage further studies on MPD-based hydroxyapatite coatings on Ti for improved orthopedic applications.

<h4>Objective</h4>A relevant part of the social and personal burden caused by Bipolar Disorder (BD) is related to depressive phases. Authors investigated the genetic impact of a set of variations located in CRY1, a gene involved in the control of the circadian rhythms, towards depressive episodes in a sample of bipolar patients from the STEP-BD sample. As a secondary analysis, CYR1 variations were analyzed as predictors of sleep disruption.<h4>Methods</h4>654 bipolar patients were included in the analysis. Data were available genome-wide. The part of the genome coding for the CRY1 was imputed and pruned according to standards in the field. 7 SNPs were available for the analysis. A correction for multitesting was applied and we had sufficient power (0.80) to detect a small-medium effect size (0.22) between two allelic frequencies each one represented by at least 300 subjects.<h4>Results</h4>Intronic rs10861688 was associated with the number of depressive events corrected for the times patients were assessed during the period of observation. In particular, AA subjects (n=21) had 4.46±3.15 events, AG (n=141) had 3.08±3.17 and GG (n=342) 2.65±2.97 (p=0.0048, beta=-0.22). No other significant associations were reported.<h4>Conclusion</h4>We bring further evidence that genes involved in the regulation of circadian rhythms may be relevant to depressive bipolar phases. Independent confirmation analyses are mandatory.

<h4>Objective</h4>The serotonin transporter (5-HTT) genes are major candidate genes for modulating the suicidal behavior. We investigated the association between serotonin transporter polymorphisms and suicidal behavior in patients with major depressive disorder (MDD).<h4>Methods</h4>Serotonin transporter intron 2 VNTR polymorphism (5-HTTVNTR) and serotonin transporter linked polymorphic region polymorphism (5-HTTLPR) were analyzed in 132 depressed patients with suicidal attempt as well as in 122 normal controls. Hamilton's 17-item Depression Rating Scale (HDRS), the Risk-Rescue rating system (RRR) and the Lethality Suicide Attempt Rating Scale updated (LSARS-II) were assessed for the depressed patients.<h4>Results</h4>Although not statistically significant, a trend was found such that the 10/10 and 10/12 alleles of 5-HTTVNTR were more common in suicidal subjects than in control subjects. Comparing allele frequency, those with a 10 allele or 10 allele carriers were higher in suicidal subjects than in control subjects. No difference was noted in 5-HTTLPR genotypes and haplotype distribution between the suicidal subjects and control subjects. The RRR scores in subjects with the 10/10 5-HTTVNTR genotype or 10 5-HTTVNTR allele were significantly lower than those in subjects with other genotypes.<h4>Conclusion</h4>These results show the possibility that 10 allele of 5-HTTVNTR is related to suicidal behavior in the suicidal subjects with MDD and suggest that 12 allele of 5-HTTVNTR might be related to more lethality in the suicidal subjects with MDD.

Phosphorus (P) is an indispensable element for all life on Earth and, during the past decade, concerns about the future of its global supply have stimulated much research on soil P and method development. This review provides an overview of advanced state-of-the-art methods currently used in soil P research. These involve bulk and spatially resolved spectroscopic and spectrometric P speciation methods (1 and 2D NMR, IR, Raman, Q-TOF MS/MS, high resolution-MS, NanoSIMS, XRF, XPS, (µ)XAS) as well as methods for assessing soil P reactions (sorption isotherms, quantum-chemical modeling, microbial biomass P, enzymes activity, DGT, ³³P isotopic exchange, ¹⁸O isotope ratios). Required experimental set-ups and the potentials and limitations of individual methods present a guide for the selection of most suitable methods or combinations.

Gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN), considered a heterogeneous neoplasia, exhibit ill-defined pathobiology and protean symptomatology and are ubiquitous in location. They are difficult to diagnose, challenging to manage, and outcome depends on cell type, secretory product, histopathologic grading, and organ of origin. A morphologic and molecular genomic review of these lesions highlights tumor characteristics that can be used clinically, such as somatostatin-receptor expression, and confirms features that set them outside the standard neoplasia paradigm. Their unique pathobiology is useful for developing diagnostics using somatostatin-receptor targeted imaging or uptake of radiolabeled amino acids specific to secretory products or metabolism. Therapy has evolved via targeting of protein kinase B signaling or somatostatin receptors with drugs or isotopes (peptide-receptor radiotherapy). With DNA sequencing, rarely identified activating mutations confirm that tumor suppressor genes are relevant. Genomic approaches focusing on cancer-associated genes and signaling pathways likely will remain uninformative. Their uniquely dissimilar molecular profiles mean individual tumors are unlikely to be easily or uniformly targeted by therapeutics currently linked to standard cancer genetic paradigms. The prevalence of menin mutations in pancreatic NEN and P27^KIP1 mutations in small intestinal NEN represents initial steps to identifying a regulatory commonality in GEP-NEN. Transcriptional profiling and network-based analyses may define the cellular toolkit. Multianalyte diagnostic tools facilitate more accurate molecular pathologic delineations of NEN for assessing prognosis and identifying strategies for individualized patient treatment. GEP-NEN remain unique, poorly understood entities, and insight into their pathobiology and molecular mechanisms of growth and metastasis will help identify the diagnostic and therapeutic weaknesses of this neoplasia.

Various single nucleotide polymorphisms have been reported to be associated with a higher risk of hepatocellular carcinoma in alcoholic cirrhotic patients. Until now, only common variants conferring a small increase in liver cancer risk have been identified. These inherited factors are able to modulate several biological pathways involved in alcohol-induced hepatocarcinogenesis, such as ethanol metabolism, inflammation, oxidative stress, or iron and lipid homeostasis. How the combination of these variants might collectively define an individual genomic risk prediction is currently being investigated. The other challenge in clinical practice lies in defining how to integrate this genetic information with other clinical parameters so as to refine selection of alcoholic cirrhotic patients according to various classes of hepatocellular carcinoma risk.

Preterm infants born before the 30th week of pregnancy are especially at risk of perinatal brain damage which is usually a result of cerebral ischemia or an ascending intrauterine infection. Prevention of preterm birth and early intervention given signs of imminent intrauterine infection can reduce the incidence of perinatal cerebral injury. It has been shown that administering magnesium intravenously to women at imminent risk of a preterm birth leads to a significant reduction in the likelihood of the infant developing cerebral palsy and motor skill dysfunction. It has also been demonstrated that delayed clamping of the umbilical cord after birth reduces the rate of brain hemorrhage among preterm infants by up to 50%. In addition, mesenchymal stem cells seem to have significant neuroprotective potential in animal experiments, as they increase the rate of regeneration of the damaged cerebral area. Clinical tests of these types of therapeutic intervention measures appear to be imminent. In the last trimester of pregnancy, the serum concentrations of estradiol and progesterone increase significantly. Preterm infants are removed abruptly from this estradiol and progesterone rich environment. It has been demonstrated in animal experiments that estradiol and progesterone protect the immature brain from hypoxic-ischemic lesions. However, this neuroprotective strategy has unfortunately not yet been subject to sufficient clinical investigation.

Peroxiredoxin 6 (PRDX6) is a bifunctional protein with both glutathione peroxidase (GPx) and iPLA2 activities. Even though several pathophysiological functions have been studied, the definitive role of PRDX6 in tumor growth is not clear. Here, we compared carcinogen-induced tumor growth in PRDX6-transgenic (Tg) mice and non-Tg mice to evaluate the roles of PRDX6 in lung tumor development. Urethane (1g/kg)-induced tumor incidence in PRDX6-Tg mice was significantly higher compared to non-Tg mice. In the tumors of PRDX6-Tg mice, the activation of JAK2/STAT3 and STAT3 DNA binding were also increased, accompanied by increased GPx and iPLA2 activities. PRDX6 was colocalized with JAK2 in tumor tissues and lung cancer cells and also showed physical interaction with JAK2. We found that increasing levels of PRDX6 increase the activation of the JAK2/STAT3 pathway. Furthermore, PRDX6-Tg mice showed altered cytokine levels in the tumors, especially leading to increased CCL5 levels. We validated that the activation of JAK2 was also decreased in lung tumors of CCR5(-/-) mice, and CCL5 increased the JAK2/STAT3 pathway in the lung cancer cells. Thus, our findings suggest that PRDX6 promotes lung tumor development via its mediated and CCL5-associated activation of the JAK2/STAT3 pathway.

To investigate the relationship between linker histone H1 and protein-protein interactions in the nucleolus, we used biochemical and proteomics approaches to characterize nucleoli purified from cultured human and mouse cells. Mass spectrometry identified 175 proteins in human T cell nucleolar extracts that bound to Sepharose-immobilized H1 in vitro. Gene ontology analysis found significant enrichment for H1 binding proteins with functions related to nucleolar chromatin structure and RNA polymerase I transcription regulation, rRNA processing, and mRNA splicing. Consistent with the affinity binding results, H1 existed in large (400 to >650kDa) macromolecular complexes in human T cell nucleolar extracts. To complement the biochemical experiments, we investigated the effects of in vivo H1 depletion on protein content and structural integrity of the nucleolus using the H1 triple isoform knockout (H1ΔTKO) mouse embryonic stem cell (mESC) model system. Proteomic profiling of purified wild-type mESC nucleoli identified a total of 613 proteins, only ~60% of which were detected in the H1 mutant nucleoli. Within the affected group, spectral counting analysis quantitated 135 specific nucleolar proteins whose levels were significantly altered in H1ΔTKO mESC. Importantly, the functions of the affected proteins in mESC closely overlapped with those of the human T cell nucleolar H1 binding proteins. Immunofluorescence microscopy of intact H1ΔTKO mESC demonstrated both a loss of nucleolar RNA content and altered nucleolar morphology resulting from in vivo H1 depletion. We conclude that H1 organizes and maintains an extensive protein-protein interaction network in the nucleolus required for nucleolar structure and integrity.

Furin is a proprotein convertase enzyme. In the liver, it cleaves prohepcidin to form active hepcidin-25, which regulates systemic iron homeostasis. Hepcidin deficiency is a component of several iron overload disorders, including β-thalassemia. Several studies have identified factors that repress hepcidin gene transcription in iron overload. However, the effect of iron overload on furin, a post-translational regulator of hepcidin, has never been evaluated. The present study aimed to investigate the changes in furin and related factors in parenteral iron-overloaded mice, including those with β-thalassemia. Wild-type (WT) and β-thalassemia intermedia (<i>th3/</i>+) C57BL/6 mice were intraperitoneally injected with 9 doses of iron dextran (1 g iron/kg body weight) over 2 weeks. In the iron overload condition, our data demonstrated a significant <i>Furin</i> mRNA reduction in WT and <i>th3/+</i> mice. In addition, the liver furin protein level in iron-overloaded WT mice was significantly reduced by 70% compared to control WT mice. However, the liver furin protein in iron-overloaded <i>th3/+</i> mice did not show a significant reduction compared to control <i>th3/+</i> mice. The hepcidin gene (hepcidin antimicrobial peptide gene, <i>Hamp1</i>) expression was increased in iron-overloaded WT and <i>th3/+</i> mice. Surprisingly, the liver hepcidin protein level and total serum hepcidin were not increased in both WT and <i>th3/+</i> mice with iron overload, regardless of the increase in <i>Hamp1</i> mRNA. In conclusion, we demonstrate furin downregulation in conjunction with <i>Hamp1</i> mRNA-unrelated pattern of hepcidin protein expression in iron-overloaded mice, particularly the WT mice, suggesting that, not only the amount of hepcidin but also the furin-mediated physiological activity may be decreased in severe iron overload condition.

Autophagy is an evolutionarily conserved process that degrades cytoplasmic components, thus contributing to cell survival and tissue homeostasis. Recent studies have demonstrated that autophagy maintains stem cells in relatively undifferentiated states (stemness) and also contributes to differentiation processes. Autophagy likewise plays a crucial role in somatic cell reprogramming, a finely regulated process that resets differentiated cells to a pluripotent state and that requires comprehensive alterations in transcriptional activities and epigenetic signatures. Autophagy assists in manifesting the functional consequences that arise from these alterations by modifying cellular protein expression profiles. The role of autophagy appears to be particularly relevant for early phases of cell reprogramming during the generation of induced pluripotent stems cells (iPSCs). In this review, we provide an overview of the core molecular machinery that constitutes the autophagic degradation system, describe the roles of autophagy in maintenance, self-renewal, and differentiation of stem cells, and discuss the autophagic process and its regulation during cell reprogramming.

<h4>Objective</h4>  Information regarding the relative risks of developing knee osteoarthritis (OA) as a result of sport participation is critical for shaping public health messages and for informing knee-OA prevention strategies. The purpose of this systematic review was to investigate the association between participation in specific sports and knee OA.<h4>Data sources</h4>  We completed a systematic literature search in September 2012 using 6 bibliographic databases (PubMed; Ovid MEDLINE; Journals@Ovid; American College of Physicians Journal Club; Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Review, Database of Abstracts of Reviews of Effects; and Ovid HealthStar), manual searches (4 journals), and reference lists (56 articles).<h4>Study selection</h4>  Studies were included if they met the following 4 criteria: (1) an aim was to investigate an association between sport participation and knee OA; (2) the outcome measure was radiographic knee OA, clinical knee OA, total knee replacement, self-reported diagnosis of knee OA, or placement on a waiting list for a total knee replacement; (3) the study design was case control or cohort; and (4) the study was written in English. Articles were excluded if the study population had an underlying condition other than knee OA.<h4>Data extraction</h4>  One investigator extracted data (eg, group descriptions, knee OA prevalence, source of nonexposed controls).<h4>Data synthesis</h4>  The overall knee-OA prevalence in sport participants (n = 3759) was 7.7%, compared with 7.3% among nonexposed controls (referent group n = 4730, odds ratio [OR] = 1.1). Specific sports with a significantly higher prevalence of knee OA were soccer (OR = 3.5), elite-level long-distance running (OR = 3.3), competitive weight lifting (OR = 6.9), and wrestling (OR = 3.8). Elite-sport (soccer or orienteering) and nonelite-sport (soccer or American football) participants without a history of knee injury had a greater prevalence of knee OA than nonexposed participants.<h4>Conclusions</h4>  Participants in soccer (elite and nonelite), elite-level long-distance running, competitive weight lifting, and wrestling had an increased prevalence of knee OA and should be targeted for risk-reduction strategies.

No abstract available.

Netrin-1, acting through its principal receptor DCC (deleted in colorectal cancer), serves as an axon guidance cue during neural development and also contributes to vascular morphogenesis, epithelial migration, and the pathogenesis of some tumors. Several lines of evidence suggest that netrin-DCC signaling can regulate and be regulated by the cAMP-dependent protein kinase, PKA, although the molecular details of this relationship are poorly understood. Specificity in PKA signaling is often achieved through differential subcellular localization of the enzyme by interaction with protein kinase A anchoring proteins (AKAPs). Here, we show that AKAP function is required for DCC-mediated activation of PKA and phosphorylation of cytoskeletal regulatory proteins of the Mena/VASP (vasodilator-stimulated phosphoprotein) family. Moreover, we show that DCC and PKA physically interact and that this association is mediated by the ezrin-radixin-moesin (ERM) family of plasma membrane-actin cytoskeleton cross-linking proteins. Silencing of ERM protein expression inhibits DCC-PKA interaction, DCC-mediated PKA activation, and phosphorylation of Mena/VASP proteins as well as growth cone morphology and neurite outgrowth. Finally, although expression of wild-type radixin partially rescued growth cone morphology and tropism toward netrin in ERM-knockdown cells, expression of an AKAP-deficient mutant of radixin did not fully rescue growth cone morphology and switched netrin tropism from attraction to repulsion. These data support a model in which ERM-mediated anchoring of PKA activity to DCC is required for proper netrin/DCC-mediated signaling.

The Hundred Person Wellness Project (HPWP) is a 10-month pilot study of 100 'well' individuals where integrated data from whole-genome sequencing, gut microbiome, clinical laboratory tests and quantified self measures from each individual are used to provide actionable results for health coaching with the goal of optimizing wellness and minimizing disease. In a commentary in BMC Medicine, Diamandis argues that HPWP and similar projects will likely result in 'unnecessary and potential harmful over-testing'. We argue that this new approach will ultimately lead to lower costs, better healthcare, innovation and economic growth. The central points of the HPWP are: 1) it is focused on optimizing wellness through longitudinal data collection, integration and mining of individual data clouds, enabling development of predictive models of wellness and disease that will reveal actionable possibilities; and 2) by extending this study to 100,000 well people, we will establish multiparameter, quantifiable wellness metrics and identify markers for wellness to early disease transitions for most common diseases, which will ultimately allow earlier disease intervention, eventually transitioning the individual early on from a disease back to a wellness trajectory.

Memory T cells are primed for rapid responses to Ag; however, the molecular mechanisms responsible for priming remain incompletely defined. CpG methylation in promoters is an epigenetic modification, which regulates gene transcription. Using targeted bisulfite sequencing, we examined methylation of 2100 genes (56,000 CpGs) mapped by deep sequencing of T cell activation in human naive and memory CD4 T cells. Four hundred sixty-six CpGs (132 genes) displayed differential methylation between naive and memory cells. Twenty-one genes exhibited both differential methylation and gene expression before activation, linking promoter DNA methylation states to gene regulation; 6 of 21 genes encode proteins closely studied in T cells, whereas 15 genes represent novel targets for further study. Eighty-four genes demonstrated differential methylation between memory and naive cells that correlated to differential gene expression following activation, of which 39 exhibited reduced methylation in memory cells coupled with increased gene expression upon activation compared with naive cells. These reveal a class of primed genes more rapidly expressed in memory compared with naive cells and putatively regulated by DNA methylation. These findings define a DNA methylation signature unique to memory CD4 T cells that correlates with activation-induced gene expression.

Huntington Disease (HD) is a progressive neurodegenerative disease caused by an elongated CAG repeat in the huntingtin (HTT) gene that encodes a polyglutamine tract in the HTT protein. Proteolysis of the mutant HTT protein (mHTT) has been detected in human and murine HD brains and is implicated in the pathogenesis of HD. Of particular importance is the site at amino acid (aa) 586 that contains a caspase-6 (Casp6) recognition motif. Activation of Casp6 occurs presymptomatically in human HD patients and the inhibition of mHTT proteolysis at aa586 in the YAC128 mouse model results in the full rescue of HD-like phenotypes. Surprisingly, Casp6 ablation in two different HD mouse models did not completely prevent the generation of this fragment, and therapeutic benefits were limited, questioning the role of Casp6 in the disease. We have evaluated the impact of the loss of Casp6 in the YAC128 mouse model of HD. Levels of the mHTT-586 fragment are reduced but not absent in the absence of Casp6 and we identify caspase 8 as an alternate enzyme that can generate this fragment. In vivo, the ablation of Casp6 results in a partial rescue of body weight gain, normalized IGF-1 levels, a reversal of the depression-like phenotype and decreased HTT levels. In the YAC128/Casp6-/- striatum there is a concomitant reduction in p62 levels, a marker of autophagic activity, suggesting increased autophagic clearance. These results implicate the HTT-586 fragment as a key contributor to certain features of HD, irrespective of the enzyme involved in its generation.

Photocrosslinked hydrogels reinforced by microfibrillated cellulose (MFC) were prepared from a methacrylate-functionalized fish elastin polypeptide and MFC dispersed in dimethylsulfoxide (DMSO). First, a water-soluble elastin peptide with a molecular weight of ca. 500 g/mol from the fish bulbus arteriosus was polymerized by N,N'-dicyclohexylcarbodiimide (DCC), a condensation reagent, and then modified with 2-isocyanatoethyl methacrylate (MOI) to yield a photocrosslinkable fish elastin polypeptide. The product was dissolved in DMSO and irradiated with UV light in the presence of a radical photoinitiator. We obtained hydrogels successfully by substitution of DMSO with water. The composite gel with MFC was prepared by UV irradiation of the photocrosslinkable elastin polypeptide mixed with dispersed MFC in DMSO, followed by substitution of DMSO with water. The tensile test of the composite gels revealed that the addition of MFC improved the tensile properties, and the shape of the stress-strain curve of the composite gel became more similar to the typical shape of an elastic material with an increase of MFC content. The rheology measurement showed that the elastic modulus of the composite gel increased with an increase of MFC content. The cell proliferation test on the composite gel showed no toxicity.

The characterization of molecular changes in diseased tissues gives insight into pathophysiological mechanisms and is important for therapeutic development. Genome-wide gene expression analysis has proven valuable for identifying biological processes in neurodegenerative diseases using post mortem human brain tissue and numerous datasets are publically available. However, many studies utilize heterogeneous tissue samples consisting of multiple cell types, all of which contribute to global gene expression values, confounding biological interpretation of the data. In particular, changes in numbers of neuronal and glial cells occurring in neurodegeneration confound transcriptomic analyses, particularly in human brain tissues where sample availability and controls are limited. To identify cell specific gene expression changes in neurodegenerative disease, we have applied our recently published computational deconvolution method, population specific expression analysis (PSEA). PSEA estimates cell-type-specific expression values using reference expression measures, which in the case of brain tissue comprises mRNAs with cell-type-specific expression in neurons, astrocytes, oligodendrocytes and microglia. As an exercise in PSEA implementation and hypothesis development regarding neurodegenerative diseases, we applied PSEA to Parkinson's and Huntington's disease (PD, HD) datasets. Genes identified as differentially expressed in substantia nigra pars compacta neurons by PSEA were validated using external laser capture microdissection data. Network analysis and Annotation Clustering (DAVID) identified molecular processes implicated by differential gene expression in specific cell types. The results of these analyses provided new insights into the implementation of PSEA in brain tissues and additional refinement of molecular signatures in human HD and PD.

MicroRNAs (miRNAs), a group of noncoding RNAs, function as post-transcriptional gene regulators and control the establishment, self-renewal and differentiation of stem cells. Vitamin C has been recognized as a reprogramming enhancer because of its ability to induce a blastocyst-like state in embryonic stem cells (ESCs). However, knowledge on the regulation of miRNAs by vitamin C in ESCs is limited. In this study, we found that vitamin C induced miRNA expression, particularly of ESC-specific miRNAs. Moreover, vitamin C maintained the miRNA expression of the Dlk1-Dio3 imprinting region. The miRNAs in this region contain identical seed sequences, which target a class of genes, including Kdm6b, Klf13, and Sox6, and are mainly related to cell differentiation and development. These genes were significantly downregulated by vitamin C. Notably, miR-143 promoted self-renewal of mouse ESCs and suppressed expression of the de novo methyltransferase gene Dnmt3a. Knockdown of miR-143 by use of its inhibitor counteracted the vitamin C-induced reduction in Dnmt3a expression, showing that vitamin C repressed Dnmt3a expression via miR-143. Vitamin C also promoted DNA demethylation, including of pluripotency gene promoters (Tbx3, Tcl1, and Esrrb) and ESC-specific miRNA promoters (miR-290-295 and miR-17-92 clusters), and DNA hydroxymethylation, including of the intergenic differentially methylated region of the Dlk1-Dio3 region. These results strongly suggested that vitamin C promoted widespread DNA demethylation in gene promoters by modulating epigenetic modifiers, including Dnmt3a, which activated pluripotency genes and ESC-specific miRNAs. Then, differentiation and development genes were repressed by ESC-enriched miRNAs, which maintained the stem cell state.

Dysfunction of the orbitofrontal cortex (OFC) impairs the ability of individuals to flexibly adapt behavior to changing stimulus-reward (S-R) contingencies. Impaired flexibility also results from interventions that alter serotonin (5-HT) and dopamine (DA) transmission in the OFC and dorsomedial striatum (DMS). However, it is unclear whether similar mechanisms underpin naturally occurring variations in behavioral flexibility. In the present study, we used a spatial-discrimination serial reversal procedure to investigate interindividual variability in behavioral flexibility in rats. We show that flexibility on this task is improved following systemic administration of the 5-HT reuptake inhibitor citalopram and by low doses of the DA reuptake inhibitor GBR12909. Rats in the upper quintile of the distribution of perseverative responses during repeated S-R reversals showed significantly reduced levels of the 5-HT metabolite, 5-hydroxy-indoleacetic acid, in the OFC. Additionally, 5-HT2A receptor binding in the OFC of mid- and high-quintile rats was significantly reduced compared with rats in the low-quintile group. These perturbations were accompanied by an increase in the expression of monoamine oxidase-A (MAO-A) and MAO-B in the lateral OFC and by a decrease in the expression of MAO-A, MAO-B, and tryptophan hydroxylase in the dorsal raphé nucleus of highly perseverative rats. We found no evidence of significant differences in markers of DA and 5-HT function in the DMS or MAO expression in the ventral tegmental area of low- vs high-perseverative rats. These findings indicate that diminished serotonergic tone in the OFC may be an endophenotype that predisposes to behavioral inflexibility and other forms of compulsive behavior.

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI+], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN+], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN+] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI+][PIN+] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI+], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN+], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI+]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI+] induction in the absence of [PIN+]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI+], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI+] aggregates in a [PIN+]-independent pathway.

Substrate dephosphorylation by the cyclin-dependent kinase (Cdk)-opposing phosphatase, Cdc14, is vital for many events during budding yeast mitotic exit. Cdc14 is sequestered in the nucleolus through inhibitory binding to Net1, from which it is released in anaphase following Net1 phosphorylation. Initial Net1 phosphorylation depends on Cdk itself, in conjunction with proteins of the Cdc14 Early Anaphase Release (FEAR) network. Later on, the Mitotic Exit Network (MEN) signaling cascade maintains Cdc14 release. An important unresolved question is how Cdc14 activity can increase in early anaphase, while Cdk activity, that is required for Net1 phosphorylation, decreases and the MEN is not yet active. Here we show that the nuclear rim protein Nur1 interacts with Net1 and, in its Cdk phosphorylated form, inhibits Cdc14 release. Nur1 is dephosphorylated by Cdc14 in early anaphase, relieving the inhibition and promoting further Cdc14 release. Nur1 dephosphorylation thus describes a positive feedback loop in Cdc14 phosphatase activation during mitotic exit, required for faithful chromosome segregation and completion of the cell division cycle.

The persistence of experience-dependent changes in brain connectivity requires RNA localization and protein synthesis. Previous studies have demonstrated a role for local translation in altering the structure and function of synapses during synapse formation and experience-dependent synaptic plasticity. In this study, we ask whether in addition to promoting local translation, local stimulation also triggers directed trafficking of RNAs from nucleus to stimulated synapses. Imaging of RNA localization and translation in cultured Aplysia sensory-motor neurons revealed that RNAs were delivered throughout the arbor of the sensory neuron, but that translation was enriched only at sites of synaptic contact and/or synaptic stimulation. Investigation of the mechanisms that trigger local translation revealed a role for calcium-dependent retrograde netrin-1/DCC receptor signaling. Spatially restricting gene expression by regulating local translation rather than by directing the delivery of mRNAs from nucleus to stimulated synapses maximizes the readiness of the entire neuronal arbor to respond to local cues.

Despite the well-documented role of remote enhancers in controlling developmental gene expression, the mechanisms that allocate enhancers to genes are poorly characterized. Here, we investigate the cis-regulatory organization of the locus containing the Tfap2c and Bmp7 genes in vivo, using a series of engineered chromosomal rearrangements. While these genes lie adjacent to one another, we demonstrate that they are independently regulated by distinct sets of enhancers, which in turn define non-overlapping regulatory domains. Chromosome conformation capture experiments reveal a corresponding partition of the locus in two distinct structural entities, demarcated by a discrete transition zone. The impact of engineered chromosomal rearrangements on the topology of the locus and the resultant gene expression changes indicate that this transition zone functionally organizes the structural partition of the locus, thereby defining enhancer-target gene allocation. This partition is, however, not absolute: we show that it allows competing interactions across it that may be non-productive for the competing gene, but modulate expression of the competed one. Altogether, these data highlight the prime role of the topological organization of the genome in long-distance regulation of gene expression.

Chronic Obstructive Pulmonary Disease (COPD) is a complex disease. Genetic, epigenetic, and environmental factors are known to contribute to COPD risk and disease progression. Therefore we developed a systematic approach to identify key regulators of COPD that integrates genome-wide DNA methylation, gene expression, and phenotype data in lung tissue from COPD and control samples. Our integrative analysis identified 126 key regulators of COPD. We identified EPAS1 as the only key regulator whose downstream genes significantly overlapped with multiple genes sets associated with COPD disease severity. EPAS1 is distinct in comparison with other key regulators in terms of methylation profile and downstream target genes. Genes predicted to be regulated by EPAS1 were enriched for biological processes including signaling, cell communications, and system development. We confirmed that EPAS1 protein levels are lower in human COPD lung tissue compared to non-disease controls and that Epas1 gene expression is reduced in mice chronically exposed to cigarette smoke. As EPAS1 downstream genes were significantly enriched for hypoxia responsive genes in endothelial cells, we tested EPAS1 function in human endothelial cells. EPAS1 knockdown by siRNA in endothelial cells impacted genes that significantly overlapped with EPAS1 downstream genes in lung tissue including hypoxia responsive genes, and genes associated with emphysema severity. Our first integrative analysis of genome-wide DNA methylation and gene expression profiles illustrates that not only does DNA methylation play a 'causal' role in the molecular pathophysiology of COPD, but it can be leveraged to directly identify novel key mediators of this pathophysiology.

<h4>Purpose</h4>We applied whole-genome sequencing (WGS) to children diagnosed with neoplasms and found to carry apparently balanced constitutional translocations to discover novel genic disruptions.<h4>Methods</h4>We applied the structural variation (SV) calling programs CREST, BreakDancer, SV-STAT, and CGAP-CNV, and we developed an annotative filtering strategy to achieve nucleotide resolution at the translocations.<h4>Results</h4>We identified the breakpoints for t(6;12)(p21.1;q24.31), disrupting HNF1A in a patient diagnosed with hepatic adenomas and maturity-onset diabetes of the young (MODY). Translocation as the disruptive event of HNF1A, a gene known to be involved in MODY3, has not been previously reported. In a subject with Hodgkin lymphoma and subsequent low-grade glioma, we identified t(5;18)(q35.1;q21.2), disrupting both SLIT3 and DCC, genes previously implicated in both glioma and lymphoma.<h4>Conclusion</h4>These examples suggest that implementing clinical WGS in the diagnostic workup of patients with novel but apparently balanced translocations may reveal unanticipated disruption of disease-associated genes and aid in prediction of the clinical phenotype.

Active DNA demethylation in plants occurs through base excision repair, beginning with removal of methylated cytosine by the ROS1/DME subfamily of 5-methylcytosine DNA glycosylases. Active DNA demethylation in animals requires the DNA glycosylase TDG or MBD4, which functions after oxidation or deamination of 5-methylcytosine, respectively. However, little is known about the steps following DNA glycosylase action in the active DNA demethylation pathways in plants and animals. We show here that the Arabidopsis APE1L protein has apurinic/apyrimidinic endonuclease activities and functions downstream of ROS1 and DME. APE1L and ROS1 interact in vitro and co-localize in vivo. Whole genome bisulfite sequencing of ape1l mutant plants revealed widespread alterations in DNA methylation. We show that the ape1l/zdp double mutant displays embryonic lethality. Notably, the ape1l+/-zdp-/- mutant shows a maternal-effect lethality phenotype. APE1L and the DNA phosphatase ZDP are required for FWA and MEA gene imprinting in the endosperm and are important for seed development. Thus, APE1L is a new component of the active DNA demethylation pathway and, together with ZDP, regulates gene imprinting in Arabidopsis.

No abstract available.

Diabetes mellitus is well known to cause bladder dysfunction; however, the molecular mechanisms governing this process and the effects on individual tissue elements within the bladder are poorly understood, particularly in type 2 diabetes. A shotgun proteomics approach was applied to identify proteins differentially expressed between type 2 diabetic (TallyHo) and control (SWR/J) mice in the bladder smooth muscle and urothelium, separately. We were able to identify 1760 nonredundant proteins from the detrusor smooth muscle and 3169 nonredundant proteins from urothelium. Pathway and network analysis of significantly dysregulated proteins was conducted to investigate the molecular processes associated with diabetes. This pinpointed ERK1/2 signaling as a key regulatory node in the diabetes-induced pathophysiology for both tissue types. The detrusor muscle samples showed diabetes-induced increased tissue remodeling-type events such as Actin Cytoskeleton Signaling and Signaling by Rho Family GTPases. The diabetic urothelium samples exhibited oxidative stress responses, as seen in the suppression of protein expression for key players in the NRF2-Mediated Oxidative Stress Response pathway. These results suggest that diabetes induced elevated inflammatory responses, oxidative stress, and tissue remodeling are involved in the development of tissue specific diabetic bladder dysfunctions. Validation of signaling dysregulation as a function of diabetes was performed using Western blotting. These data illustrated changes in ERK1/2 phosphorylation as a function of diabetes, with significant decreases in diabetes-associated phosphorylation in urothelium, but the opposite effect in detrusor muscle. These data highlight the importance of understanding tissue specific effects of disease process in understanding pathophysiology in complex disease and pave the way for future studies to better understand important molecular targets in reversing bladder dysfunction.

Implantation requires coordinated interactions between the conceptus and surrounding decidual cells, but the involvement of clock genes in this process is incompletely understood. Circadian oscillations are predicated on transcriptional-translational feedback loops, which balance the activities of the transcriptional activators CLOCK (circadian locomotor output cycles kaput) and brain muscle arnt-like 1 and repressors encoded by PER (Period) and Cryptochrome genes. We show that loss of PER2 expression silences circadian oscillations in decidualizing human endometrial stromal cells (HESCs). Down-regulation occurred between 12 and 24 hours following differentiation and coincided with reduced CLOCK binding to a noncanonical E-box enhancer in the PER2 promoter. RNA sequencing revealed that premature inhibition of PER2 by small interfering RNA knockdown leads to a grossly disorganized decidual response. Gene ontology analysis highlighted a preponderance of cell cycle regulators among the 1121 genes perturbed upon PER2 knockdown. Congruently, PER2 inhibition abrogated mitotic expansion of differentiating HESCs by inducing cell cycle block at G2/M. Analysis of 70 midluteal endometrial biopsies revealed an inverse correlation between PER2 transcript levels and the number of miscarriages in women suffering reproductive failure (Spearman rank test, ρ = -0.3260; P = 0.0046). Thus, PER2 synchronizes endometrial proliferation with initiation of aperiodic decidual gene expression; uncoupling of these events may cause recurrent pregnancy loss.

The CAG repeat expansion in the Huntington's disease gene HTT extends a polyglutamine tract in mutant huntingtin that enhances its ability to facilitate polycomb repressive complex 2 (PRC2). To gain insight into this dominant gain of function, we mapped histone modifications genome-wide across an isogenic panel of mouse embryonic stem cell (ESC) and neuronal progenitor cell (NPC) lines, comparing the effects of Htt null and different size Htt CAG mutations. We found that Htt is required in ESC for the proper deposition of histone H3K27me3 at a subset of 'bivalent' loci but in NPC it is needed at 'bivalent' loci for both the proper maintenance and the appropriate removal of this mark. In contrast, Htt CAG size, though changing histone H3K27me3, is prominently associated with altered histone H3K4me3 at 'active' loci. The sets of ESC and NPC genes with altered histone marks delineated by the lack of huntingtin or the presence of mutant huntingtin, though distinct, are enriched in similar pathways with apoptosis specifically highlighted for the CAG mutation. Thus, the manner by which huntingtin function facilitates PRC2 may afford mutant huntingtin with multiple opportunities to impinge upon the broader machinery that orchestrates developmentally appropriate chromatin status.

Intestinal epithelial stem cells (IESCs) control the intestinal homeostatic response to inflammation and regeneration. The underlying mechanisms are unclear. Cytokine-STAT5 signaling regulates intestinal epithelial homeostasis and responses to injury. We link STAT5 signaling to IESC replenishment upon injury by depletion or activation of Stat5 transcription factor. We found that depletion of Stat5 led to deregulation of IESC marker expression and decreased LGR5(+) IESC proliferation. STAT5-deficient mice exhibited worse intestinal histology and impaired crypt regeneration after γ-irradiation. We generated a transgenic mouse model with inducible expression of constitutively active Stat5. In contrast to Stat5 depletion, activation of STAT5 increased IESC proliferation, accelerated crypt regeneration, and conferred resistance to intestinal injury. Furthermore, ectopic activation of STAT5 in mouse or human stem cells promoted LGR5(+) IESC self-renewal. Accordingly, STAT5 promotes IESC proliferation and regeneration to mitigate intestinal inflammation. STAT5 is a functional therapeutic target to improve the IESC regenerative response to gut injury.

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that can induce various clinical tumors and has caused severe economic losses in China. To improve our understanding of the host cellular responses to virus infection and the pathogenesis of ALV-J infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with multidimensional liquid chromatography-tandem mass spectrometry to detect the protein changes in DF-1 cells infected and mock-infected with ALV-J. A total of 75 cellular proteins were significantly changed, including 33 upregulated proteins and 42 downregulated proteins. The reliability of iTRAQ-LC MS/MS was confirmed via real-time PCR. Most of these proteins were related to the physiological functions of metabolic processes, biosynthetic processes, responses to stimuli, protein binding, signal transduction, cell cytoskeleton, and so forth. We also found some proteins that play important roles in apoptosis and oncogenicity. The differentially expressed proteins identified may provide valuable information to elucidate the pathogenesis of virus infection and virus-host interactions.

The chromatin fiber undergoes significant structural changes during the cell's life cycle to modulate DNA accessibility. Detailed mechanisms of such structural transformations of chromatin fibers as affected by various internal and external conditions such as the ionic conditions of the medium, the linker DNA length, and the presence of linker histones, constitute an open challenge. Here we utilize Monte Carlo (MC) simulations of a coarse grained model of chromatin with nonuniform linker DNA lengths as found in vivo to help explain some aspects of this challenge. We investigate the unfolding mechanisms of chromatin fibers with alternating linker lengths of 26-62 bp and 44-79 bp using a series of end-to-end stretching trajectories with and without linker histones and compare results to uniform-linker-length fibers. We find that linker histones increase overall resistance of nonuniform fibers and lead to fiber unfolding with superbeads-on-a-string cluster transitions. Chromatin fibers with nonuniform linker DNA lengths display a more complex, multi-step yet smoother process of unfolding compared to their uniform counterparts, likely due to the existence of a more continuous range of nucleosome-nucleosome interactions. This finding echoes the theme that some heterogeneity in fiber component is biologically advantageous.

A reference genome is a high quality individual genome that is used as a coordinate system for the genomes of a population, or genomes of closely related subspecies. Given a set of genomes partitioned by homology into alignment blocks we formalize the problem of ordering and orienting the blocks such that the resulting ordering maximally agrees with the underlying genomes' ordering and orientation, creating a pan-genome reference ordering. We show this problem is NP-hard, but also demonstrate, empirically and within simulations, the performance of heuristic algorithms based upon a cactus graph decomposition to find locally maximal solutions. We describe an extension of our Cactus software to create a pan-genome reference for whole genome alignments, and demonstrate how it can be used to create novel genome browser visualizations using human variation data as a test. In addition, we test the use of a pan-genome for describing variations and as a reference for read mapping.

Membrane remodeling in the early stages of phagocytosis enables the engulfment of particles or pathogens and receptor signaling to activate innate immune responses. Members of the Rab GTPase family and their disparate effectors are recruited sequentially to regulate steps throughout phagocytosis. Rab31 (Rab22b) is known for regulating post-Golgi trafficking, and here we show in macrophages that Rab31-GTP is additionally and specifically recruited to early-stage phagosomes. At phagocytic cups, Rab31 is first recruited during the phosphoinositide transition from PI(4,5)P2 to PI(3,4,5)P3, and it persists on PI(3)P-enriched phagosomes. During early phagocytosis, we find that Rab31 recruits the signaling adaptor APPL2. siRNA depletion of either Rab31 or APPL2 reduces FcγR-mediated phagocytosis. Mechanistically, this corresponds with a delay in the transition to PI(3,4,5)P3 and phagocytic cup closure. APPL2 depletion also reduced PI3K/Akt signaling and enhanced p38 signaling from FcγR. We thus conclude that Rab31/APPL2 is required for key roles in phagocytosis and prosurvival responses of macrophages. Of interest, in terms of localization and function, this Rab31/APPL2 complex is distinct from the Rab5/APPL1 complex, which is also involved in phagocytosis and signaling.

Polo-like kinase 1 (PLK1) is highly expressed in many cancers and therefore a biomarker of transformation and potential target for the development of cancer-specific small molecule drugs. RO3280 was recently identified as a novel PLK1 inhibitor; however its therapeutic effects in leukemia treatment are still unknown. We found that the PLK1 protein was highly expressed in leukemia cell lines as well as 73.3% (11/15) of pediatric acute myeloid leukemia (AML) samples. PLK1 mRNA expression was significantly higher in AML samples compared with control samples (82.95 ± 110.28 vs. 6.36 ± 6.35; p < 0.001). Kaplan-Meier survival analysis revealed that shorter survival time correlated with high tumor PLK1 expression (p = 0.002). The 50% inhibitory concentration (IC50) of RO3280 for acute leukemia cells was between 74 and 797 nM. The IC50 of RO3280 in primary acute lymphocytic leukemia (ALL) and AML cells was between 35.49 and 110.76 nM and 52.80 and 147.50 nM, respectively. RO3280 induced apoptosis and cell cycle disorder in leukemia cells. RO3280 treatment regulated several apoptosis-associated genes. The regulation of DCC, CDKN1A, BTK, and SOCS2 was verified by western blot. These results provide insights into the potential use of RO3280 for AML therapy; however, the underlying mechanisms remain to be determined.

Antimicrobial drug resistance is a major human health threat. Among the many attempts to tackle this problem, the synthesis of antimicrobial compounds that mimic natural antimicrobial peptides appears as a promising approach. Peptide-based dendrimers can be designed to have higher potency than natural antimicrobial peptides and at the same time they can evade the bacterial defense system. Novel dendrimers with similar chemical structure but varying potency in terms of minimum inhibitory concentration were designed. The dependency between dendrimer structure and antibacterial activity as well as their capacity to attack model cell membranes was studied. The data suggests that supramolecular structure in terms of charge distribution and amphiphilicity, rather than net charge, is the main driver for disruption of cellular membranes and this correlates well with dendrimer hemolytic activity.

Plumbagin (PLB) has exhibited a potent anticancer effect in preclinical studies, but the molecular interactome remains elusive. This study aimed to compare the quantitative proteomic responses to PLB treatment in human prostate cancer PC-3 and DU145 cells using the approach of stable-isotope labeling by amino acids in cell culture (SILAC). The data were finally validated using Western blot assay. First, the bioinformatic analysis predicted that PLB could interact with 78 proteins that were involved in cell proliferation and apoptosis, immunity, and signal transduction. Our quantitative proteomic study using SILAC revealed that there were at least 1,225 and 267 proteins interacting with PLB and there were 341 and 107 signaling pathways and cellular functions potentially regulated by PLB in PC-3 and DU145 cells, respectively. These proteins and pathways played a critical role in the regulation of cell cycle, apoptosis, autophagy, epithelial to mesenchymal transition (EMT), and reactive oxygen species generation. The proteomic study showed substantial differences in response to PLB treatment between PC-3 and DU145 cells. PLB treatment significantly modulated the expression of critical proteins that regulate cell cycle, apoptosis, and EMT signaling pathways in PC-3 cells but not in DU145 cells. Consistently, our Western blotting analysis validated the bioinformatic and proteomic data and confirmed the modulating effects of PLB on important proteins that regulated cell cycle, apoptosis, autophagy, and EMT in PC-3 and DU145 cells. The data from the Western blot assay could not display significant differences between PC-3 and DU145 cells. These findings indicate that PLB elicits different proteomic responses in PC-3 and DU145 cells involving proteins and pathways that regulate cell cycle, apoptosis, autophagy, reactive oxygen species production, and antioxidation/oxidation homeostasis. This is the first systematic study with integrated computational, proteomic, and functional analyses revealing the networks of signaling pathways and differential proteomic responses to PLB treatment in prostate cancer cells. Quantitative proteomic analysis using SILAC represents an efficient and highly sensitive approach to identify the target networks of anticancer drugs like PLB, and the data may be used to discriminate the molecular and clinical subtypes, and to identify new therapeutic targets and biomarkers, for prostate cancer. Further studies are warranted to explore the potential of quantitative proteomic analysis in the identification of new targets and biomarkers for prostate cancer.

We demonstrate that the natural product brartemicin, a newly discovered inhibitor of cancer cell invasion, is a high-affinity ligand of the carbohydrate-recognition domain (CRD) of the C-type lectin mincle. Recent studies have revealed that mincle is a key macrophage receptor for the mycobacterial virulence factor trehalose dimycolate (TDM), which is a glycolipid component of the mycobacterial cell wall. Major uncertainties, however, remain concerning the mechanism of TDM-binding and subsequent signal transduction as well as interplay of potential co-receptors. Due to the lipid nature of TDM, functional studies are difficult and soluble mincle-ligands are therefore of significant interest. Brartemicin, together with designed analogs also presented in this paper, may thus serve as useful molecular probes for future studies of mincle. Through computational studies, we further provide an insight into the probable mode of binding of brartemicin.

Serotonin critically affects the neural processing of emotionally salient stimuli, including indices of threat; however, how alterations in serotonin signaling contribute to changes in brain function is not well understood. Recently, we showed in a placebo-controlled study of 32 healthy males that brain serotonin 4 receptor (5-HT4) binding, assessed with [(11)C]SB207145 PET, was sensitive to a 3-week intervention with the selective serotonin reuptake inhibitor fluoxetine, supporting it as an in vivo model for fluctuations in central serotonin levels. Participants also underwent functional magnetic resonance imaging while performing a gender discrimination task of fearful, angry, and neutral faces. This offered a unique opportunity to evaluate whether individual fluctuations in central serotonin levels, indexed by change in [(11)C]SB207145 binding, predicted changes in threat-related reactivity (ie, fear and angry vs neutral faces) within a corticolimbic circuit including the amygdala and medial prefrontal and anterior cingulate cortex. We observed a significant association such that decreased brain-wide [(11)C]SB207145 binding (ie, increased brain serotonin levels) was associated with lower threat-related amygdala reactivity, whereas intervention group status did not predict change in corticolimbic reactivity. This suggests that in the healthy brain, interindividual responses to pharmacologically induced and spontaneously occurring fluctuations in [(11)C]SB207145 binding, a putative marker of brain serotonin levels, affect amygdala reactivity to threat. Our finding also supports that change in brain [(11)C]SB207145 binding may be a relevant marker for evaluating neurobiological mechanisms underlying sensitivity to threat and serotonin signaling.

Huntington's disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington's disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.

A Gram-staining-negative, catalase- and oxidase-positive, obligately aerobic, motile by gliding, bright yellow and rod-shaped bacterial strain was isolated from soil of Daemo Mountain (Daemosan) in Seoul, Republic of Korea. Its taxonomic position was investigated by using a polyphasic study. On the basis of 16S rRNA gene sequence comparisons, strain THG-DJ7(T) was found to be most closely related to Flavobacterium denitrificans ED5(T) (97.9 % sequence similarity), Flavobacterium ginsenosidimutans THG 01(T) (97.6 %), Flavobacterium kyungheensis THG-107(T) (97.5 %), Flavobacterium anhuiense D3(T) (97.1 %) and Flavobacterium ginsengisoli DCY54(T) (97.0 %). The DNA-DNA relatedness between strain THG-DJ7(T) and its phylogenetically closest neighbours was below 40.0 %. The G+C content of the genomic DNA was determined to be 32.6 mol%. The only isoprenoid quinone detected in strain THG-DJ7(T) was menaquinone-6 (MK-6). The major component in the polyamine pattern was sym-homospermidine. The major polar lipids were found to be phosphatidylethanolamine, phosphatidyldimethylethanolamine, an unidentified aminophosphoglycolipid, phosphatidylserine and an unidentified lipid. The major fatty acids were identified as iso-C15 : 0, iso-C15 : 0 3-OH and summed feature 3 (comprising C16 : 1ω7c and/or C16 : 1ω6c). These data support the affiliation of strain THG-DJ7(T) to the genus Flavobacterium. We report the phenotypic, genotypic and phylogenetic characterization of strain THG-DJ7(T). Based on the findings presented, a novel species of the genus Flavobacterium, Flavobacterium daemonensis sp. nov. is proposed, with THG-DJ7(T) ( = KACC 17651(T) = JCM 19455(T)) as the type strain.

Cell-based bone regeneration strategies offer promise for traumatic bone injuries, congenital defects, non-union fractures and other skeletal pathologies. Postnatal bone remodeling and fracture healing provide evidence that an osteochondroprogenitor cell is present in adult life that can differentiate to remodel or repair the fractured bone. However, cell-based skeletal repair in the clinic is still in its infancy, mostly due to poor characterization of progenitor cells and lack of knowledge about their in vivo behavior. Here, we took a combined approach of high-throughput screening, flow-based cell sorting and in vivo transplantation to isolate markers that identify osteochondroprogenitor cells. We show that the presence of tetraspanin CD9 enriches for osteochondroprogenitors within CD105(+) mesenchymal cells and that these cells readily form bone upon transplantation. In addition, we have used Thy1.2 and the ectonucleotidase CD73 to identify subsets within the CD9(+) population that lead to endochondral or intramembranous-like bone formation. Utilization of this unique cell surface phenotype to enrich for osteochondroprogenitor cells will allow for further characterization of the molecular mechanisms that regulate their osteogenic properties.

Hepcidin is a key hormone that induces the degradation of ferroportin (FPN), a protein that exports iron from reticuloendothelial macrophages and enterocytes. The aim of the present study was to experimentally evaluate if the obesity induced by a high-fat diet (HFD) modifies the expression of FPN in macrophages and enterocytes, thus altering the iron bioavailability. In order to directly examine changes associated with iron metabolism in vivo, C57BL/6J mice were fed either a control or a HFD. Serum leptin levels were evaluated. The hepcidin, divalent metal transporter-1 (DMT1), FPN and ferritin genes were analyzed by real-time polymerase chain reaction. The amount of iron present in both the liver and spleen was determined by flame atomic absorption spectrometry. Ferroportin localization within reticuloendothelial macrophages was observed by immunofluorescence microscopy. Obese animals were found to exhibit increased hepcidin gene expression, while iron accumulated in the spleen and liver. They also exhibited changes in the sublocation of splenic cellular FPN and a reduction in the FPN expression in the liver and the spleen, while no changes were observed in enterocytes. Possible explanations for the increased hepcidin expression observed in HFD animals may include: increased leptin levels, the liver iron accumulation or endoplasmic reticulum (ER) stress. Together, the results indicated that obesity promotes changes in iron bioavailability, since it altered the iron recycling function.

Suboptimal intrauterine development has been linked to predisposition to cardiovascular disease in adulthood, a concept termed 'developmental origins of health and disease'. Although the exact mechanisms underlying this developmental programming are unknown, a growing body of evidence supports the involvement of epigenetic regulation. Epigenetic mechanisms such as DNA methylation, histone modifications and micro-RNA confer added levels of gene regulation without altering DNA sequences. These modifications are relatively stable signals, offering possible insight into the mechanisms underlying developmental origins of health and disease. This review will discuss the role of epigenetic mechanisms in heart development as well as aberrant epigenetic regulation contributing to cardiovascular disease. Additionally, we will address recent advances targeting epigenetic mechanisms as potential therapeutic approaches to cardiovascular disease.

TDP-43 pathology is a disease hallmark that characterizes amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). Although a critical role for TDP-43 as an RNA-binding protein has emerged, the regulation of TDP-43 function is poorly understood. Here, we identify lysine acetylation as a novel post-translational modification controlling TDP-43 function and aggregation. We provide evidence that TDP-43 acetylation impairs RNA binding and promotes accumulation of insoluble, hyper-phosphorylated TDP-43 species that largely resemble pathological inclusions in ALS and FTLD-TDP. Moreover, biochemical and cell-based assays identify oxidative stress as a signalling cue that promotes acetylated TDP-43 aggregates that are readily engaged by the cellular defense machinery. Importantly, acetylated TDP-43 lesions are found in ALS patient spinal cord, indicating that aberrant TDP-43 acetylation and loss of RNA binding are linked to TDP-43 proteinopathy. Thus, modulating TDP-43 acetylation represents a plausible strategy to fine-tune TDP-43 activity, which could provide new therapeutic avenues for TDP-43 proteinopathies.

Molecular changes occurring during mammalian oocyte maturation are partly regulated by cytoplasmic polyadenylation (CP) and affect oocyte quality, yet the extent of CP activity during oocyte maturation remains unknown. Single bovine oocyte RNA sequencing (RNA-Seq) was performed to examine changes in transcript abundance during in vitro oocyte maturation in cattle. Polyadenylated RNA from individual germinal-vesicle and metaphase-II oocytes was amplified and processed for Illumina sequencing, producing approximately 30 million reads per replicate for each sample type. A total of 10,494 genes were found to be expressed, of which 2,455 were differentially expressed (adjusted P < 0.05 and fold change >2) between stages, with 503 and 1,952 genes respectively increasing and decreasing in abundance. Differentially expressed genes with complete 3'-untranslated-region sequence (279 increasing and 918 decreasing in polyadenylated transcript abundance) were examined for the presence, position, and distribution of motifs mediating CP, revealing enrichment (85%) and lack thereof (18%) in up- and down-regulated genes, respectively. Examination of total and polyadenylated RNA abundance by quantitative PCR validated these RNA-Seq findings. The observed increases in polyadenylated transcript abundance within the RNA-Seq data are likely due to CP, providing novel insight into targeted transcripts and resultant differential gene expression profiles that contribute to oocyte maturation.

Deleted in colorectal cancer (DCC), a large transmembrane receptor of netrin-1, is critical for mediating netrin-1's cardioprotective function. In the present study we investigated novel mechanisms underlying netrin-1-induced, rapid, and feed-forward up-regulation of DCC, which is believed to sustain nitric oxide (NO) production to potentiate cardioprotection. Intriguingly, NO markedly reduced expression of the E3 ubiquitin ligase seven in absentia homolog (SIAH) that is specific for regulation of protesome-dependent DCC degradation, resulting in accumulation of DCC. The two SIAH isoforms compensate for each other when one is repressed; inhibition of both SIAH1 and SIAH2 using combined siRNAs significantly reduced infarct size while improving cardiac function after ischemia/reperfusion injury of the heart. This effect was absent in DCC-deficient mice. Moreover, in vivo RNAi inhibition of SIAH1/2 further augmented netrin-1's cardioprotective function. In summary, these data identify a novel therapeutic target of SIAH in facilitating NO/netrin-1-dependent cardioprotection, using the DCC receptor. Combination of netrin-1 and SIAH RNAi may prove to be a substantially effective therapy for myocardial infarction.

Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera. We analyzed human proteins present in the small intestine of patients infected with V. cholerae O1 to characterize the host response to this pathogen. We collected duodenal biopsy specimens from patients with acute cholera after stabilization and again 30 days after initial presentation. Peptides extracted from biopsy specimens were sequenced and quantified using label-free mass spectrometry and SEQUEST. Twenty-seven host proteins were differentially abundant between the acute and convalescent stages of infection; the majority of these have known roles in innate defense, cytokine production, and apoptosis. Immunostaining confirmed that two proteins, WARS and S100A8, were more abundant in lamina propria cells during the acute stage of cholera. Analysis of the differentially abundant proteins revealed the activation of key regulators of inflammation by the innate immune system, including Toll-like receptor 4, nuclear factor kappa-light-chain-enhancer of activated B cells, mitogen-activated protein kinases, and caspase-dependent inflammasomes. Interleukin-12β (IL-12β) was a regulator of several proteins that were activated during cholera, and we confirmed that IL-12β was produced by lymphocytes recovered from duodenal biopsy specimens of cholera patients. Our study shows that a broad inflammatory response is generated in the gut early after onset of cholera, which may be critical in the development of long-term mucosal immunity against V. cholerae O1.

<h4>Background and aims</h4>Screening patients at risk for hepatocellular carcinoma (HCC) facilitates early detection of disease, with improved outcome. The most common causes of HCC include chronic viral hepatitis infection-namely hepatitis B, hepatitis C, and cirrhosis. The aim of this study was to assess the awareness of screening among physicians involved in the management of patients at risk for HCC.<h4>Methods</h4>Three hundred physicians from three academic centers were invited to participate in a mailed survey questionnaire. The main outcome measure was physicians' knowledge of the current HCC screening guidelines. Demographic and clinical variables were obtained from the survey questionnaire.<h4>Results</h4>A total of 177 (59.0%) out of the 300 invited physicians responded to the survey questionnaire, including faculty members (n = 129), residents (n = 46), and fellows (n = 2). The specialty areas of the responding physicians were internal medicine (62.1%), family medicine (16.4%), gastroenterology (15.3%), oncology (3.4%) and others (2.8%). The number of physicians who performed HCC screening in patients with cirrhosis secondary to chronic hepatitis B and chronic hepatitis C infection were 163 (92.1%) and 167 (94.4%), respectively; 35.0% of them used alpha-fetoprotein (AFP) every 6 months, while 22.0% used imaging modalities every 6 months to screen for HCC. Further, 22 physicians (12.4%) did not check for serum AFP levels and 33 (18.6%) never used imaging to screen for HCC.<h4>Conclusion</h4>The majority of the participating physicians screen high-risk patients for HCC. However, the most appropriate modality of screening (i.e. imaging) is not employed by most physicians and there is greater reliance on AFP levels.

Poly(lactic acid) (PLA) was modified using collagen through a grafting method to improve its biocompatibility and degradability. The carboxylic group at the open end of PLA was transferred into the reactive acylchlorided group by a reaction with phosphorus pentachloride. Then, collagen-modified PLA (collagen-PLA) was prepared by the reaction between the reactive acylchlorided group and amino/hydroxyl groups on collagen. Subsequently, the structure of collagen-PLA was confirmed by Fourier transform infrared spectroscopy, fluorescein isothiocyanate-labeled fluorescence spectroscopy, X-ray photoelectron spectroscopy, and DSC analyses. Finally, some properties of collagen-PLA, such as hydrophilicity, cell compatibility and degradability were characterized. Results showed that collagen had been grafted onto the PLA with 5% graft ratio. Water contact angle and water absorption behavior tests indicated that the hydrophilicity of collagen-PLA was significantly higher than that of PLA. The cell compatibility of collagen-PLA with mouse embryonic fibroblasts (3T3) was also significantly better than PLA in terms of cell morphology and cell proliferation, and the degradability of PLA was also improved after introducing collagen. Results suggested that collagen-PLA was a promising candidate for biomedical applications.

One consequence of modern cancer therapy is chemotherapy related cognitive dysfunction or "chemobrain", the subjective experience of cognitive deficits at any point during or following chemotherapy. Chemobrain, a well-established clinical syndrome, has become an increasing concern because the number of long-term cancer survivors is growing dramatically. There is strong evidence that correlates changes in peripheral cytokines with the development of chemobrain in commonly used chemotherapeutic drugs for different types of cancer. However, the mechanisms by which these cytokines elicit change in the central nervous system are still unclear. In this review, we hypothesize that the administration of chemotherapy agents initiates a cascade of biological changes, with short-lived alterations in the cytokine milieu inducing persistent epigenetic alterations. These epigenetic changes lead to changes in gene expression, alterations in metabolic activity and neuronal transmission that are responsible for generating the subjective experience of cognition. This speculative but testable hypothesis should help to gain a comprehensive understanding of the mechanism underlying cognitive dysfunction in cancer patients. Such knowledge is critical to identify pharmaceutical targets with the potential to prevent and treat cancer-treatment related cognitive dysfunction and similar disorders.