Gene Literature Dashboard

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Neuroinflammation is a prominent component of several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease, Parkinson's disease, tauopathies, amyotrophic lateral sclerosis and prion diseases. In such conditions, the ability to decrease neuroinflammation by drug therapy may influence disease progression. Statins have been used to treat hyperlipidemia as well as reduce neuroinflammation and oxidative stress in various tissues. In previous studies, treatment of scrapie-infected mice with the type 1 statins, simvastatin or pravastatin, showed a small beneficial effect on survival time. In the current study, to increase the effectiveness of statin therapy, we treated infected mice with atorvastatin, a type 2 statin that has improved pharmacokinetics over many type 1 statins. Treatments with either simvastatin or pravastatin were tested for comparison. We evaluated scrapie-infected mice for protease-resistant PrP (PrPres) accumulation, gliosis, neuroinflammation and time until advanced clinical disease requiring euthanasia. All three statin treatments reduced total serum cholesterol ≥40 % in mice. However, gliosis and PrPres deposition were similar in statin-treated and untreated infected mice. Time to euthanasia due to advanced clinical signs was not changed in statin-treated mice relative to untreated mice, a finding at odds with previous reports. Expression of 84 inflammatory genes involved in neuroinflammation was also quantitated. Seven genes were reduced by pravastatin, and one gene was reduced by atorvastatin. In contrast, simvastatin therapy did not reduce any of the tested genes, but did slightly increase the expression of Ccl2 and Cxcl13. Our studies indicate that none of the three statins tested were effective in reducing scrapie-induced neuroinflammation or neuropathogenesis.

Microglia are the main resident macrophage population of the CNS and perform numerous functions required for CNS development, homeostasis, immunity, and repair. Many lines of evidence also indicate that dysregulation of microglia contributes to the pathogenesis of neurodegenerative and behavioral diseases. These observations provide a compelling argument to more clearly define the mechanisms that control microglia identity and function in health and disease. In this Review, we present a conceptual framework for how different classes of transcription factors interact to select and activate regulatory elements that control microglia development and their responses to internal and external signals. We then describe functions of specific transcription factors in normal and pathological contexts and conclude with a consideration of open questions to be addressed in the future.

To maintain cellular homeostasis, subcellular organelles communicate with each other and form physical and functional networks through membrane contact sites coupled by protein tethers. In particular, endoplasmic reticulum (ER)-mitochondrial contacts (EMC) regulate diverse cellular activities such as metabolite exchange (Ca²⁺ and lipids), intracellular signaling, apoptosis, and autophagy. The significance of EMCs has been highlighted by reports indicating that EMC dysregulation is linked to neurodegenerative diseases. Therefore, obtaining a better understanding of the physical and functional components of EMCs should provide new insights into the pathogenesis of several neurodegenerative diseases. Here, we applied engineered ascorbate peroxidase (APEX) to map the proteome at EMCs in live HEK293 cells. APEX was targeted to the outer mitochondrial membrane, and proximity-labeled proteins were analyzed by stable isotope labeling with amino acids in culture (SILAC)-LC/MS-MS. We further refined the specificity of the proteins identified by combining biochemical subcellular fractionation to the protein isolation method. We identified 405 proteins with a 2.0-fold cutoff ratio (log base 2) in SILAC quantification from replicate experiments. We performed validation screening with a Split-Rluc8 complementation assay that identified reticulon 1A (RTN1A), an ER-shaping protein localized to EMCs as an EMC promoter. Proximity mapping augmented with biochemical fractionation and additional validation methods reported here could be useful to discover other components of EMCs, identify mitochondrial contacts with other organelles, and further unravel their communication.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

Cardiovascular calcification (CVC) is a progressive complication of chronic kidney disease and a predictor of CV events and mortality. The use of biomarkers to predict CV risk and activities of potential or current treatment drugs in these patients could have a crucial impact on therapeutic approaches. Our aim was to develop a novel assay for measurement of the rate of calcium phosphate crystallization in human plasma and provide a tool to evaluate the effects of crystallization inhibitors. The efficacy of inhibitors was determined by adding inhibitory compounds (polyphosphates, fetuin-A, sodium thiosulfate or citrate) to control samples. The assay was additionally validated for SNF472, an experimental formulation of phytate being developed for the treatment of calciphylaxis and CVC in patients with end-stage renal disease (ESRD) undergoing hemodialysis (HD). The method was repeatable and reproducible. The plasma crystallization rate was reduced up to 80% in a concentration-dependent manner following treatment with inhibitors in vitro, among which SNF472 was the most potent. This method appears beneficial in evaluating and discriminating between inhibitory activities of compounds such as polyphosphates on calcium phosphate crystallization, which present a novel therapeutic approach to treat CVC in ESRD patients.

Autoimmune type 1 diabetes (T1D) is thought to be caused by a defective immune regulation with regulatory T (Treg) cells playing a fundamental role in this process. Tolerance mechanisms depend on tunable responses that are sensitive to minor perturbations in the expression of molecules that can be carried out by multiple epigenetic mechanisms, including regulation by microRNAs. In this study, microRNA expression profile was investigated in Treg cells isolated from peripheral blood (PB) and from pancreatic draining lymph nodes (PLN) of T1D patients and non-diabetic subjects. Among 72 microRNAs analyzed, miR-125a-5p resulted specifically hyper-expressed in Treg cells purified from PLN of T1D patients. TNFR2 and CCR2 were identified as miR-125a-5p target genes. Elevated miR-125a-5p was detected in Treg cells isolated from PLN but not from PB of donors with T1D and was associated with reduced CCR2 expression. A specific beta-cell expression of the CCR2-ligand (CCL2) was observed in the pancreata of cadaveric donors, suggesting that beta-cells are prone to attract CCR2⁺ Treg cells. These novel data propose a mechanism, occurring in PLNs of T1D patients, involving increased expression of miR-125a-5p on Treg cells which results into reduced expression of CCR2, thus limiting their migration and eventual function in the pancreas.

Fusion gene derived from genomic rearrangement plays a key role in cancer initiation. The discovery of novel gene fusions may be of significant importance in cancer diagnosis and treatment. Meanwhile, next generation sequencing technology provide a sensitive and efficient way to identify gene fusions in genomic levels. However, there are still many challenges and limitations remaining in the existing methods which only rely on unmapped reads or discordant alignment fragments. In this work we have developed GFusion, a novel method using RNA-Seq data, to identify the fusion genes. This pipeline performs multiple alignments and strict filtering algorithm to improve sensitivity and reduce the false positive rate. GFusion successfully detected 34 from 43 previously reported fusions in four cancer datasets. We also demonstrated the effectiveness of GFusion using 24 million 76 bp paired-end reads simulation data which contains 42 artificial fusion genes, among which GFusion successfully discovered 37 fusion genes. Compared with existing methods, GFusion presented higher sensitivity and lower false positive rate. The GFusion pipeline can be accessed freely for non-commercial purposes at: https://github.com/xiaofengsong/GFusion .

There is evidence supporting an association between the serotonin-transporter-linked polymorphic region (5-HTTLPR) and the Five Factor Model (FFM) of human personality. 5-HTTLPR has also been found to interact with stressful life events to increase risk of psychopathology. In the present study, by taking into account stressful life events in the form of childhood adversity, we examined the association between 5-HTTLPR and FFM traits using an adult Swedish cohort (N = 3112). We found that 5-HTTLPR was significantly associated with openness (to experience). Specifically, homozygote carriers of the short allele had lower levels of openness compared to carriers of the long allele. In addition, childhood adversity was found to influence openness. These findings support a previously reported association of 5-HTTLPR with openness in a younger cohort and may provide insights into the neurobiological basis of human personality.

<h4>Background</h4>Some studies showed a relation between chondrocalcinosis and osteoarthritis (OA). Hence, considering the importance of chondrocalcinosis diagnosis andnecessity for its integration with OA, the current study aims at investigating prevalence of chondrocalcinosis in patients above 50 years admitted to Isfahan Al-Zahra Medical Center and its relationship with OA.<h4>Materials and methods</h4>In a cross-sectional study, 600 patients who referred to the radiology units of Al-Zahra Hospital for radiography of different joints were studied during 2013-2014. The patients images were studied for chondrocalcinosis and OA by a radiologist and also examined clinically and results of imaging by an expert rheumatologist. The prevalence of chondrocalcinosis and it relation with OA was determined by Statistical Package for Social Sciences software and using of Chi-square and <i>t</i>-test.<h4>Results</h4>23 patients under study had chondrocalcinosis (3.83%). patients with chondrocalcinosis had higher age average, and they were in age group of 70 years and older, but no significant difference was observed regarding the prevalence of the disease in both genders. Chondrocalcinosis prevalence in terms of body mass index showed significant differences (<i>P</i> = 0.001). All patients with chondrocalcinosis had a history of joint disease and prevalence of chondrocalcinosis in terms of joint disease history showed a significant difference (<i>P</i> < 0.001).<h4>Conclusion</h4>Prevalence of chondrocalcinosis is relatively high in the Iranian population of 70 years and older. Hence, more investigation considering to the diagnosis of chondrocalcinosis among patients with OA is very important.

Stable isotope analysis has been used extensively to establish trophic relationships in many ecosystems. Present study utilised stable isotope signatures of carbon and nitrogen to identify trophic structure of aquatic food web in river and rice field ecosystems in Perak, northern peninsular Malaysia. The mean δ¹³C values of all producers ranged from -35.29 ± 0.21 to -26.00 ± 0.050‰. The greatest δ¹⁵N values noted was in zenarchopterid fish with 9.68 ± 0.020‰. The δ¹⁵N values of aquatic insects ranged between 2.59 ± 0.107 in Elmidae (Coleoptera) and 8.11 ± 0.022‰ in Nepidae (Hemiptera). Correspondingly, with all the δ¹³C and δ¹⁵N values recorded, it can be deduced that there are four trophic levels existed in the freshwater ecosystems which started with the producer (plants), followed by primary consumer (aquatic insects and non-predatory fish), secondary consumer (invertebrate predators) and lastly tertiary consumer (vertebrate predators).

<h4>Background</h4>Atopic dermatitis (AD) is a common skin disorder with elevated prevalence. Cataract induced by AD rarely occurs in adolescent and young adult patients, which is also called atopic cataract. Using whole exome sequencing, we aimed to explore genetic alterations among AD and atopic cataract.<h4>Result</h4>We recruited a 19 year-old Chinese male with AD accompanied with cataracts, his father with AD and his mother without AD or cataract. Through analysis of the exomic sequence of the 3 individuals from the same family, we identified that with respect to AD, there were 162 genes mutated in both this patient and his father but not in his mother. In addition, we found 10 genes mutated in this patient only without in his parents according to cataract.<h4>Conclusion</h4>This research suggests that coinheritance of mutations in these genes may correlate with AD, and the pathogenesis of AD complicated with cataracts was related to genetic factors.

<h4>Objectives</h4>Understanding how loci identified by genome wide association studies (GWAS) contribute to pathogenesis requires new mechanistic insights. Variants within CDKAL1 are strongly linked to an increased risk of developing type 2 diabetes and obesity. Investigations in mouse models have focused on the function of Cdkal1 as a tRNA^Lys modifier and downstream effects of Cdkal1 loss on pro-insulin translational fidelity in pancreatic β-cells. However, Cdkal1 is broadly expressed in other metabolically relevant tissues, including adipose tissue. In addition, the Cdkal1 homolog Cdk5rap1 regulates mitochondrial protein translation and mitochondrial function in skeletal muscle. We tested whether adipocyte-specific Cdkal1 deletion alters systemic glucose homeostasis or adipose mitochondrial function independently of its effects on pro-insulin translation and insulin secretion.<h4>Methods</h4>We measured mRNA levels of type 2 diabetes GWAS genes, including Cdkal1, in adipose tissue from lean and obese mice. We then established a mouse model with adipocyte-specific Cdkal1 deletion. We examined the effects of adipose Cdkal1 deletion using indirect calorimetry on mice during a cold temperature challenge, as well as by measuring cellular and mitochondrial respiration in vitro. We also examined brown adipose tissue (BAT) mitochondrial morphology by electron microscopy. Utilizing co-immunoprecipitation followed by mass spectrometry, we performed interaction mapping to identify new CDKAL1 binding partners. Furthermore, we tested whether Cdkal1 loss in adipose tissue affects total protein levels or accurate Lys incorporation by tRNA^Lys using quantitative mass spectrometry.<h4>Results</h4>We found that Cdkal1 mRNA levels are reduced in adipose tissue of obese mice. Using adipose-specific Cdkal1 KO mice (A-KO), we demonstrated that mitochondrial function is impaired in primary differentiated brown adipocytes and in isolated mitochondria from A-KO brown adipose tissue. A-KO mice displayed decreased energy expenditure during 4 °C cold challenge. Furthermore, mitochondrial morphology was highly abnormal in A-KO BAT. Surprisingly, we found that lysine codon representation was unchanged in Cdkal1 A-KO adipose tissue. We identified novel protein interactors of CDKAL1, including SLC25A4/ANT1, an inner mitochondrial membrane ADP/ATP translocator. ANT proteins can account for the UCP1-independent basal proton leak in BAT mitochondria. Cdkal1 A-KO mice had increased ANT1 protein levels in their white adipose tissue.<h4>Conclusions</h4>Cdkal1 is necessary for normal mitochondrial morphology and function in adipose tissue. These results suggest that the type 2 diabetes susceptibility gene CDKAL1 has novel functions in regulating mitochondrial activity.

<h4>Background</h4>Reverse phase protein array (RPPA) analysis, allows investigation of potential targets at the functional protein level,. We identified TNBC subtypes at the protein level using RPPA and compared them with mRNA molecular subtypes (TNBCtype, TNBCtype-4, and PAM50) that is unique in its availability of both RPPA and mRNA analyses.<h4>Methods</h4>We classified the samples from 80 TNBC patients using both k-means and hierarchical consensus clustering analysis and performed Ingenuity Pathway Analysis. We also investigated whether we could reproduce the mRNA molecular subtypes using the RPPA dataset.<h4>Results</h4>Both clustering methods divided all samples into 2 clusters that were biologically the same. The top canonical pathways included inflammation, hormonal receptors, and MAPK signaling pathways for the first cluster ["inflammation and hormonal-related (I/H) subtype"] and the GADD45, DNA damage, and p53 signaling pathways for the second cluster ["DNA damage (DD)-related subtype"]. Further k-means cluster analysis identified 5 TNBC clusters. Comparison between sample classification using the 5 RPPA-based k-means cluster subtypes and 6 gene-expression-based TNBCtype molecular subtypes showed significant association between the 2 classifications (p = 0.017).<h4>Conclusions</h4>The I/H and DD subtypes identified by RPPA advance our understanding of TNBC's heterogeneity from the functional proteomic perspective.

In adult rats, the administration of lithium-pilocarpine (LiPilo) reproduces most clinical and neuropathological features of human temporal lobe epilepsy (TLE). Carisbamate (CRS) possesses the property of modifying epileptogenesis in this model. Indeed, about 50% of rats subjected to LiPilo status epilepticus (SE) develop non-convulsive seizures (NCS) instead of motor seizures when treated with CRS. However, the mechanisms underlying these effects remain unknown. The aim of this study was to perform a proteomic analysis in the hippocampus of rats receiving LiPilo and developing motor seizures or NCS following CRS treatment. Fifteen adult male Sprague-Dawley rats were used. SE was induced by LiPilo injection. CRS treatment was initiated at 1 h and 9 h after SE onset and maintained for 7 days, twice daily. Four groups were studied after video-EEG control of the occurrence of motor seizures: a control group receiving saline (CT <i>n</i> = 3) and three groups that underwent SE: rats treated with diazepam (DZP <i>n</i> = 4), rats treated with CRS displaying NCS (CRS-NCS <i>n</i> = 4) or motor seizures (CRS-TLE <i>n</i> = 4). Proteomic analysis was conducted by 2D-SDS-PAGE. Twenty-four proteins were found altered. In the CRS-NCS group, proteins related to glycolysis and ATP synthesis were down-regulated while proteins associated with pyruvate catabolism were up-regulated. Moreover, among the other proteins differentially expressed, we found proteins related to inflammatory processes, protein folding, tissue regeneration, response to oxidative stress, gene expression, biogenesis of synaptic vesicles, signal transduction, axonal transport, microtubule formation, cell survival, and neuronal plasticity. Our results suggest a global reduction of glycolysis and cellular energy production that might affect brain excitability. In addition, CRS seems to modulate proteins related to many other pathways that could significantly participate in the epileptogenesis-modifying effect observed.

Tibolone is a synthetic steroid with estrogenic, androgenic and progestogenic activity, but the evidence regarding its effects on fibrinogen and antithrombin III (ATIII) has not been conclusive. We assessed the impact of tibolone on fibrinogen and ATIII through a systematic review and meta-analysis of available randomized controlled trials (RCTs). The search included PUBMED, Web of Science, Scopus, and Google Scholar (up to January 31st, 2016) to identify controlled clinical studies investigating the effects of oral tibolone treatment on fibrinogen and ATIII. Overall, the impact of tibolone on plasma fibrinogen concentrations was reported in 10 trials comprising 11 treatment arms. Meta-analysis did not suggest a significant reduction of fibrinogen levels following treatment with tibolone (WMD: -5.38%, 95% CI: -11.92, +1.16, p=0.107). This result was robust in the sensitivity analysis and not influenced after omitting each of the included studies from meta-analysis. When the studies were categorized according to the duration of treatment, there was no effect in the subsets of trials lasting either <12months (WMD: -7.64%, 95% CI: -16.58, +1.29, p=0.094) or ≥12months (WMD: -0.62%, 95% CI: -8.40, +7.17, p=0.876). With regard to ATIII, there was no change following treatment with tibolone (WMD: +0.74%, 95% CI: -1.44, +2.93, p=0.505) and this effect was robust in sensitivity analysis. There was no differential effect of tibolone on plasma ATIII concentrations in trials with either <12months (WMD: +2.26%, 95% CI: -3.14, +7.66, p=0.411) or≥12months (WMD: +0.06%, 95% CI: -1.16, +1.28, p=0.926) duration. Consistent with the results of subgroup analysis, meta-regression did not suggest any significant association between the changes in plasma concentrations of fibrinogen (slope: +0.40; 95% CI: -0.39, +1.19; p=0.317) and ATIII (slope: -0.17; 95% CI: -0.54, +0.20; p=0.374) with duration of treatment. In conclusion, meta-analysis did not suggest a significant reduction of fibrinogen and ATIII levels following treatment with tibolone.

The differentiation status of tumors is used as a prognostic indicator, with tumors comprised of less differentiated cells exhibiting higher levels of aggressiveness that correlate with a poor prognosis. Although oncogenes contribute to blocking differentiation, it is not clear how they globally alter miRNA expression during differentiation to achieve this result. The pediatric sarcoma Alveolar Rhabdomyosarcoma, which is primarily characterized by the expression of the PAX3-FOXO1 oncogenic fusion protein, consists of undifferentiated muscle cells. However, it is unclear what role PAX3-FOXO1 plays in promoting the undifferentiated state. We demonstrate that expression of PAX3-FOXO1 globally alters the expression of over 80 individual miRNA during early myogenic differentiation, resulting in three primary effects: 1) inhibition of the expression of 51 miRNA essential for promoting myogenesis, 2) promoting the aberrant expression of 43 miRNA not normally expressed during myogenesis, and 3) altering the expression pattern of 39 additional miRNA. Combined, these changes are predicted to have an overall negative effect on myogenic differentiation. This is one of the first studies describing how an oncogene globally alters miRNA expression to block differentiation and has clinical implications for the development of much needed multi-faceted tumor-specific therapeutic regimens.

No abstract available.

BACKGROUND Rituximab is commonly used to reduce the agglutinin titer in ABO-incompatible liver transplant recipients. Although well-tolerated, rituximab infusion therapy may result in severe pulmonary adverse effects such as drug-induced pneumonitis, leading to acute respiratory distress syndrome (ARDS), which has a high mortality rate. Management of such rare cases in an ABO-incompatible patient has never been described before. Herein, we present successful use of extracorporeal membrane oxygenation (ECMO) support for rituximab-induced ARDS in an ABO-incompatible living donor liver transplantation (LDLT) recipient. CASE REPORT A 57-year-old man patient presented with acute-on-chronic hepatic failure. Due to worsening clinical condition and unavailability of a deceased donor organ, ABO-incompatible LDLT was considered. The patient received rituximab therapy and plasmapheresis 1 week before the transplantation to reduce the B cell count. However, he suddenly developed acute respiratory distress-like symptoms, with a chest X-ray suggesting organized pneumonia. Infectious etiology was excluded as evidenced from negative sputum and blood culture, which were repeated after 48 h. LDLT was performed and ECMO support was instituted in the immediate postoperative period due to worsening of the ARDS. The pulmonary signs improved, with a chest X-ray showing clear lung fields on the 5th postoperative day. The patient recovered well and was discharged with normal liver functions in the 4th postoperative month. CONCLUSIONS This is first reported experience of successful use of ECMO in an ABO-incompatible liver transplant recipient with rituximab-induced ARDS. This experience shows the feasibility and effectiveness of ECMO support in liver transplant recipients with poor respiratory functions.

<h4>Objectives</h4>The nature of land use and mobility during the transition to agriculture has often been debated. Here, we use isotope analysis of tooth enamel from human populations buried in two different Neolithic burial monuments, Penywyrlod and Ty Isaf, in south-east Wales, to examine patterns of land use and to evaluate where individuals obtained their childhood diet.<h4>Materials and methods</h4>We employ strontium (⁸⁷ Sr/⁸⁶ Sr) and oxygen (δ¹⁸ O) and carbon (δ¹³ C) isotope analysis of enamel from adjacent molars. We compare strontium isotope values measured in enamel to locally bioavailable ⁸⁷ Sr/⁸⁶ Sr values. We combine discussion of these results with evaluation of new radiocarbon dates obtained from both sites.<h4>Results</h4>The majority of enamel samples from Penywyrlod have strontium isotope ratios above 0.7140. In contrast, the majority of those from Ty Isaf have ⁸⁷ Sr/⁸⁶ Sr values below 0.7140. At Penywyrlod oxygen isotope ratios range between 25.9 and 28.2 ‰ (mean 26.7 ± 0.6 ‰, 1σ, n = 15) and enamel δ¹³ C_carbonate values range between -18.0 and -15.0 ‰ (mean -16.0 ± 0.8 ‰, 1σ, n = 15). At Ty Isaf oxygen isotope ratios exhibited by Neolithic individuals range between 25.4 and 27.7 ‰ (mean 26.7 ± 0.6 ‰, 1σ, n = 15) and enamel δ¹³ C_carbonate values range between -16.9 and -14.9 ‰ (mean -16.0 ± 0.6 ‰, 1σ, n = 15).<h4>Discussion</h4>The strontium isotope results suggest that the majority of individuals buried at Penywyrlod did not source their childhood diet locally. One individual in this group has strontium isotope ratios that exceed all current known biosphere values within England and Wales. This individual is radiocarbon dated to the first few centuries of the 4^th millennium BC, consistent with the period in which agriculture was initiated in Wales: the results therefore provide evidence for migration during the transition to farming in Wales. In contrast, all individuals sampled from Ty Isaf post-date the period in which agriculture is considered to have been initiated and could have sourced their childhood diet from the local region in which they were buried.

<h4>Background</h4>Normal serum alanine aminotransferase (ALT) levels differ with age, gender, and body mass index. Adjusting the upper limits of normal (ULN) for ALT needs further research in different populations. Aim of this work was to monitor the effect of successful chronic hepatitis C (CHC) treatment on the ALT levels in patients with normal pretreatment ALT.<h4>Methods</h4>Data of 1160 CHC patients with persistent pretreatment normal liver enzymes were retrospectively analyzed. Treatment response to direct acting antiviral agents (DAAs) therapy was recorded. Changes in ALT levels before and after treatment were analyzed by patients' demographic, laboratory, and radiologic characteristics. Areas under the receiver operating characteristic curve (AUROC) of ALT after treatment were used to generate a new ALT ULN.<h4>Results</h4>Males were 568 (49%) and females 592 (51%) with a mean age of 50.7 years. After treatment, mean (±SD) of ALT levels significantly decreased from (26.3±7.8) to (19.1±10.9). This reduction was more significant in interferon-free than interferon-based regimens. ROC curve analyses suggested a new ALT ULN cut off (26.4 IU/L) in the treated patients (sensitivity=78.6%, specificity=83.8%, AUROC=0.89. This cutoff dropped to 14.7 IU/L in cirrhotic patients (sensitivity=77.4%, specificity=44.7%, AUROC=0.612). The identified cutoffs were 16.3 IU/L (sensitivity=66.7%, specificity=47.5%, AUROC=0.499) and 15.5 IU/L (sensitivity=76.5%, specificity=51.3%, AUROC=0.576) in males and females, respectively.<h4>Conclusion</h4>The current ALT ULN needs readjustment to identify new normal cutoffs in CHC patients. Posttreatment cutoffs differ according to gender, pretreatment liver affection, and treatment regimen.

A growing body of circumstantial evidence suggests that ice nucleation active (Ice⁺) bacteria contribute to the initiation of precipitation by heterologous freezing of super-cooled water in clouds. However, little is known about the concentration of Ice⁺ bacteria in precipitation, their genetic and phenotypic diversity, and their relationship to air mass trajectories and precipitation chemistry. In this study, 23 precipitation events were collected over 15 months in Virginia, USA. Air mass trajectories and water chemistry were determined and 33 134 isolates were screened for ice nucleation activity (INA) at -8 °C. Of 1144 isolates that tested positive during initial screening, 593 had confirmed INA at -8 °C in repeated tests. Concentrations of Ice⁺ strains in precipitation were found to range from 0 to 13 219 colony forming units per liter, with a mean of 384±147. Most Ice⁺ bacteria were identified as members of known and unknown Ice⁺ species in the Pseudomonadaceae, Enterobacteriaceae and Xanthomonadaceae families, which nucleate ice employing the well-characterized membrane-bound INA protein. Two Ice⁺ strains, however, were identified as Lysinibacillus, a Gram-positive genus not previously known to include Ice⁺ bacteria. INA of the Lysinibacillus strains is due to a nanometer-sized molecule that is heat resistant, lysozyme and proteinase resistant, and secreted. Ice⁺ bacteria and the INA mechanisms they employ are thus more diverse than expected. We discuss to what extent the concentration of culturable Ice⁺ bacteria in precipitation and the identification of a new heat-resistant biological INA mechanism support a role for Ice⁺ bacteria in the initiation of precipitation.

<h4>Background</h4>Neurodevelopment is a complex process involving both genetic and environmental factors. Prenatal exposure to lead (Pb) has been associated with lower performance on neurodevelopmental tests. Adverse neurodevelopmental outcomes are more frequent and/or more severe when toxic exposures interact with genetic susceptibility.<h4>Methods</h4>To explore possible loci associated with increased susceptibility to prenatal Pb exposure, we performed a genome-wide gene-environment interaction study (GWIS) in young children from Mexico (n = 390) and Bangladesh (n = 497). Prenatal Pb exposure was estimated by cord blood Pb concentration. Neurodevelopment was assessed using the Bayley Scales of Infant Development.<h4>Results</h4>We identified a locus on chromosome 8, containing UNC5D, and demonstrated evidence of its genome-wide significance with mental composite scores (rs9642758, p _meta = 4.35 × 10^-6). Within this locus, the joint effects of two independent single nucleotide polymorphisms (SNPs, rs9642758 and rs10503970) had a p-value of 4.38 × 10^-9 for mental composite scores. Correlating GWIS results with in vitro transcriptomic profiles identified one common gene, SLC1A5, which is involved in synaptic function, neuronal development, and excitotoxicity. Further analysis revealed interconnected interactions that formed a large network of 52 genes enriched with oxidative stress genes and neurodevelopmental genes.<h4>Conclusions</h4>Our findings suggest that certain genetic polymorphisms within/near genes relevant to neurodevelopment might modify the toxic effects of Pb exposure via oxidative stress.

Trophoblast stem cells (TSCs) give rise to specialized cell types within the placenta. However, the regulatory mechanisms that guide trophoblast cell fate decisions during placenta development remain ill defined. Here we exploited ATAC-seq and transcriptional profiling strategies to describe dynamic changes in gene expression and chromatin accessibility during TSC differentiation. We detect significantly increased chromatin accessibility at key genes upregulated as TSCs exit from the stem cell state. However, downregulated gene expression is not simply due to the loss of chromatin accessibility in proximal regions. Additionally, transcriptional targets recognized by the zinc finger transcriptional repressor Prdm1/Blimp1, an essential regulator of placenta development, were identified in ChIP-seq experiments. Comparisons with previously reported ChIP-seq datasets for primordial germ cell-like cells and E18.5 small intestine, combined with functional annotation analysis revealed that Blimp1 has broadly shared as well as cell type-specific functional activities unique to the trophoblast lineage. Importantly, Blimp1 not only silences TSC gene expression but also prevents aberrant activation of divergent developmental programmes. Overall the present study provides new insights into the chromatin landscape and Blimp1-dependent regulatory networks governing trophoblast gene expression.

Heterogeneity of planarian stem cells has been categorised on the basis of single cell expression analyses and subsequent experiments to demonstrate lineage relationships. Some data suggest that despite heterogeneity in gene expression amongst cells in the cell cycle, in fact only one sub-population, known as sigma neoblasts, can self-renew. Without the tools to perform live in vivo lineage analysis, we instead took an alternative approach to provide independent evidence for defining the self-renewing stem cell population. We exploited the role of highly conserved condensin family genes to functionally assay neoblast self-renewal properties. Condensins are involved in forming properly condensed chromosomes to allow cell division to proceed during mitosis, and their abrogation inhibits mitosis and can lead to repeated endoreplication of the genome in cells that make repeated attempts to divide. We find that planarians possess only the condensin I complex, and that this is required for normal stem cell function. Abrogation of condensin function led to rapid stem cell depletion accompanied by the appearance of 'giant' cells with increased DNA content. Using previously discovered markers of heterogeneity we show that enlarged cells are always from the sigma-class of the neoblast population and we never observe evidence for endoreplication for the other neoblast subclasses. Overall, our data establish that condensins are essential for stem cell maintenance and provide independent evidence that only sigma-neoblasts are capable of multiple rounds of cell division and hence self-renewal.

The provision of essential and non-essential amino acids for breast-fed infants is the major function of milk proteins. In addition, breast-fed infants might benefit from bioactivities of milk proteins, which are exhibited in the intestine during the digestive phase and by absorption of intact proteins or derived peptides. For lactoferrin, osteopontin and milk fat globule membrane proteins/lipids, which have not until recently been included in substantial amounts in infant formulas, in vitro experiments and animal models provide a convincing base of evidence for bioactivities, which contribute to the protection of the infant from pathogens, improve nutrient absorption, support the development of the immune system and provide components for optimal neurodevelopment. Technologies have become available to obtain these compounds from cow´s milk and the bovine compounds also exhibit bioactivities in humans. Randomized clinical trials with experimental infant formulas incorporating lactoferrin, osteopontin, or milk fat globule membranes have already provided some evidence for clinical benefits. This review aims to compare findings from laboratory and animal experiments with outcomes of clinical studies. There is good justification from basic science and there are promising results from clinical studies for beneficial effects of lactoferrin, osteopontin and the milk fat globule membrane complex of proteins and lipids. Further studies should ideally be adequately powered to investigate effects on clinically relevant endpoints in healthy term infants.

Under physiological conditions, the function of astrocytes in providing brain metabolic support is compromised under pathophysiological conditions caused by human immunodeficiency virus (HIV) and opioids. Herein, we examined the role of autophagy, a lysosomal degradation pathway important for cellular homeostasis and survival, as a potential regulatory mechanism during pathophysiological conditions in primary human astrocytes. Blocking autophagy with small interfering RNA (siRNA) targeting <i>BECN1</i>, but not the Autophagy-related 5 (<i>ATG5</i>) gene, caused a significant decrease in HIV and morphine-induced intracellular calcium release. On the contrary, inducing autophagy pharmacologically with rapamycin further enhanced calcium release and significantly reverted HIV and morphine-decreased glutamate uptake. Furthermore, siBeclin1 caused an increase in HIV-induced nitric oxide (NO) release, while viral-induced NO in astrocytes exposed to rapamycin was decreased. HIV replication was significantly attenuated in astrocytes transfected with siRNA while significantly induced in astrocytes exposed to rapamycin. Silencing with siBeclin1, but not siATG5, caused a significant decrease in HIV and morphine-induced interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α) release, while secretion of IL-8 was significantly induced with rapamycin. Mechanistically, the effects of siBeclin1 in decreasing HIV-induced calcium release, viral replication, and viral-induced cytokine secretion were associated with a decrease in activation of the nuclear factor kappa B (NF-κB) pathway.

The present study aimed to identify the genes associated with the histologic tumor grade of patients with esophageal squamous cell carcinoma (ESCC) and to provide valuable information for the identification of potential diagnostic biomarkers in ESCC. Tumor samples of ESCC patients retrieved from <i>The Cancer Genome Atlas</i> were divided into Grade 1 (well-differentiated; G1), Grade 2 (moderately-differentiated; G2) and Grade 3 (poorly-differentiated; G3) groups in accordance with the clinical record of the tumor grade of ESCC patients. The genes associated with tumor grade were identified. The signaling pathways of identified genes were enriched from the <i>Kyoto Encyclopedia of Genes and Genomes</i> (KEGG). The diagnostic value of candidate genes was assessed by receiver operating characteristic analysis. We used the GSE23400 dataset generated from the Gene Expression Omnibus to examine the expression levels of candidate genes in ESCC tissues compared to matched mucosa tissues. In total, 440 genes positively correlated with tumor grade and 882 genes negatively correlated with tumor grade were identified. There were 310 differentially expressed genes (DEGs) between G1 and G2, 184 DEGs between G2 and G3, and 710 DEGs between G1 and G3. There were 1322 genes associated with tumor grade that were significantly enriched in pathways in cancer and the phospholipase D signaling pathway. Cyclin-dependent kinase inhibitor 1A, golgin A7 family member B and transforming growth factor B1-induced anti-apoptotic factor 1 (<i>TIAF1</i>) had potential diagnostic value for discriminating ESCC patients with G1 from those with G3. <i>TIAF1</i> was significantly down-regulated in ESCC. The results of the present study comprise useful groundwork with respect to determining the tumorigenesis mechanism in ESCC and discovering potential diagnostic biomarkers for ESCC.

<h4>Background and objective</h4>Current surveillance strategies for colorectal cancer following polypectomy are determined by endoscopic and histopathological factors. Such a distinction has been challenged. The present study was designed to identify molecular parameters in colonic polyps potentially defining new sub-groups at risk.<h4>Methods</h4>One hundred patients were enrolled in this multicentre study. Polyps biopsies underwent formalin-free processing (PAXgene, PreAnalytiX) and targeted next generation sequencing (38 genes (QIAGEN), NextSeq 500 platform (Illumina)). Genetic and histopathological analyses were done blinded to other data.<h4>Results</h4>In 100 patients, 224 polyps were removed. Significant associations of genetic alterations with endoscopic or histological polyp characteristics were observed for <i>BRAF</i>, <i>KRAS</i>, <i>TCF7L2</i>, <i>FBXW7</i> and <i>CTNNB1</i> mutations. Multivariate analysis revealed that polyps ≥ 10 mm have a significant higher relative risk for harbouring oncogene mutations (relative risk 3.467 (1.742-6.933)). Adenomas and right-sided polyps are independent risk factors for <i>CTNNB1</i> mutations (relative risk 18.559 (2.371-145.245) and 12.987 (1.637-100.00)).<h4>Conclusions</h4>Assessment of the mutational landscape of polyps can be integrated in the workflow of current colonoscopy practice. There are distinct genetic patterns related to polyp size and location. These results suffice to optimise individual risk calculation and may help to better define surveillance intervals.

Healthy volunteers are crucial for biomedical research. Inadvertent inclusion of subjects with nonalcoholic fatty liver disease (NAFLD) as controls can compromise study validity and subject safety. Given the rising prevalence of NAFLD in the general population, we sought to identify its prevalence and potential impact in volunteers for clinical trials. We conducted a cross-sectional study of subjects who were classified as healthy volunteers between 2011 and 2015 and had no known liver disease. Subjects were classified as presumed NAFLD (pNF; alanine aminotransferase [ALT] level ≥ 20 for women or ≥ 31 for men and body mass index [BMI] > 25 kg/m² ), healthy non-NAFLD controls (normal ALT and BMI), or indeterminate. A total of 3160 subjects participated as healthy volunteers in 149 clinical trials (1-29 trials per subject); 1732 of these subjects (55%) had a BMI > 25 kg/m² and 1382 (44%) had abnormal ALT. pNF was present in 881 subjects (27.9%), and these subjects were older than healthy control subjects and had higher triglycerides, low-density lipoprotein cholesterol, and HbA1c and lower high-density lipoprotein cholesterol (P < 0.001 for all). The 149 trials included 101 non-interventional, 33 interventional, and 15 vaccine trials. The impact on study validity of recruiting NAFLD subjects as controls was estimated as likely, probable, and unlikely in 10, 41, and 98 trials, respectively. The proportion of pNF subjects (28%-29%) did not differ by impact. Only 14% of trials used both BMI and ALT for screening. ALT cutoffs for screening were based on local reference values. Grade 3-4 ALT elevations during the study period were rare but more common in pNF subjects than in healthy control subjects (4 versus 1).<h4>Conclusion</h4>NAFLD is common and often overlooked in volunteers for clinical trials, despite its potential impact on subject safety and validity of study findings. Increased awareness of NAFLD prevalence and stricter ALT cutoffs may ameliorate this problem. (Hepatology 2017;66:825-833).

<h4>Background</h4>Implants or implantable devices should integrate into the host tissue faster than fibrous capsule formation, in which the design of the interface is one of the biggest challenges. Generally, bioactive materials are not viable for load-bearing applications, so inert biomaterials are proposed. However, the surface must be modified through techniques such as coating with bioactive materials, roughness and sized pores. The aim of this research was to validate an approach for the evaluation of the tissue growth on implants of porous alumina coated with bioactive materials.<h4>Methods</h4>Porous alumina implants were coated with 45S5 Bioglass® (BG) and hydroxyapatite (HA) and implanted in rat tibiae for a period of 28 days. Ex vivo resections were performed to analyze osseointegration, along with histological analysis, Scanning Electron Microscopy with Energy Dispersive X-Ray spectroscopy (SEM-EDX) line scanning, radiography and biomechanical testing.<h4>Results</h4>Given that the process of implant integration needs with the bone tissue to be accelerated, it was then seen that BG acted to start the rapid integration, and HA acted to sustaining the process.<h4>Conclusions</h4>Inert materials coated with bioglass and HA present a potential for application as bone substitutes, preferably with pores of diameters between 100 μm and 400 μm and, restrict for smaller than 100 μm, because it prevents pores without organized tissue formation or vacant. Designed as functional gradient material, stand out for applications in bone tissue under load, where, being the porous surface responsible for the osseointegration and the inner material to bear and to transmit the loads.

MicroRNAs (miRNAs) regulate critical cell processes, such as apoptosis, proliferation, and development. However, the role of miRNAs in embryonic stem cell (ESC) neural differentiation induced by retinoic acid (RA) and factors that govern neural directional differentiation remain poorly understood. In this study, we demonstrated that miR-219 is sufficient in promoting mouse ESCs to undergo neural differentiation. We discovered that Foxj3 and Zbtb18, two target genes of miR-219, are not able to determine the process of RA-induced differentiation, however they prevent ESCs from differentiating into neural cells. We identified four downstream genes, namely, Olig1, Zic5, Erbb2, and Olig2, which are essential to the gene interaction networks for neural differentiation. These data explain the mechanism of RA-induced neural differentiation of mESCs on the basis of miRNAs and support the crucial role of miR-219 in neurodevelopment.

Glycoengineered therapeutic antibodies and glycosite-specific antibody-drug conjugates (gsADCs) have generated great interest among researchers because of their therapeutic potential. Endoglycosidase-catalyzed in vitro glycoengineering technology is a powerful tool for IgG Fc (fragment cystallizable) N-glycosylation remodeling. In this protocol, native heterogeneously glycosylated IgG N-glycans are first deglycosylated with a wild-type endoglycosidase. Next, a homogeneous N-glycan substrate, presynthesized as described here, is attached to the remaining N-acetylglucosamine (GlcNAc) of IgG, using a mutant endoglycosidase (also called endoglycosynthase) that lacks hydrolytic activity but possesses transglycosylation activity for glycoengineering. Compared with in vivo glycoengineering technologies and the glycosyltransferase-enabled in vitro engineering method, the current approach is robust and features quantitative yield, homogeneous glycoforms of produced antibodies and ADCs, compatibility with diverse natural and non-natural glycan structures, convenient exploitation of native IgG as the starting material, and a well-defined conjugation site for antibody modifications. Potential applications of this method cover a broad scope of antibody-related research, including the development of novel glycoengineered therapeutic antibodies with enhanced efficacy, site-specific antibody-drug conjugation, and site-specific modification of antibodies for fluorescent labeling, PEGylation, protein cross-linking, immunoliposome formation, and so on, without loss of antigen-binding affinity. It takes 5-8 d to prepare the natural or modified N-glycan substrates, 3-4 d to engineer the IgG N-glycosylation, and 2-5 d to synthesize the small-molecule toxins and prepare the gsADCs.

We present the synthesis and biological evaluation of the prototype of a new class of cephalosporins, containing an additional isolated beta lactam ring with two phenyl substituents. This new compound is effective against Gram positive microorganisms, with a potency similar to that of ceftriaxone, a cephalosporin widely used in clinics and taken as a reference, and with no cytotoxicity against two different human cell lines, even at a concentration much higher than the minimal inhibitory concentration tested. Additionally, a deep computational analysis has been conducted with the aim of understanding the contribution of its moieties to the binding energy towards several penicillin-binding proteins from both Gram positive and Gram negative bacteria. All these results will help us developing derivatives of this compound with improved chemical and biological properties, such as a broader spectrum of action and/or an increased affinity towards their molecular targets.

<h4>Objective</h4>We performed a comprehensive review and meta-analysis to evaluate the diagnostic values of serum single and multiplex tumor-associated autoantibodies (TAAbs) in patients with lung cancer (LC).<h4>Methods</h4>We searched the MEDLINE and EMBASE databases for relevant studies investigating serum TAAbs for the diagnosis of LC. The primary outcomes included sensitivity, specificity and accuracy of the test.<h4>Results</h4>The systematic review and meta-analysis included 31 articles with single autoantibody and 39 with multiplex autoantibodies. Enzyme-linked immunosorbent assay (ELISA) was the most common detection method. For the diagnosis of patients with all stages and early-stage LC, different single or combinations of TAAbs demonstrated different diagnostic values. Although individual TAAbs showed low diagnostic sensitivity, the combination of multiplex autoantibodies offered relatively high sensitivity. For the meta-analysis of a same panel of autoantibodies in patients at all stages of LC, the pooled results of the panel of 6 TAAbs (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2) were: sensitivity 38% (95% CI 0.35-0.40), specificity 89% (95% CI 0.86-0.91), diagnostic accuracy 65.9% (range 62.5-81.8%), AUC 0.52 (0.48-0.57), while the summary estimates of 7 TAAbs (p53, CAGE, NY-ESO-1, GBU4-5, SOX2, MAGE A4 and Hu-D) were: sensitivity 47% (95% CI 0.34-0.60), specificity 90% (95% CI 0.89-0.92), diagnostic accuracy 78.4% (range 67.5-88.8%), AUC 0.90 (0.87-0.93). For the meta-analysis of the same panel of autoantibodies in patients at early-stage of LC, the sensitivities of both panels of 7 TAAbs and 6 TAAbs were 40% and 29.7%, while their specificities were 91% and 87%, respectively.<h4>Conclusions</h4>Serum single or combinations of multiplex autoantibodies can be used as a tool for the diagnosis of LC patients at all stages or early-stage, but the combination of multiplex autoantibodies shows a higher detection capacity; the diagnostic value of the panel of 7 TAAbs is higher than the panel of 6 TAAbs, which may be used as potential biomarkers for the early detection of LC.

<h4>Background</h4>Human follicular fluid (HFF) provides a key environment for follicle development and oocyte maturation, and contributes to oocyte quality and in vitro fertilization (IVF) outcome.<h4>Methods</h4>To better understand folliculogenesis in the ovary, a proteomic strategy based on dual reverse phase high performance liquid chromatography (RP-HPLC) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (LC-MALDI TOF/TOF MS) was used to investigate the protein profile of HFF from women undergoing successful IVF.<h4>Results</h4>A total of 219 unique high-confidence (False Discovery Rate (FDR) < 0.01) HFF proteins were identified by searching the reviewed Swiss-Prot human database (20,183 sequences), and MS data were further verified by western blot. PANTHER showed HFF proteins were involved in complement and coagulation cascade, growth factor and hormone, immunity, and transportation, KEGG indicated their pathway, and STRING demonstrated their interaction networks. In comparison, 32% and 50% of proteins have not been reported in previous human follicular fluid and plasma.<h4>Conclusions</h4>Our HFF proteome research provided a new complementary high-confidence dataset of folliculogenesis and oocyte maturation environment. Those proteins associated with innate immunity, complement cascade, blood coagulation, and angiogenesis might serve as the biomarkers of female infertility and IVF outcome, and their pathways facilitated a complete exhibition of reproductive process.

<b>Purpose:</b><i>FGFR1</i> amplification occurs in approximately 15% of estrogen receptor-positive (ER⁺) human breast cancers. We investigated mechanisms by which <i>FGFR1</i> amplification confers antiestrogen resistance to ER⁺ breast cancer.<b>Experimental Design:</b> ER⁺ tumors from patients treated with letrozole before surgery were subjected to Ki67 IHC, FGFR1 FISH, and RNA sequencing (RNA-seq). ER⁺/<i>FGFR1</i>-amplified breast cancer cells, and patient-derived xenografts (PDX) were treated with FGFR1 siRNA or the FGFR tyrosine kinase inhibitor lucitanib. Endpoints were cell/xenograft growth, FGFR1/ERα association by coimmunoprecipitation and proximity ligation, ER genomic activity by ChIP sequencing, and gene expression by RT-PCR.<b>Results:</b> ER⁺/<i>FGFR1</i>-amplified tumors in patients treated with letrozole maintained cell proliferation (Ki67). Estrogen deprivation increased total and nuclear FGFR1 and FGF ligands expression in ER⁺/<i>FGFR1-</i>amplified primary tumors and breast cancer cells. In estrogen-free conditions, FGFR1 associated with ERα in tumor cell nuclei and regulated the transcription of ER-dependent genes. This association was inhibited by a kinase-dead FGFR1 mutant and by treatment with lucitanib. ChIP-seq analysis of estrogen-deprived ER⁺/<i>FGFR1</i>-amplified cells showed binding of FGFR1 and ERα to DNA. Treatment with fulvestrant and/or lucitanib reduced FGFR1 and ERα binding to DNA. RNA-seq data from <i>FGFR1</i>-amplified patients' tumors treated with letrozole showed enrichment of estrogen response and E2F target genes. Finally, growth of ER⁺/<i>FGFR1-</i>amplified cells and PDXs was more potently inhibited by fulvestrant and lucitanib combined than each drug alone.<b>Conclusion</b>s<b>:</b> These data suggest the ERα pathway remains active in estrogen-deprived ER⁺/<i>FGFR1</i>-amplified breast cancers. Therefore, these tumors are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. <i>Clin Cancer Res; 23(20); 6138-50. ©2017 AACR</i>.

The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.

Mitochondrial protein synthesis is essential for the production of components of the oxidative phosphorylation system. RNA modifications in the mammalian mitochondrial translation apparatus play key roles in facilitating mitochondrial gene expression as they enable decoding of the non-conventional genetic code by a minimal set of tRNAs, and efficient and accurate protein synthesis by the mitoribosome. Intriguingly, recent transcriptome-wide analyses have also revealed modifications in mitochondrial mRNAs, suggesting that the concept of dynamic regulation of gene expression by the modified RNAs (the "epitranscriptome") extends to mitochondria. Furthermore, it has emerged that defects in RNA modification, arising from either mt-DNA mutations or mutations in nuclear-encoded mitochondrial modification enzymes, underlie multiple mitochondrial diseases. Concomitant advances in the identification of the mitochondrial RNA modification machinery and recent structural views of the mitochondrial translation apparatus now allow the molecular basis of such mitochondrial diseases to be understood on a mechanistic level.

DHHC enzymes catalyze palmitoylation, a major post-translational modification that regulates a number of key cellular processes. There are up to 24 DHHCs in mammals and hundreds of substrate proteins that get palmitoylated. However, how DHHC enzymes engage with their substrates is still poorly understood. There is currently no structural information about the interaction between any DHHC enzyme and protein substrates. In this study we have investigated the structural and thermodynamic bases of interaction between the ankyrin repeat domain of human DHHC17 (ANK17) and Snap25b. We solved a high-resolution crystal structure of the complex between ANK17 and a peptide fragment of Snap25b. Through structure-guided mutagenesis, we discovered key residues in DHHC17 that are critically important for interaction with Snap25b. We further extended our finding by showing that the same residues are also crucial for the interaction of DHHC17 with Huntingtin, one of its most physiologically relevant substrates.

Huntington's disease (HD) is a fatal genetic disorder characterized by cell death of medium-sized spiny neurons (MSNs) in the striatum, traditionally attributed to excessive glutamate inputs and/or receptor sensitivity. While changes in corticostriatal projections have typically been studied in mouse models of HD, morphological and functional alterations in thalamostriatal projections have received less attention. In this study, an adeno-associated virus expressing channelrhodopsin-2 under the calcium/calmodulin-dependent protein kinase IIα promoter was injected into the sensorimotor cortex or the thalamic centromedian-parafascicular nuclear complex in the R6/2 mouse model of HD, to permit selective activation of corticostriatal or thalamostriatal projections, respectively. In symptomatic R6/2 mice, peak amplitudes and areas of corticostriatal glutamate AMPA and NMDA receptor-mediated responses were reduced. In contrast, although peak amplitudes of AMPA and NMDA receptor-mediated thalamostriatal responses also were reduced, the areas remained unchanged due to an increase in response decay times. Blockade of glutamate reuptake further increased response areas and slowed rise and decay times of NMDA responses. These effects appeared more pronounced at thalamostriatal synapses of R6/2 mice, suggesting increased activation of extrasynaptic NMDA receptors. In addition, the probability of glutamate release was higher at thalamostriatal than corticostriatal synapses, particularly in R6/2 mice. Morphological studies indicated that the density of all excitatory synaptic contacts onto MSNs was reduced, which matches the basic electrophysiological findings of reduced amplitudes. There was a consistent reduction in the area of spines but little change in presynaptic terminal size, indicating that the postsynaptic spine may be more significantly affected than presynaptic terminals. These results highlight the significant and differential contribution of the thalamostriatal projection to glutamate excitotoxicity in HD.

PurposeAs genome science advances, people receiving personalized genetic information may receive reinterpretations of pathogenicity. Little is known about responses to adjusted results. We examined how reinterpretations might affect attitudes about genetic testing and intentions to share results with family.MethodsData were collected from high-socioeconomic-status participants (n = 58) in a genome sequencing study. Twenty-nine originally learned they were carriers of Duarte variant galactosemia, based on a variant that was reclassified as benign. Positive testers (n = 19) had a newly identified causative variant and remained carriers. Negative testers (n = 10) learned they were no longer carriers. Twenty-nine controls were carriers for a disease of comparable severity with no reclassification. Participants completed baseline, immediate, and 3-month follow-up surveys.ResultsApproximately 80% of participants demonstrated complete or partially accurate recall of their results and reported positive or neutral reactions to their result and about genetic information more generally. Positive testers reported lower intentions to share the change in their result with family. Controls reported the lowest intentions to learn future results. There were no significant group differences or changes over time in perceived ambiguity or negative emotions.ConclusionThe results suggest that high-socioeconomic-status participants understand reinterpretations conferring a neutral change or a change from carrier to noncarrier status. Participants' responses to changes in carrier results for a low-risk condition indicated minimal adverse effects.

Maternal diabetes is known to cause neural tube defects (NTDs) in embryos and neuropsychological deficits in infants. Several metabolic pathways and a plethora of genes have been identified to be deregulated in developing brain of embryos by maternal diabetes, although the exact mechanism remains unknown. Recently, miRNAs have been shown to regulate genes involved in brain development and maturation. Therefore, we hypothesized that maternal diabetes alters the expression of miRNAs that regulate genes involved in biological pathways critical for neural tube development and closure during embryogenesis. To address this, high throughput miRNA expression profiling in neural stem cells (NSCs) isolated from the forebrain of embryos from normal or streptozotocin-induced diabetic pregnancy was carried out. It is known that maternal diabetes results in fetal hypoglycemia/hyperglycemia or hypoxia. Hence, NSCs from embryos of control pregnant mice were exposed to low or high glucose or hypoxia <i>in vitro</i>. miRNA pathway analysis revealed distinct deregulation of several biological pathways, including axon guidance pathway, which are critical for brain development in NSCs exposed to different treatments. Among the differentially expressed miRNAs, the miRNA-30 family members which are predicted to target genes involved in brain development was upregulated in NSCs from embryos of diabetic pregnancy when compared to control. miRNA-30b was found to be upregulated while its target gene Sirtuin 1 (<i>Sirt1</i>), as revealed by luciferase assay, was down regulated in NSCs from embryos of diabetic pregnancy. Further, overexpression of miRNA-30b in NSCs, resulted in decreased expression of Sirt1 protein, and altered the neuron/glia ratio. On the other hand, siRNA mediated knockdown of Sirt1 in NSCs promoted astrogenesis, indicating that miRNA-30b alters lineage specification via Sirt1. Overall, these results suggest that maternal diabetes alters the genes involved in neural tube formation via regulating miRNAs.

A straightforward strategy for the synthesis of dihydroquinazolines is presented, which allows for the preparation of 3,4- and 1,4-dihydroquinazolines with different substitution patterns from 2-aminobenzylamine (2-ABA) as common precursor. The required functionalization of both amino groups present in 2-ABA was achieved by different routes involving selective <i>N-</i>acylation and cesium carbonate-mediated <i>N-</i>alkylation reactions, avoiding protection/deprotection steps. The heterocycles were efficiently synthesized in short reaction times by microwave-assisted ring closure of the corresponding aminoamides promoted by ethyl polyphosphate (PPE).

Accumulating evidence shows the important role of long non-coding RNAs (lncRNAs) in competing endogenous RNA (ceRNA) networks for predicting survival in tumor patients. However, prognostic biomarkers for lung squamous cell carcinoma (LUSC) are still lacking. The objective of this study is to identify a lncRNA signature for evaluation of overall survival (OS) in 474 LUSC patients from The Cancer Genome Atlas (TCGA) database. A total of 474 RNA sequencing profiles in LUSC patients with clinical data were obtained, providing a large sample of RNA sequencing data, and 83 LUSC-specific lncRNAs, 26 miRNAs, and 85 mRNAs were identified to construct the ceRNA network (fold change>2, P<0.05). Among these above 83 LUSC-specific lncRNAs, 22 were assessed as closely related to OS in LUSC patients using a univariate Cox proportional regression model. Meanwhile, two (FMO6P and PRR26) of the above 22 OS-related lncRNAs were identified using a multivariate Cox regression model to construct a risk score as an independent indicator of the prognostic value of the lncRNA signature in LUSC patients. LUSC patients with low-risk scores were more positively correlated with OS (P<0.001). The present study provides a deeper understanding of the lncRNA-related ceRNA network in LUSC and suggests that the two-lncRNA signature could serve as an independent biomarker for prognosis of LUSC.

Pb is a pervasive environmental threat to human health. Although remarkable progress has been made in its neurotoxicity, the precise molecular mechanisms underlying this widespread toxicant still remain elusive. In this study, the detailed roles of EZH2, a transcriptional repressor, in the regulation of Pb-led neurotoxicity were investigated, highlighting its sub-functionalization, compartmentalization, functional chaperones and downstream partners. Based on the findings, EZH2's protein levels were significantly reduced in response to Pb treatment; EZH2's gain-of-function trials recovered the dampened neurite outgrowth; EZH2' recruitment to ploycomb complex, as well as its interaction with cytosolic Vav1, was altered in a distinct manner, suggesting that EZH2's multiple roles were markedly redistributed in this context; EZH2's cytosolic and nuclear presence differed in their respective response towards Pb treatment; EZH2 directly occupied the promoters of <i>EGR2</i>, <i>NGFR</i> and <i>CaMKK2</i>, genes responsible for various nerve functions and repair mechanisms, and essentially contributed to their aberrant expression. It indicated that EZH2 mediated the dynamic changes of a cascade of key molecules and consequently the related neurological impairments. In summary, EZH2 emerges as a central player to regulate Pb-led neurotoxicity in a transcriptionally dependent and independent manner, and thereby provided a promising molecular target for medical intervention.

Damaging inflammation is a hallmark of <i>Mycobacterium tuberculosis</i> infection, and understanding how this is regulated is important for the development of new therapies to limit excessive inflammation. The E3 ubiquitin ligase, Roquin, is involved in immune regulation; however, its role in immunity to <i>M. tuberculosis</i> is unknown. To address this, we infected mice with a point mutation in <i>Roquin1/Rc3h1</i> (<i>sanroque</i>). Aerosol-infected <i>sanroque</i> mice showed enhanced control of <i>M. tuberculosis</i> infection associated with delayed bacterial dissemination and upregulated TNF production in the lungs after 2 wk. However, this early control of infection was not maintained, and by 8 wk postinfection <i>sanroque</i> mice demonstrated an increased bacterial burden and dysregulated inflammation in the lungs. As the inflammation in the lungs of the <i>sanroque</i> mice could have been influenced by emerging autoimmune conditions that are characteristic of the mice aging, the function of Roquin was examined in immune cell subsets in the absence of autoimmune complications. <i>M. bovis</i> bacillus Calmette-Guérin-primed <i>sanroque</i> T cells transferred into <i>Rag1^-/-</i> mice provided equivalent protection in the spleen and liver. Interestingly, the transfer of mycobacteria-specific (P25 CD4⁺ TCR transgenic) wild-type spleen cells into <i>sanroque</i><i>Rag1</i>^-/- mice actually led to enhanced protection with reduced bacterial load, decreased chemokine expression, and reduced inflammation in the lungs compared with transfers into <i>Rag1^-</i>^/- mice expressing intact Roquin. These studies suggest that modulation of Roquin in myeloid cells may reduce both inflammation and bacterial growth during the chronic phase of <i>M. tuberculosis</i> infection.

Small-diameter sensory dysfunction resulting from diabetes has received much attention in the literature, whereas the impact of diabetes on α-motoneurons (MN) has not. In addition, the chance of developing insulin resistance and diabetes is increased in obesity. No study has examined the impact of obesity or diabetes on the biophysical properties of MN. Lean Zucker rats and Zucker diabetic fatty (ZDF) rats were separated into lean, obese (ZDF fed standard chow), and diabetic (ZDF fed high-fat diet that led to diabetes) groups. Glass micropipettes recorded hindlimb MN properties from identified flexor and extensor MN. MN were separated within their groups on the basis of input conductance, which created high- and low-input conductance subpopulations for each. A significant shorter (20%) afterhyperpolarization half-decay (AHP_1/2) was found in low-conductance MN for the diabetic group only, whereas AHP½ tended to be shorter in the obese group (19%). Significant positive correlations were found among rheobase and input conductance for both lean and obese animals. No differences were found between the groups for afterhyperpolarization amplitude (AHP_amp), input conductance, rheobase, or any of the rhythmic firing properties (frequency-current slope and spike-frequency adaptation index). MN properties continue to be heterogeneous in obese and diabetic animals. Obesity does not seem to influence lumbar MN. Despite the resistance of MN to the impact of diabetes, the reduced AHP_1/2 decay and the tendency for a reduction in AHP_amp may be the first sign of change to MN function.<b>NEW & NOTEWORTHY</b> Knowledge about the impact of obesity and diabetes on the biophysical properties of motoneurons is lacking. We found that diabetes reduces the duration of the afterhyperpolarization and that motoneuron function is unchanged by obesity. A reduced afterhyperpolarization may impact discharge characteristics and may be the first sign of change to motoneuron function.

Brain metastases (BMs) are the most common malignancy of the central nervous system. Recently it has been demonstrated that plasminogen activator inhibitor serpins promote brain metastatic colonization, suggesting that mutations in serpins or other members of the coagulation cascade can provide critical advantages during BM formation. We performed whole-exome sequencing on matched samples of breast cancer and BMs and found mutations in the coagulation pathway genes in 5 out of 10 BM samples. We then investigated the mutational status of 33 genes belonging to the coagulation cascade in a panel of 29 BMs and we identified 56 Single Nucleotide Variants (SNVs). The frequency of gene mutations of the pathway was significantly higher in BMs than in primary tumours, and SERPINI1 was the most frequently mutated gene in BMs. These findings provide direction in the development of new strategies for the treatment of BMs.

We have previously shown that OX40L/OX40 interaction is critical for TCR-independent selective proliferation of Foxp3⁺ Tregs, but not Foxp3^- effector T-cells (Teff), when CD4⁺ T-cells are co-cultured with GM-CSF derived bone marrow dendritic cells (G-BMDCs). Events downstream of OX40L/OX40 interaction in Tregs responsible for this novel mechanism are not understood. Earlier, OX40L/OX40 interaction has been shown to stimulate CD4⁺ T-cells through the formation of a signalosome involving TRAF2/PKC-Ѳ leading to NF-kB activation. In this study, using CD4⁺ T-cells from WT and OX40^-/- mice we first established that OX40 mediated activation of NF-kB was critical for this Treg proliferation. Although CD4⁺ T-cells from PKC-Ѳ^-/- mice were also defective in G-BMDC induced Treg proliferation ex vivo, this defect could be readily corrected by adding exogenous IL-2 to the co-cultures. Furthermore, by treating WT, OX40^-/-, and PKC-Ѳ^-/- mice with soluble OX40L we established that OX40L/OX40 interaction was required and sufficient to induce Treg proliferation in vivo independent of PKC-Ѳ status. Although PKC-Ѳ is dispensable for TCR-independent Treg proliferation per se, it is essential for optimum IL-2 production by Teff cells. Finally, our findings suggest that OX40L binding to OX40 likely results in recruitment of TRAF1 for downstream signalling.

The intestinal epithelium has two distinct two stem cell populations, namely, crypt base columnar (CBC) cells and +4 cells. Several specific markers have been identified for each stem cell population. In this study, we examined the expression profiles of these markers in colitis-associated carcinogenesis (CAC) to investigate whether they can be used as biomarkers for the early detection of dysplasia. The expression of intestinal stem cell (ISC) markers was measured by real-time polymerase chain reaction during CAC that was induced by azoxymethane and dextran sodium sulfate treatment. CBC stem cell markers increased continuously with tumor development, whereas a +4 cell expression profile was not present. CBC stem cell population was suppressed in the acute colitis and then expanded to repopulate the crypts during the regeneration period. Notably, RNA in situ hybridization revealed that all dysplasia and cancer samples showed increased expression of CBC stem cell markers in more than one-third of the tumor height, whereas regenerative glands had CBC stem cell markers confined to the lower one-third of the crypt. These results suggest that CBC stem cell markers could be a useful tool for the early detection of colitis-induced tumors.

Breast cancer is a heterogeneous disease due to its clinico-pathological features and response to therapy. The classification of breast tumors based on their hormone receptor status and pathologic features. Post-translational histone modifications come into prominence for regulation of gene expression in cancer pathogenesis. Here, we analyzed dysregulation of H3K9ac and H3K27me3-enriched subtype-specific genes using ChIP-on-chip assay in breast cancer tumors and matched normal tissue samples. Breast cancer tumors were classified according to St Gallen Consensus 2013. Our results indicated that the promoter regions of genes modified by H3K9ac epi-mark are commonly associated with tumors with HER2-positive and TNBC subtype. H3K27me3-enriched genes were comprised of Luminal A and B1 subtypes. We constructed a network structure to elicit epigenetically regulated genes related with breast cancer progression. The central genes of the network (RUNX1, PAX3, GATA4 and DLX5) were subjected for epigenetically dysregulation in association with different breast cancer subtypes. Our study submits epigenetic mechanisms are crucial to elicit subtype-specific regulation in breast cancer and ChIP-on-chip assay provides a better understanding for breast tumorigenesis and new approaches for prevention and treatment.

Protein homeostasis, or proteostasis, is essential for cell function, development, and organismal viability. The composition of the proteome is adjusted to the specific requirements of a particular cell type and status. Moreover, multiple metabolic and environmental conditions challenge the integrity of the proteome. To maintain the quality of the proteome, the proteostasis network monitors proteins from their synthesis through their degradation. Whereas somatic stem cells lose their ability to maintain proteostasis with age, immortal pluripotent stem cells exhibit a stringent proteostasis network associated with their biological function and intrinsic characteristics. Moreover, growing evidence indicates that enhanced proteostasis mechanisms play a central role in immortality and cell fate decisions of pluripotent stem cells. Here, we will review new insights into the melding fields of proteostasis and pluripotency and their implications for the understanding of organismal development and survival.

<h4>Background</h4>Cancer is one of the leading causes of mortality. The neoplastic transformation of normal cells to cancer cells is caused by a progressive accumulation of genetic and epigenetic alterations in oncogenes, tumor suppressor genes and epigenetic regulators, providing cells with new properties, collectively known as the hallmarks of cancer. During the process of neoplastic transformation cells progressively acquire novel characteristics such as unlimited growth potential, increased motility and the ability to migrate and invade adjacent tissues, the ability to spread from the tumor of origin to distant sites, and increased resistance to various types of stresses, mostly attributed to the activation of genetic stress-response programs. Accumulating evidence indicates a crucial role of microRNAs (miRNAs or miRs) in the initiation and progression of cancer, acting either as oncogenes (oncomirs) or as tumor suppressors via several molecular mechanisms. MiRNAs comprise a class of small ~22 bp long noncoding RNAs that play a key role in the regulation of gene expression at the post-transcriptional level, acting as negative regulators of mRNA translation and/or stability. MiRNAs are involved in the regulation of a variety of biological processes including cell cycle progression, DNA damage responses and apoptosis, epithelial-to-mesenchymal cell transitions, cell motility and stemness through complex and interactive transcription factor-miRNA regulatory networks.<h4>Conclusions</h4>The impact and the dynamic potential of miRNAs with oncogenic or tumor suppressor properties in each stage of the multistep process of tumorigenesis, and in the adaptation of cancer cells to stress, are discussed. We propose that the balance between oncogenic versus tumor suppressive miRNAs acting within transcription factor-miRNA regulatory networks, influences both the multistage process of neoplastic transformation, whereby normal cells become cancerous, and their stress responses. The role of specific tumor-derived exosomes containing miRNAs and their use as biomarkers in diagnosis and prognosis, and as therapeutic targets, are also discussed.

The serotonin transporter (5-HTT) regulates serotonin homeostasis and has been used as a target for different drugs in depression treatment. Although the serotonergic system has received a lot of attention, little is known about the effects of these drugs over serotonin transporters. In this work, we investigated the expression pattern of 5-HTT during development of the visual system and the influence of fluoxetine on different signaling pathways. Our data showed that the expression of 5-HTT has a gradual increase from postnatal day 0 until 42 and decrease afterwards. Moreover, chronic fluoxetine treatment both in childhood and adolescence induces down regulation of 5-HTT expression and phosphorylation of ERK and AKT signaling pathways. Together these data suggest that the levels of 5-HTT protein could be important for the development of the central nervous system and suggest that the ERK and AKT are involved in the molecular pathways of antidepressants drugs, acting in concert to improve serotonergic signaling.

The Fish Plasma Model (FPM) was applied to water exposure and tissue concentrations in fish collected from two wastewater treatment plant impacted estuarine sites. In this study we compared predicted fish plasma concentrations to Cmax values for humans, which represents the maximum plasma concentration for the minimum therapeutic dose. The results of this study show that predictions of plasma concentrations for a variety of pharmaceutical and personal care products (PPCPs) from effluent concentrations resulted in 37 compounds (54%) exceeding the response ratio (RR = Fish [Plasma]/1%Cmax_total) of 1 compared to 3 compounds (14%) detected with values generated with estuarine receiving water concentrations. When plasma concentrations were modeled from observed whole-body tissue residues, 16 compounds out of 24 detected for Chinook (67%) and 7 of 14 (50%) for sculpin resulted in an RR_tissue value greater than 1, which highlights the importance of this dose metric over that using estuarine water. Because the tissue residue approach resulted in a high percentage of compounds with calculated response ratios exceeding a value of unity, we believe this is a more accurate representation for exposure in the field. Predicting plasma concentrations from tissue residues improves our ability to assess the potential for adverse effects in fish because exposure from all sources is captured. Tissue residues are also more likely to represent steady-state conditions compared to those from water exposure because of the inherent reduction in variability usually observed for field data and the time course for bioaccumulation. We also examined the RR in a toxic unit approach to highlight the importance of considering multiple compounds exhibiting a similar mechanism of action.

Programmed death ligand-1 (PD-L1), which is highly expressed in gastric cancers, interacts with programmed death-1 (PD-1) on T cells and is involved in T-cell immune resistance. To increase the therapeutic safety and accuracy of PD-1/PD-L1 blockade, RNA interference through targeted gene delivery was performed in our study. We developed folic acid (FA)- and disulfide (SS)-polyethylene glycol (PEG)-conjugated polyethylenimine (PEI) complexed with superparamagnetic iron oxide Fe₃O₄ nanoparticles (SPIONs) as a siRNA-delivery system for PD-L1 knockdown. The characterization, binding ability, cytotoxicity, transfection efficiency, and cellular internalization of the polyplex were determined. At nitrogen:phosphate (N:P) ratios of 10 or above, the FA-PEG-SS-PEI-SPIONs bound to PD-L1 siRNA to form a polyplex with a diameter of approximately 120 nm. Cell-viability assays showed that the polyplex had minimal cytotoxicity at low N:P ratios. The FA-conjugated polyplex showed higher transfection efficiency and cellular internalization in the folate receptor-overexpressing gastric cancer cell line SGC-7901 than a non-FA-conjugated polyplex. Subsequently, we adopted the targeted FA-PEG-SS-PEI-SPION/siRNA polyplexes at an N:P ratio of 10 for function studies. Cellular magnetic resonance imaging (MRI) showed that the polyplex could also act as a <i>T</i>₂-weighted contrast agent for cancer MRI. Furthermore, one of four PD-L1 siRNAs exhibited effective PD-L1 knockdown in PD-L1-overexpressing SGC-7901. To determine the effects of the functionalized polyplex on T-cell function, we established a coculture model of activated T cells and SGC-7901 cells and demonstrated changes in secreted cytokines. Our findings highlight the potential of this class of multifunctional theranostic nanoparticles for effective targeted PD-L1-knockdown therapy and MRI diagnosis in gastric cancers.

Based on a previous study, glabridin displayed a dose-dependent increase in estrogenic activity and cell proliferative activity in Ishikawa cells. However, when treated in combination with 17β-E₂, synergistic estrogenic effect was observed but without the same synergistic increase in cell proliferative effect. This study aimed to identify the estrogen and nonestrogen-regulated activities induced by glabridin and in combination with 17β-E₂ in comparison with 17β-E₂. The results showed that 10 µM glabridin and the combination treatment of 100 nM glabridin with 1 nM 17β-E₂ regulated both the genomic and nongenomic estrogen pathways to possibly provide benefits of estrogens in cardiovascular, circulatory, and vasculature systems. Meanwhile, the combination of 100 nM glabridin with 1 nM 17β-E₂ seems to be more suitable to be used as an estrogen replacement. Finally, the results of this study have added on to the present knowledge of glabridin's function as a phytoestrogen and suggested new ideas for the usage of glabridin.

Optimal therapy for infections with carbapenem resistant GNB is not well established due to the weakness of data. Patients presenting with bloodstream infections caused by multidrug resistant <i>Klebsiella pneumoniae</i> were treated with a combination treatment. Optimal therapy for infections with carbapenem resistant Gram-negative bacteria is a serious problem in pediatric patients. We presented three cases who were successfully treated with addition of ertapenem to the combination treatment for bacteremia with multidrug resistant <i>Klebsiella pneumoniae</i>. Dual carbapenem treatment approach is a new approach for these infections and requires more data in children.

Huntington's disease (HD) is a monogenic neurodegenerative disorder characterized by the progressive decay of motor and cognitive abilities accompanied by psychiatric episodes. Tracking and modeling the progression of the multi-faceted clinical symptoms of HD is a challenging problem that has important implications for staging of HD patients and the development of improved enrollment criteria for future HD studies and trials. In this paper, we describe the first steps towards this goal. We begin by curating data from four recent observational HD studies, each containing a diverse collection of clinical assessments. The resulting dataset is unprecedented in size and contains data from 19,269 study participants. By analyzing this large dataset, we are able to discover hidden low dimensional structure in the data that correlates well with surrogate measures of HD progression. The discovered structures are promising candidates for future consumption by downstream statistical HD progression models.

Pelizaeus-Merzbacher disease (PMD) is a rare Mendelian disorder characterised by central nervous system hypomyelination. PMD typically manifests in infancy or early childhood and is caused by mutations in proteolipid protein-1 (PLP1). However, variants in several other genes including gap junction protein gamma 2 (GJC2) can also cause a similar phenotype and are referred to PMD-like disease (PMLD). Whole-exome sequencing in two siblings presenting with clinical symptoms of PMD revealed a homozygous variant in the arginyl-tRNA synthetase (RARS) gene: NM_002887.3: c.[5A>G] p.(Asp2Gly). Subsequent screening of a PMD cohort without a genetic diagnosis identified an unrelated individual with novel compound heterozygous variants including a missense variant c.[1367C>T] p.(Ser456Leu) and a de novo deletion c.[1846_1847delTA] p.(Tyr616Leufs*6). Protein levels of RARS and the multi-tRNA synthetase complex into which it assembles were found to be significantly reduced by 80 and 90% by western blotting and Blue native-PAGE respectively using patient fibroblast extracts. As RARS is involved in protein synthesis whereby it attaches arginine to its cognate tRNA, patient cells were studied to determine their ability to proliferate with limiting amounts of this essential amino acid. Patient fibroblasts cultured in medium with limited arginine at 30 °C and 40 °C, showed a significant decrease in fibroblast proliferation (P<0.001) compared to control cells, suggestive of inefficiency of protein synthesis in the patient cells. Our functional studies provide further evidence that RARS is a PMD-causing gene.

Expression of ZFAS1, a newly identified long noncoding RNA (lncRNA), is dysregulated in several types of cancer. Here we assessed the prognostic value of ZFAS1 in solid tumors. A comprehensive literature search was performed by screening the PubMed, EMBASE, MEDLINE, Cochrane Library, CNKI, and Wanfang databases. A total of 874 patients from 10 studies were included. The pooled analysis demonstrated that patients with high ZFAS1 expression had a significantly shorter overall survival (OS) (HR, 1.58; 95% CI, 1.28-1.97; <i>P</i> < 0.001) and recurrence-free survival (RFS) (HR, 1.90; 95% CI, 1.29-2.79; <i>P</i> = 0.001). Moreover, elevated ZFAS1 expression correlated with tumor size, tumor-node-metastasis (TNM) stage, and lymph node metastasis (LNM). These results demonstrate that increased ZFAS1 expression correlates with a poor prognosis in cancer patients, which suggests ZFAS1 might be useful as a potential prognostic biomarker in patients with solid tumors.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common chronic liver disease in developed countries because of the obesity epidemic. The disease increases liver-related morbidity and mortality, and often increases the risk for other comorbidities, such as type 2 diabetes and cardiovascular disease. Insulin resistance related to metabolic syndrome is the main pathogenic trigger that, in association with adverse genetic, humoral, hormonal and lifestyle factors, precipitates development of NAFLD. Biochemical markers and radiological imaging, along with liver biopsy in selected cases, help in diagnosis and prognostication. Intense lifestyle changes aiming at weight loss are the main therapeutic intervention to manage cases. Insulin sensitizers, antioxidants, lipid lowering agents, incretin-based drugs, weight loss medications, bariatric surgery and liver transplantation may be necessary for management in some cases along with lifestyle measures. This review summarizes the latest evidence on the epidemiology, natural history, pathogenesis, diagnosis and management of NAFLD.

We report a case of acute massive pulmonary embolism in a patient with antithrombin III deficiency. The patient was treated with rivaroxaban. The patient responded well to the therapy, and contrast-enhanced computed tomography showed nearly complete disappearance of the pulmonary embolism. Patients with low antithrombin III activity may have resistance to heparin therapy, leading to insufficient anticoagulation during the acute phase of thromboembolism. This case suggests that direct oral anticoagulants, such as rivaroxaban, may be effective first-line agents for treating venous thromboembolism in patients with antithrombin III deficiency. <<b>Learning objective:</b> Recently, direct oral anticoagulants represent a novel treatment option for venous thromboembolism with several practical advantages over conventional therapy. Antithrombin-III deficiency may lead to insufficient anticoagulation during the acute phase of thromboembolism. The present case suggests that rivaroxaban is a direct Factor Xa inhibitor and does not require cofactors such as antithrombin-III, thus it is suitable for anticoagulation therapy in patients with low antithrombin-III activity.>.

In rodent and human brains, the small GTP-binding protein Rhes is highly expressed in virtually all dopaminoceptive striatal GABAergic medium spiny neurons, as well as in large aspiny cholinergic interneurons, where it is thought to modulate dopamine-dependent signaling. Consistent with this knowledge, and considering that dopaminergic neurotransmission is altered in neurological and psychiatric disorders, here we sought to investigate whether Rhes mRNA expression is altered in brain regions of patients with Parkinson's disease (PD), Schizophrenia (SCZ), and Bipolar Disorder (BD), when compared to healthy controls (about 200 post-mortem samples). Moreover, we performed the same analysis in the putamen of non-human primate Macaca Mulatta, lesioned with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Overall, our data indicated comparable Rhes mRNA levels in the brain of patients with SCZ and BD, and their respective healthy controls. In sharp contrast, the putamen of patients suffering from PD showed a significant 35% reduction of this transcript, compared to healthy subjects. Interestingly, in line with observations obtained in humans, we found 27% decrease in Rhes mRNA levels in the putamen of MPTP-treated primates. Based on the established inhibitory influence of Rhes on dopamine-related responses, we hypothesize that its striatal downregulation in PD patients and animal models of PD might represent an adaptive event of the dopaminergic system to functionally counteract the reduced nigrostriatal innervation.

<h4>Background</h4>Persons living with HIV/AIDS (PLWHA) are at an increased risk of suicide. Increased suicidal risk is a predictor of future attempted and completed suicides and has been associated with poor quality of life and poor adherence with antiretroviral therapy. Clinical risk factors have low predictive value for suicide, hence the interest in potential neurobiological correlates and specific heritable markers of suicide vulnerability. The serotonin transporter gene has previously been implicated in the aetiology of increased suicidal risk in non-HIV infected study populations and its variations may provide a platform for identifying genetic risk for suicidality among PLWHA. The present cross-sectional study aimed at identifying two common genetic variants of the serotonin transporter gene and their association with increased suicidal risk among human immunodeficiency virus (HIV)-positive adults in Uganda.<h4>Results</h4>The prevalence of increased suicidal risk (defined as moderate to high risk suicidality on the suicidality module of the Mini Neuropsychiatric Interview (M.I.N.I) was 3.3% (95% CI, 2.0-5.3). The 5-HTTLPR was found to be associated with increased suicidal risk before Bonferroni correction (p-value = 0.0174). A protective effect on increased suicidal risk was found for the 5-HTTLPR/rs25531 S _A allele (p-value = 0.0046)- which directs reduced expression of the serotonin transporter gene (5-HTT).<h4>Conclusion</h4>The S _A allele at the 5-HTTLPR/rs25531 locus is associated with increased suicidal risk among Ugandan PLWHA. Further studies are needed to validate this finding in Ugandan and other sub-Saharan samples.

<h4>Background</h4>Fusion genes generated from chromosomal translocation play an important role in hematological malignancies. Detection of fusion genes currently employ use of either conventional RT-PCR methods or fluorescent in situ hybridization (FISH), where both methods involve tedious methodologies and require prior characterization of chromosomal translocation events as determined by cytogenetic analysis. In this study, we describe a real-time quantitative reverse transcription PCR (qRT-PCR)-based multi-fusion gene screening method with the capacity to detect 22 fusion genes commonly found in leukemia. This method does not require pre-characterization of gene translocation events, thereby facilitating immediate diagnosis and therapeutic management.<h4>Methods</h4>We performed fluorescent qRT-PCR (F-qRT-PCR) using a commercially-available multi-fusion gene detection kit on a patient cohort of 345 individuals comprising 108 cases diagnosed with acute myeloid leukemia (AML) for initial evaluation; remaining patients within the cohort were assayed for confirmatory diagnosis. Results obtained by F-qRT-PCR were compared alongside patient analysis by cytogenetic characterization.<h4>Results</h4>Gene translocations detected by F-qRT-PCR in AML cases were diagnosed in 69.4% of the patient cohort, which was comparatively similar to 68.5% as diagnosed by cytogenetic analysis, thereby demonstrating 99.1% concordance. Overall gene fusion was detected in 53.7% of the overall patient population by F-qRT-PCR, 52.9% by cytogenetic prediction in leukemia, and 9.1% in non-leukemia patients by both methods. The overall concordance rate was calculated to be 99.0%. Fusion genes were detected by F-qRT-PCR in 97.3% of patients with CML, followed by 69.4% with AML, 33.3% with acute lymphoblastic leukemia (ALL), 9.1% with myelodysplastic syndromes (MDS), and 0% with chronic lymphocytic leukemia (CLL).<h4>Conclusions</h4>We describe the use of a F-qRT-PCR-based multi-fusion gene screening method as an efficient one-step diagnostic procedure as an effective alternative to lengthy conventional diagnostic procedures requiring both cytogenetic analysis followed by targeted quantitative reverse transcription (qRT-PCR) methods, thus allowing timely patient management.

Antithrombin mainly inhibits factor Xa and thrombin. The reactive center loop (RCL) is crucial for its interactions with its protease targets and is fully inserted into the A-sheet after its cleavage, causing translocation of the covalently linked protease to the opposite end of the A-sheet. Antithrombin variants with altered RCL hinge residues behave as substrates rather than inhibitors, resulting in stoichiometries of inhibition greater than one. Other antithrombin residues have been suggested to interfere with RCL insertion or the stability of the antithrombin-protease complex, but available crystal structures or mutagenesis studies have failed to identify such residues. Here, we characterized two mutations, S365L and I207T, present in individuals with type II antithrombin deficiency and identified a new antithrombin functional domain. S365L did not form stable complexes with thrombin or factor Xa, and the I207T/I207A variants inhibited both proteases with elevated stoichiometries of inhibition. Close proximity of Ile-207 and Ser-365 to the inserted RCL suggested that the preferred reaction of these mutants as protease substrates reflects an effect on the rate of the RCL insertion and protease translocation. However, both residues lie within the final docking site for the protease in the antithrombin-protease complex, supporting the idea that the enhanced substrate reactions may result from an increased dissociation of the final complexes. Our findings demonstrate that the distal end of the antithrombin A-sheet is crucial for the last steps of protease inhibition either by affecting the rate of RCL insertion or through critical interactions with proteases at the end of the A-sheet.

Mineralocorticoid receptor (MR) mediates the sodium-retaining action of aldosterone in the distal nephron. Herein, we decipher mechanisms by which hypotonicity increases MR expression in renal principal cells. We identify HuR (human antigen R), an mRNA-stabilizing protein, as an important posttranscriptional regulator of MR expression. Hypotonicity triggers a rapid and reversible nuclear export of HuR in renal KC3AC1 cells, as quantified by high-throughput microscopy. We also identify a key hairpin motif in the 3'-untranslated region of MR transcript, pivotal for the interaction with HuR and its stabilizing function. Next, we show that hypotonicity increases MR recruitment onto Sgk1 promoter, a well-known MR target gene, thereby enhancing aldosterone responsiveness. Our data shed new light on the crucial role of HuR as a stabilizing factor for the MR transcript and provide evidence for a short autoregulatory loop in which expression of a nuclear receptor transcriptionally regulating water and sodium balance is controlled by osmotic tone.

The prevalence of neuropsychiatric disorders following traumatic brain injury (TBI) is 20%-50%, and disorders of mood and cognition may remain even after recovery of neurologic function is achieved. Selective serotonin reuptake inhibitors (SSRI) block the reuptake of serotonin in presynaptic cells to lead to increased serotonergic activity in the synaptic cleft, constituting first-line treatment for a variety of neurocognitive and neuropsychiatric disorders. This review investigates the utility of SSRIs in treating post-TBI disorders. In total, 37 unique reports were consolidated from the Cochrane Central Register and PubMed (eight randomized-controlled trials (RCTs), nine open-label studies, 11 case reports, nine review articles). SSRIs are associated with improvement of depressive but not cognitive symptoms. Pooled analysis using the Hamilton Depression Rating Scale demonstrate a significant mean decrease of depression severity following sertraline compared to placebo-a result supported by several other RCTs with similar endpoints. Evidence from smaller studies demonstrates mood improvement following SSRI administration with absent or negative effects on cognitive and functional recovery. Notably, studies on SSRI treatment effects for post-traumatic stress disorder after TBI remain absent, and this represents an important direction of future research. Furthermore, placebo-controlled studies with extended follow-up periods and concurrent biomarker, neuroimaging and behavioral data are necessary to delineate the attributable pharmacological effects of SSRIs in the TBI population.

In the clinic selective serotonin reuptake inhibitors (SSRIs), like Fluoxetine, remain the primary treatment for major depression. It has been suggested that miR-16 regulates serotonin transporters (SERT) via raphe nuclei and hippocampal responses to antidepressants. However, the underlying mechanism and regulatory pathways are still obtuse. Here, a chronic unpredicted mild stress (CUMS) depression model in rats was established, and then raphe nuclei miR-16 and intragastric Fluoxetine injections were administered for a duration of 3 weeks. An open field test and sucrose preference quantification displayed a significant decrease in the CUMS groups when compare to the control groups, however these changes were attenuated by both miR-16 and Fluoxetine treatments. A dual-luciferase reporter assay system verified that hsa-miR-16 inhibitory effects involve the targeting of 3'UTR on the 5-HTT gene. Expression levels of miR-16 and BDNF in the hippocampus were examined with RT-PCR, and it was found that increased 5-HT2a receptor expression induced by CUMS can be decreased by miR-16 and Fluoxetine administration. Immunofluorescence showed that expression levels of neuron NeuN and MAP-2 in CUMS rats were lower. Apoptosis and autophagy levels were evaluated separately through relative expression of Bcl-2, Caspase-3, Beclin-1, and LC3II. Furthermore, CUMS was found to decrease levels of hippocampal mTOR, PI3K, and AKT. These findings indicate that apoptosis and autophagy related pathways could be involved in the effectiveness of antidepressants, in which miR-16 participates in the regulation of, and is likely to help integrate rapid therapeutic strategies to alleviate depression clinically. These findings indicate that miR-16 participates in the regulation of apoptosis and autophagy and could account for some part of the therapeutic effect of SSRIs. This discovery has the potential to further the understanding of SSRIs and accelerate the development of new treatments for depression.

Nucleotides play a central role in life-form metabolism, by interacting with proteins and mediating the function of proteins. It is estimated that nucleotides constitute about 15% of the biologically relevant ligands included in PDB. Prediction of binding sites of nucleotides is useful in understanding the function of proteins and can facilitate the in silico design of drugs. In this study, we propose a nucleotide-binding site predictor, namely, NSiteMatch. The NSiteMatch algorithm integrates three different strategies: geometrical analysis, energy calculation, and template comparison. Unlike a traditional template-based predictor, which identifies global similarity between target structure and template, NSiteMatch concerns the local similarity between a surface patch of the target protein and the binding sites of template. To this end, NSiteMatch identifies more templates than traditional template-based predictors. The NSiteMatch predictor is compared with three representative methods, Findsite, Q-SiteFinder, and MetaPocket. An extensive evaluation demonstrates that NSiteMatch achieves higher success rates than Findsite, Q-SiteFinder, and MetaPocket, in prediction of binding sites of ATP, ADP, and AMP.

Cellular senescence is a complex biological process that leads to irreversible cell-cycle arrest. Various extrinsic and intrinsic insults are associated with the onset of cellular senescence and frequently accompany genomic or epigenomic alterations. Cellular senescence is believed to contribute to tumor suppression, immune response, and tissue repair as well as aging and age-related diseases. Long noncoding RNAs (lncRNAs) are >200 nucleotides long, poorly conserved, and transcribed in a manner similar to that of mRNAs. They are tightly regulated during various cellular and physiological processes. Although many lncRNAs and their functional roles are still undescribed, the importance of lncRNAs in a variety of biological processes is widely recognized. RNA-binding proteins (RBPs) have a pivotal role in posttranscriptional regulation as well as in mRNA transport, storage, turnover, and translation. RBPs interact with mRNAs, other RBPs, and noncoding RNAs (ncRNAs) including lncRNAs, and they are involved in the regulation of a broad spectrum of cellular processes. Like other cell fate regulators, lncRNAs and RBPs, separately or cooperatively, are implicated in initiation and maintenance of cellular senescence, aging, and age-related diseases. Here, we review the current understanding of both lncRNAs and RBPs and their association with oxidative stress, senescence, and age-related diseases.

Glioblastoma (GBM), the most malignant of the brain tumors, has been classified on the basis of molecular signature into four subtypes: classical, mesenchymal, proneural and neural, among which the mesenchymal and proneural subtypes have the shortest and longest survival, respectively. Here we show that the transcription factor <i>PATZ1</i> gene is upregulated in gliomas compared to normal brain and, among GBMs, is particularly enriched in the proneural subtype and co-localize with stemness markers. Accordingly, in GBM-derived glioma-initiating stem cells (GSCs) PATZ1 is overexpressed compared to differentiated tumor cells and its expression significantly correlates with the characteristic stem cell capacity to grow as neurospheres <i>in vitro</i>. Interestingly, survival analysis demonstrated that PATZ1 lower levels informed poor prognosis in GBM and, specifically, in the proneural subgroup, suggesting it may serve a role as diagnostic and prognostic biomarker for intra-subtype heterogeneity of proneural GBM. We also show that PATZ1 suppresses the expression of the mesenchyme-inducer CXCR4, and that PATZ1 and CXCR4 are inversely correlated in GSC and proneural GBM. Overall these findings support a central role of PATZ1 in regulating malignancy of GBM.

The mineral component of bone and other biological calcifications is primarily a carbonate substituted calcium apatite. Integration of carbonate into two sites, substitution for phosphate (B-type carbonate) and substitution for hydroxide (A-type carbonate), influences the crystal properties which relate to the functional properties of bone. In the present work, a series of AB-type carbonated apatites (AB-CAp) having varying A-type and B-type carbonate weight fractions were prepared and analyzed by Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), and carbonate analysis. A detailed characterization of A-site and B-site carbonate assignment in the FTIR ν₃ region is proposed. The mass fractions of carbonate in A-site and B- site of AB-CAp correlate differently with crystal axis length and crystallite domain size. In this series of samples reduction in crystal domain size correlates only with A-type carbonate which indicates that carbonate in the A-site is more disruptive to the apatite structure than carbonate in the B-site. High temperature methods were required to produce significant A-type carbonation of apatite, indicating a higher energy barrier for the formation of A-type carbonate than for B-type carbonate. This is consistent with the dominance of B-type carbonate substitution in low temperature synthetic and biological apatites.

Particulate therapeutics fabricated from polymeric materials have become increasingly popular over the past several decades. Generally, polymeric systems are easy to synthesize and have tunable parameters, giving them significant potential for wide use in the clinic. They come in many different forms, including as nanoparticles, microparticles, and colloidal gels. In this review, we discuss the current preparation methods for each type of platform, as well as some representative applications. To achieve enhanced performance, lipid coatings and other surface modification techniques for introducing additional functionality are also mentioned. We hope that, by outlining the various methods and techniques for their preparation, it will be possible to provide insights into the utility of these polymeric platforms and further encourage their development for biomedical applications.

Cerebellins are secreted hexameric proteins that form tripartite complexes with the presynaptic cell-adhesion molecules neurexins or 'deleted-in-colorectal-cancer', and the postsynaptic glutamate-receptor-related proteins GluD1 and GluD2. These tripartite complexes are thought to regulate synapses. However, cerebellins are expressed in multiple isoforms whose relative distributions and overall functions are not understood. Three of the four cerebellins, Cbln1, Cbln2, and Cbln4, autonomously assemble into homohexamers, whereas the Cbln3 requires Cbln1 for assembly and secretion. Here, we show that Cbln1, Cbln2, and Cbln4 are abundantly expressed in nearly all brain regions, but exhibit strikingly different expression patterns and developmental dynamics. Using newly generated knockin reporter mice for Cbln2 and Cbln4, we find that Cbln2 and Cbln4 are not universally expressed in all neurons, but only in specific subsets of neurons. For example, Cbln2 and Cbln4 are broadly expressed in largely non-overlapping subpopulations of excitatory cortical neurons, but only sparse expression was observed in excitatory hippocampal neurons of the CA1- or CA3-region. Similarly, Cbln2 and Cbln4 are selectively expressed, respectively, in inhibitory interneurons and excitatory mitral projection neurons of the main olfactory bulb; here, these two classes of neurons form dendrodendritic reciprocal synapses with each other. A few brain regions, such as the nucleus of the lateral olfactory tract, exhibit astoundingly high Cbln2 expression levels. Viewed together, our data show that cerebellins are abundantly expressed in relatively small subsets of neurons, suggesting specific roles restricted to subsets of synapses.

It is now recognized that several rounds of whole genome duplication (WGD) have occurred during the evolution of vertebrates, but the link between WGDs and phenotypic diversification remains unsolved. We have investigated in this study the impact of the teleost-specific WGD on the evolution of the sox gene family in teleostean fishes. The sox gene family, which encodes for transcription factors, has essential role in morphology, physiology and behavior of vertebrates and teleosts, the current largest group of vertebrates. We have first redrawn the evolution of all sox genes identified in eleven teleost genomes using a comparative genomic approach including phylogenetic and synteny analyses. We noticed, compared to tetrapods, an important expansion of the sox family: 58% (11/19) of sox genes are duplicated in teleost genomes. Furthermore, all duplicated sox genes, except sox17 paralogs, are derived from the teleost-specific WGD. Then, focusing on five sox genes, analyzing the evolution of coding and non-coding sequences, as well as the expression patterns in fish embryos and adult tissues, we demonstrated that these paralogs followed lineage-specific evolutionary trajectories in teleost genomes. This work, based on whole genome data from multiple teleostean species, supports the contribution of WGDs to the expansion of gene families, as well as to the emergence of genomic differences between lineages that might promote genetic and phenotypic diversity in teleosts.

XRCC1 is a scaffold protein involved in base excision repair and single strand break repair. It is a phosphoprotein that contains more than 45 phosphorylation sites, however only a few of these have been characterized and connected to specific kinases and functions. Mitogen activated protein kinases (MAPK) are mediators of cellular stress responses, and here we demonstrate that p38 MAPK signaling is involved in phosphorylation of XRCC1 and regulation of recruitment to oxidative stress. Inhibition of p38 MAPK caused a marked pI shift of XRCC1 towards a less phosphorylated state. Inhibition of p38 also increased the immediate accumulation of XRCC1 at site of DNA damage in a poly(ADP)-ribose (PAR) dependent manner. These results suggest a link between PARylation, p38 signaling and XRCC1 recruitment to DNA damage. Additionally, we characterized two phosphorylation sites, T358 and T367, located within, or close to, the phosphate-binding pocket of XRCC1, which is important for interaction with PAR. Mutation of these sites impairs recruitment of XRCC1 to DNA damage and binding to PARP1/PAR. Collectively, our data suggest that phosphorylation of T358 and T367 and p38 signaling are important for proper regulation of XRCC1 recruitment to DNA damage and thereby avoidance of potential toxic and mutagenic BER-intermediates.

Genome-wide association studies have identified risk loci for SLE, but a large proportion of the genetic contribution to SLE still remains unexplained. To detect novel risk genes, and to predict an individual's SLE risk we designed a random forest classifier using SNP genotype data generated on the "Immunochip" from 1,160 patients with SLE and 2,711 controls. Using gene importance scores defined by the random forest classifier, we identified 15 potential novel risk genes for SLE. Of them 12 are associated with other autoimmune diseases than SLE, whereas three genes (ZNF804A, CDK1, and MANF) have not previously been associated with autoimmunity. Random forest classification also allowed prediction of patients at risk for lupus nephritis with an area under the curve of 0.94. By allele-specific gene expression analysis we detected cis-regulatory SNPs that affect the expression levels of six of the top 40 genes designed by the random forest analysis, indicating a regulatory role for the identified risk variants. The 40 top genes from the prediction were overrepresented for differential expression in B and T cells according to RNA-sequencing of samples from five healthy donors, with more frequent over-expression in B cells compared to T cells.

Macroautophagy mediates the selective degradation of proteins and non-proteinaceous cellular constituents. Here, we show that the N-end rule pathway modulates macroautophagy. In this mechanism, the autophagic adapter p62/SQSTM1/Sequestosome-1 is an N-recognin that binds type-1 and type-2 N-terminal degrons (N-degrons), including arginine (Nt-Arg). Both types of N-degrons bind its ZZ domain. By employing three-dimensional modeling, we developed synthetic ligands to p62 ZZ domain. The binding of Nt-Arg and synthetic ligands to ZZ domain facilitates disulfide bond-linked aggregation of p62 and p62 interaction with LC3, leading to the delivery of p62 and its cargoes to the autophagosome. Upon binding to its ligand, p62 acts as a modulator of macroautophagy, inducing autophagosome biogenesis. Through these dual functions, cells can activate p62 and induce selective autophagy upon the accumulation of autophagic cargoes. We also propose that p62 mediates the crosstalk between the ubiquitin-proteasome system and autophagy through its binding Nt-Arg and other N-degrons.Soluble misfolded proteins that fail to be degraded by the ubiquitin proteasome system (UPS) are redirected to autophagy via specific adaptors, such as p62. Here the authors show that p62 recognises N-degrons in these proteins, acting as a N-recognin from the proteolytic N-end rule pathway, and targets these cargos to autophagosomal degradation.

<h4>Rationale</h4>3,4-Methylenedioxymethamphetamine (MDMA) persistently improves symptoms of post-traumatic stress disorder (PTSD) when combined with psychotherapy. Studies in rodents suggest that these effects can be attributed to enhancement of fear memory extinction. Therefore, MDMA may improve the effects of exposure-based therapy for PTSD, particularly in treatment-resistant patients. However, given MDMA's broad pharmacological profile, further investigation is warranted before moving to a complex clinical population.<h4>Objectives</h4>We aimed to inform clinical research by providing a translational model of MDMA's effect, and elucidating monoaminergic mechanisms through which MDMA enhances fear extinction.<h4>Methods</h4>We explored the importance of monoamine transporters targeted by MDMA to fear memory extinction, as measured by reductions in conditioned freezing and fear-potentiated startle (FPS) in mice. Mice were treated with selective inhibitors of individual monoamine transporters prior to combined MDMA treatment and fear extinction training.<h4>Results</h4>MDMA enhanced the lasting extinction of FPS. Acute and chronic treatment with a 5-HT transporter (5-HTT) inhibitor blocked MDMA's effect on fear memory extinction. Acute inhibition of dopamine (DA) and norepinephrine (NE) transporters had no effect. 5-HT release alone did not enhance extinction. Blockade of MDMA's effect by 5-HTT inhibition also downregulated 5-HT_2A-mediated behavior, and 5-HT_2A antagonism disrupted MDMA's effect on extinction.<h4>Conclusions</h4>We validate enhancement of fear memory extinction by MDMA in a translational behavioral model, and reveal the importance of 5-HTT and 5-HT_2A receptors to this effect. These observations support future clinical research of MDMA as an adjunct to exposure therapy, and provide important pharmacological considerations for clinical use in a population frequently treated with 5-HTT inhibitors.

The establishment of polarity is a critical process in pathogenic fungi, mediating infection-related morphogenesis and host tissue invasion. Here, we report the identification of TPC1 (Transcription factor for Polarity Control 1), which regulates invasive polarized growth in the rice blast fungus Magnaporthe oryzae. TPC1 encodes a putative transcription factor of the fungal Zn(II)2Cys6 family, exclusive to filamentous fungi. Tpc1-deficient mutants show severe defects in conidiogenesis, infection-associated autophagy, glycogen and lipid metabolism, and plant tissue colonisation. By tracking actin-binding proteins, septin-5 and autophagosome components, we show that Tpc1 regulates cytoskeletal dynamics and infection-associated autophagy during appressorium-mediated plant penetration. We found that Tpc1 interacts with Mst12 and modulates its DNA-binding activity, while Tpc1 nuclear localisation also depends on the MAP kinase Pmk1, consistent with the involvement of Tpc1 in this signalling pathway, which is critical for appressorium development. Importantly, Tpc1 directly regulates NOXD expression, the p22phox subunit of the fungal NADPH oxidase complex via an interaction with Mst12. Tpc1 therefore controls spatial and temporal regulation of cortical F-actin through regulation of the NADPH oxidase complex during appressorium re-polarisation. Consequently, Tpc1 is a core developmental regulator in filamentous fungi, linking the regulated synthesis of reactive oxygen species and the Pmk1 pathway, with polarity control during host invasion.

Proteolysis in eukaryotic cells is mainly mediated by the ubiquitin (Ub)-proteasome system (UPS) and the autophagylysosome system (hereafter autophagy). The UPS is a selective proteolytic system in which substrates are recognized and tagged with ubiquitin for processive degradation by the proteasome. Autophagy is a bulk degradative system that uses lysosomal hydrolases to degrade proteins as well as various other cellular constituents. Since the inception of their discoveries, the UPS and autophagy were thought to be independent of each other in components, action mechanisms, and substrate selectivity. Recent studies suggest that cells operate a single proteolytic network comprising of the UPS and autophagy that share notable similarity in many aspects and functionally cooperate with each other to maintain proteostasis. In this review, we discuss the mechanisms underlying the crosstalk and interplay between the UPS and autophagy, with an emphasis on substrate selectivity and compensatory regulation under cellular stresses.

Hereditary hyperferritinemia-cataract syndrome is an autosomal dominant genetic disorder associated with mutations in the 5'UTR region of the ferritin light chain gene. These mutations cause the ferritin levels to increase even in the absence of iron overload. Patients also develop bilateral cataract early due to accumulation of ferritin in the lens, and many are misdiagnosed as having hemochromatosis and thus not properly treated. The first cases were described in 1995 and several mutations have already been identified. However, this syndrome is still a poorly understood. We report two cases of unrelated Brazilian families with clinical suspicion of the syndrome, which were treated in our department. For the definitive diagnosis, the affected patients, their parents and siblings were submitted to Sanger sequencing of the 5'UTR region for detection of the ferritin light gene mutation. Single nucleotide polymorphism-like mutations were found in the affected patients, previously described. The test assisted in making the accurate diagnosis of the disease, and its description is important so that the test can be incorporated into clinical practice.

The δ-protocadherins comprise a small family of homophilic cell adhesion molecules within the larger cadherin superfamily. They are essential for neural development as mutations in these molecules give rise to human neurodevelopmental disorders, such as schizophrenia and epilepsy, and result in behavioral defects in animal models. Despite their importance to neural development, a detailed understanding of their mechanisms and the ways in which their loss leads to changes in neural function is lacking. However, recent results have begun to reveal roles for the δ-protocadherins in both regulation of neurogenesis and lineage-dependent circuit assembly, as well as in contact-dependent motility and selective axon fasciculation. These evolutionarily conserved mechanisms could have a profound impact on the robust assembly of the vertebrate nervous system. Future work should be focused on unraveling the molecular mechanisms of the δ-protocadherins and understanding how this family functions broadly to regulate neural development.

The nuclear factor erythroid 2 like 2 (Nfe2l2/Nrf2) is a master regulator of antioxidant gene transcription. We recently identified that constitutive activation of Nrf2 (CaNrf2) caused reductive stress (RS) in the myocardium. Here we investigate how chronic Nrf2 activation alters myocardial mRNA transcriptome in the hearts of CaNrf2 transgenic (TG-low and TG-high) mice using an unbiased integrated systems approach and next generation RNA sequencing followed by qRT-PCR methods. A total of 246 and 1031 differentially expressed genes (DEGs) were identified in the heart of TGL and TGH in relation to NTG littermates at ~ 6 months of age. Notably, the expression and validation of the transcripts were gene-dosage dependent and statistically significant. Ingenuity Pathway Analysis identified enriched biological processes and canonical pathways associated with myocardial RS in the CaNrf2-TG mice. In addition, an overrepresentation of xenobiotic metabolic signaling, glutathione-mediated detoxification, unfolded protein response, and protein ubiquitination was observed. Other, non-canonical signaling pathways identified include: eNOS, integrin-linked kinase, glucocorticoid receptor, PI3/AKT, actin cytoskeleton, cardiac hypertrophy, and the endoplasmic reticulum stress response. In conclusion, this mRNA profiling identified a "biosignature" for pro-reductive (TGL) and reductive stress (TGH) that can predict the onset, rate of progression, and clinical outcome of Nrf2-dependent myocardial complications. We anticipate that this global sequencing analysis will illuminate the undesirable effect of chronic Nrf2 signaling leading to RS-mediated pathogenesis besides providing important guidance for the application of Nrf2 activation-based cytoprotective strategies.

<h4>Introduction</h4>Hereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated.<h4>Materials and methods</h4>Thirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation.<h4>Results</h4>Unprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans‑Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality.<h4>Conclusions</h4>AT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

Neurodegenerative diseases in humans are frequently associated with prominent accumulation of toxic protein inclusions and defective organelles. Autophagy is a process of bulk lysosomal degradation that eliminates these harmful substances and maintains the subcellular environmental quality. In support of autophagy's importance in neuronal homeostasis, several genetic mutations that interfere with autophagic processes were found to be associated with familial neurodegenerative disorders. In addition, genetic mutations in autophagy-regulating genes provoked neurodegenerative phenotypes in animal models. The Drosophila model significantly contributed to these recent developments, which led to the theory that autophagy dysregulation is one of the major underlying causes of human neurodegenerative disorders. In the current review, we discuss how studies using Drosophila enhanced our understanding of the relationship between autophagy and neurodegenerative processes.

Huntington's disease is caused by polyglutamine (polyQ)-expansion mutations in the CAG tandem repeat of the Huntingtin gene. The central feature of Huntington's disease pathology is the aggregation of mutant Huntingtin (Htt) protein into micrometer-sized inclusion bodies. Soluble mutant Htt states are most proteotoxic and trigger an enhanced risk of death whereas inclusions confer different changes to cellular health, and may even provide adaptive responses to stress. Yet the molecular mechanisms underpinning these changes remain unclear. Using the flow cytometry method of pulse-shape analysis (PulSA) to sort neuroblastoma (Neuro2a) cells enriched with mutant or wild-type Htt into different aggregation states, we clarified which transcriptional signatures were specifically attributable to cells before versus after inclusion assembly. Dampened CREB signalling was the most striking change overall and invoked specifically by soluble mutant Httex1 states. Toxicity could be rescued by stimulation of CREB signalling. Other biological processes mapped to different changes before and after aggregation included NF-kB signalling, autophagy, SUMOylation, transcription regulation by histone deacetylases and BRD4, NAD+ biosynthesis, ribosome biogenesis and altered HIF-1 signalling. These findings open the path for therapeutic strategies targeting key molecular changes invoked prior to, and subsequently to, Httex1 aggregation.

Ferroptosis is a newly discovered form of non-apoptotic cell death in multiple human diseases. However, the epigenetic mechanisms underlying ferroptosis remain poorly defined. First, we demonstrated that lymphoid-specific helicase (LSH), which is a DNA methylation modifier, interacted with WDR76 to inhibit ferroptosis by activating lipid metabolism-associated genes, including GLUT1, and ferroptosis related genes SCD1 and FADS2, in turn, involved in the Warburg effect. WDR76 targeted these genes expression in dependent manner of LSH and chromatin modification in DNA methylation and histone modification. These effects were dependent on iron and lipid reactive oxygen species. We further demonstrated that EGLN1 and c-Myc directly activated the expression of LSH by inhibiting HIF-1α. Finally, we demonstrated that LSH functioned as an oncogene in lung cancer <i>in vitro</i> and <i>in vivo</i>. Therefore, our study elucidates the molecular basis of the c-Myc/EGLN1-mediated induction of LSH expression that inhibits ferroptosis, which can be exploited for the development of therapeutic strategies targeting ferroptosis for the treatment of cancer.

No abstract available.

The nervous system is populated by diverse types of neurons, each of which has dendritic trees with strikingly different morphologies. These neuron-specific morphologies determine how dendritic trees integrate thousands of synaptic inputs to generate different firing properties. To ensure proper neuronal function and connectivity, it is necessary that dendrite patterns are precisely controlled and coordinated with synaptic activity. Here, we summarize the molecular and cellular mechanisms that regulate the formation of cell type-specific dendrite patterns during development. We focus on different aspects of vertebrate dendrite patterning that are particularly important in determining the neuronal function; such as the shape, branching, orientation and size of the arbors as well as the development of dendritic spine protrusions that receive excitatory inputs and compartmentalize postsynaptic responses. Additionally, we briefly comment on the implications of aberrant dendritic morphology for nervous system disease.

A disintegrin and metalloproteinases (ADAMs) are a family of mSultidomain, membrane-anchored proteases that regulate diverse cellular functions, including cell adhesion, migration, proteolysis and other cell signaling events. Catalytically-active ADAMs act as ectodomain sheddases that proteolytically cleave type I and type II transmembrane proteins and some GPI-anchored proteins from the cellular surface. ADAMs can also modulate other cellular signaling events through a process known as regulated intramembrane proteolysis (RIP). Through their proteolytic activity, ADAMs can rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g. inflammation) and play a central role in coordinating intercellular communication. Dysregulation of these processes through aberrant expression, or sustained ADAM activity, is linked to chronic inflammation, inflammation-associated cancer and tumorigenesis. ADAM10 was the first disintegrin-metalloproteinase demonstrated to have proteolytic activity and is the prototypic ADAM associated with RIP activity (e.g. sequential Notch receptor processing). ADAM10 is abundantly expressed throughout the gastrointestinal tract and during normal intestinal homeostasis ADAM10 regulates many cellular processes associated with intestinal development, cell fate specification and maintenance of intestinal stem cell/progenitor populations. In addition, several signaling pathways that undergo ectodomain shedding by ADAM10 (e.g. Notch, EGFR/ErbB, IL-6/sIL-6R) help control intestinal injury/regenerative responses and may drive intestinal inflammation and colon cancer initiation and progression. Here, I review some of the proposed functions of ADAM10 associated with intestinal crypt homeostasis and tumorigenesis within the gastrointestinal tract in vivo. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Several studies have suggested that propiconazole (PROP) may be an endocrine disruptor; possibly altering the activity of the CYP51 enzyme, which is part of the cholesterol biosynthesis pathway required for the production of sexual steroid hormones. Another PROP effect is inhibition of the aromatase enzyme that converts androgens into estrogens, which could lead to negative effects on reproductive parameters. Therefore, the present study evaluated the reproductive and developmental toxicity of PROP by exposing two generations (F1 and F2) of male rats to this fungicide, since a previous study from our lab reported that PROP has anti-estrogenic and anti-androgenic activities (Costa et al., 2015) in the male parental (P) generation. The F1 males were exposed to PROP (4 or 20mg/kg) through germ cells (via the P generation), intra uterus, and lactation, following treatment by gavage from post-natal day (PND) 21 to 120, while the F2 generation was exposed through germ cells, intra uterus, and lactation. The parameters observed in both F1 and F2 generations were: body weight, anogenital distance (PND 0 and 21), ontogenic reflex, testosterone plasmatic levels, testis weight, and testicular histomorphology (PND 21); and in the F1 generation only: preputial separation (PND 40), sexual behavior, organ weights, testosterone and estradiol plasmatic levels (PND 120), sperm count and morphology, and testicular histomorphology at adulthood. In the F1 and F2 generations, PROP (4mg/kg) presented a decrease in testosterone levels, and in the F1 decreases in the vas deferens weight, without hormonal and functional changes of the reproductive organs, either at 4mg/kg or at 20mg/kg, in adulthood. Based on the results of this work, PROP did not alter the gonadal-endocrine parameters under these exposure conditions in rats.

HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies.

Individual variation in alcohol consumption in human populations is determined by genetic, environmental, social and cultural factors. In contrast to humans, genetic contributions to complex behavioral phenotypes can be readily dissected in Drosophila, where both the genetic background and environment can be controlled and behaviors quantified through simple high-throughput assays. Here, we measured voluntary consumption of ethanol in ∼3000 individuals of each sex from an advanced intercross population derived from 37 lines of the Drosophila melanogaster Genetic Reference Panel. Extreme quantitative trait loci mapping identified 385 differentially segregating allelic variants located in or near 291 genes at P < 10^-8 . The effects of single nucleotide polymorphisms associated with voluntary ethanol consumption are sex-specific, as found for other alcohol-related phenotypes. To assess causality, we used RNA interference knockdown or P{MiET1} mutants and their corresponding controls and functionally validated 86% of candidate genes in at least one sex. We constructed a genetic network comprised of 23 genes along with a separate trio and a pair of connected genes. Gene ontology analyses showed enrichment of developmental genes, including development of the nervous system. Furthermore, a network of human orthologs showed enrichment for signal transduction processes, protein metabolism and developmental processes, including nervous system development. Our results show that the genetic architecture that underlies variation in voluntary ethanol consumption is sexually dimorphic and partially overlaps with genetic factors that control variation in feeding behavior and alcohol sensitivity. This integrative genetic architecture is rooted in evolutionarily conserved features that can be extrapolated to human genetic interaction networks.

Peroxisomes are a critical rheostat of reactive oxygen species (ROS), yet their role in drug sensitivity and resistance remains unexplored. Gene expression analysis of clinical lymphoma samples suggests that peroxisomes are involved in mediating drug resistance to the histone deacetylase inhibitor (HDACi) Vorinostat (Vor), which promotes ROS-mediated apoptosis. Vor augments peroxisome numbers in cultured lymphoma cells, concomitant with increased levels of peroxisomal proteins PEX3, PEX11B, and PMP70. Genetic inhibition of peroxisomes, using PEX3 knockdown, reveals that peroxisomes protect lymphoma cells against Vor-mediated cell death. Conversely, Vor-resistant cells were tolerant to elevated ROS levels and possess upregulated levels of (1) catalase, a peroxisomal antioxidant, and (2) plasmalogens, ether glycerophospholipids that represent peroxisome function and serve as antioxidants. Catalase knockdown induces apoptosis in Vor-resistant cells and potentiates ROS-mediated apoptosis in Vor-sensitive cells. These findings highlight the role of peroxisomes in resistance to therapeutic intervention in cancer, and provide a novel modality to circumvent drug resistance.

Approximately 7,000 rare diseases affect millions of individuals in the United States. Although rare diseases taken together have an enormous impact, there is a significant gap between basic research and clinical interventions. Opportunities now exist to accelerate drug development for the treatment of rare diseases. Disease foundations and research centers worldwide focus on better understanding rare disorders. Here, the state-of-the-art drug discovery strategies for small molecules and biological approaches for orphan diseases are reviewed. Rare diseases are usually genetic diseases; hence, employing pharmacogenetics to develop treatments and using whole genome sequencing to identify the etiologies for such diseases are appropriate strategies to exploit. Beginning with high throughput screening of small molecules, the benefits and challenges of target-based and phenotypic screens are discussed. Explanations and examples of drug repurposing are given; drug repurposing as an approach to quickly move programs to clinical trials is evaluated. Consideration is given to the category of biologics which include gene therapy, recombinant proteins, and autologous transplants. Disease models, including animal models and induced pluripotent stem cells (iPSCs) derived from patients, are surveyed. Finally, the role of biomarkers in drug discovery and development, as well as clinical trials, is elucidated.

Previous studies have identified the odd-skipped related 2 (Osr2) transcription factor as a key intrinsic regulator of palatal shelf growth and morphogenesis. However, little is known about the molecular program acting downstream of Osr2 in the regulation of palatogenesis. In this study, we isolated palatal mesenchyme cells from embryonic day 12.5 (E12.5) and E13.5 Osr2^RFP/+ and Osr2^RFP/- mutant mouse embryos and performed whole transcriptome RNA sequencing analyses. Differential expression analysis of the RNA sequencing datasets revealed that expression of 70 genes was upregulated and expression of 61 genes was downregulated by >1.5-fold at both E12.5 and E13.5 in the Osr2^RFP/- palatal mesenchyme cells, in comparison with Osr2^RFP/+ littermates. Gene ontology analysis revealed enrichment of signaling molecules and transcription factors crucial for skeletal development and osteoblast differentiation among those significantly upregulated in the Osr2 mutant palatal mesenchyme. Using quantitative real-time polymerase chain reaction (RT-PCR)and in situ hybridization assays, we validated that the Osr2^-/- embryos exhibit significantly increased and expanded expression of many osteogenic pathway genes, including Bmp3, Bmp5, Bmp7, Mef2c, Sox6, and Sp7 in the developing palatal mesenchyme. Furthermore, we demonstrate that expression of Sema3a, Sema3d, and Sema3e, is ectopically activated in the developing palatal mesenchyme in Osr2^-/- embryos. Through chromatin immunoprecipitation, followed by RT-PCR analysis, we demonstrate that endogenous Osr2 protein binds to the promoter regions of the Sema3a and Sema3d genes in the embryonic palatal mesenchyme. Moreover, Osr2 expression repressed the transcription from the Sema3a and Sema3d promoters in cotransfected cells. Since the Sema3 subfamily of signaling molecules plays diverse roles in the regulation of cell proliferation, migration, and differentiation, these data reveal a novel role for Osr2 in regulation of palatal morphogenesis through preventing aberrant activation of Sema3 signaling. Together, these data indicate that Osr2 controls multiple molecular pathways, including BMP and Sema3 signaling, in palate development.

The genetic factors that give rise to variation in susceptibility to environmental toxins remain largely unexplored. Studies on genetic variation in susceptibility to environmental toxins are challenging in human populations, due to the variety of clinical symptoms and difficulty in determining which symptoms causally result from toxic exposure; uncontrolled environments, often with exposure to multiple toxicants; and difficulty in relating phenotypic effect size to toxic dose, especially when symptoms become manifest with a substantial time lag. Drosophila melanogaster is a powerful model that enables genome-wide studies for the identification of allelic variants that contribute to variation in susceptibility to environmental toxins, since the genetic background, environmental rearing conditions and toxic exposure can be precisely controlled. Here, we used extreme QTL mapping in an outbred population derived from the D. melanogaster Genetic Reference Panel to identify alleles associated with resistance to lead and/or cadmium, two ubiquitous environmental toxins that present serious health risks. We identified single nucleotide polymorphisms (SNPs) associated with variation in resistance to both heavy metals as well as SNPs associated with resistance specific to each of them. The effects of these SNPs were largely sex-specific. We applied mutational and RNAi analyses to 33 candidate genes and functionally validated 28 of them. We constructed networks of candidate genes as blueprints for orthologous networks of human genes. The latter not only provided functional contexts for known human targets of heavy metal toxicity, but also implicated novel candidate susceptibility genes. These studies validate Drosophila as a translational toxicogenomics gene discovery system.

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

The role of hepatitis C virus (HCV) in insulin resistance (IR) is not fully understood. The aim of this study was to determine the impact of amino acid (aa) substitutions in the <i>core</i> region of HCV according to IR and to identify clinical and laboratory associations. Ninety-two treatment-naive HCV patients were recruited to determine laboratory data and blood cell count. IR was determined using Homeostasis Model Assessment (HOMA) index where IR was defined as HOMA ≥2. HCV RNA load and genotype were determined by Abbott Real time HCV. HCV <i>core</i> region was determined by direct nucleotide sequencing. Bivariate analysis was conducted using HOMA IR ≥2 as a dependent factor. IR prevalence was 43.5% (<i>n</i> = 40), vitamin D sufficiency was found in 76.1% (<i>n</i> = 70) and 72.8% (<i>n</i> = 67) had advanced liver fibrosis. In the bivariate analyses, elevated values of γGT (<i>p</i> = 0.024) and fibrosis staging (<i>p</i> = 0.004) were associated with IR, but IR was not related to core mutations. The presence of glutamine in position 70 was associated with low vitamin D concentration (<i>p</i> = 0.005). In the multivariate analysis, no variable was independently associated with HOMA-IR. In conclusion, lack of association between IR and HCV core mutations in positions 70 and 91 suggests that genetic variability of this region has little impact on IR.

Neurodegenerative diseases are a heterogeneous, mostly age-associated group of disorders characterized by progressive neuronal loss, the most prevalent being Alzheimer disease. It is anticipated that, with continuously increasing life expectancy, these diseases will pose a serious social and health problem in the near feature. Meanwhile, however, their etiology remains largely obscure even though all possible novel clues are being thoroughly examined. In this regard, a concept has been proposed that p53, as a transcription factor controlling many vital cellular pathways including apoptosis, may contribute to neuronal death common to all neurodegenerative disorders. In this work, we review the research devoted to the possible role of p53 in the pathogenesis of these diseases. We not only describe aberrant changes in p53 level/activity observed in CNS regions affected by particular diseases but, most importantly, put special attention to the complicated reciprocal regulatory ties existing between p53 and proteins commonly regarded as pathological hallmarks of these diseases, with the ultimate goal to identify the primary element of their pathogenesis.

The spores of Lygodium japonicum (Thunb.) Sw. (L. japonicum) have been used in traditional Chinese medicine for the treatment of various inflammatory diseases. However, the molecular mechanisms underlying their anti‑inflammatory effects have yet to be elucidated. In the present study, we investigated the anti‑inflammatory effects of ethanol extracts of L. japonicum spores (ELJ) by measuring the production of inflammatory mediators, and explored the molecular mechanisms underlying the effects of ELJ in murine macrophages in vitro using immunoblotting analyses. At non‑cytotoxic concentrations of (50‑300 µg/ml), ELJ was revealed to significantly suppress the production of nitric oxide (NO) and tumor necrosis factor (TNF)‑α in lipopolysaccharide (LPS)‑stimulated murine RAW 264.7 macrophages; ELJ repressed the production of interleukin (IL)‑6 only at high concentrations (≥200 µg/ml). The ELJ‑mediated decrease in NO production was demonstrated to depend on the downregulation of inducible NO synthase mRNA and protein expression. Conversely, the mRNA and protein expression of cyclooxygenase‑2 were not affected by ELJ. In addition, ELJ was revealed to inhibit the mRNA expression of IL‑6, IL‑1β, and TNF‑α in LPS‑stimulated RAW 264.7 macrophages. The effects of ELJ on proinflammatory mediators may have been due to the stabilization of inhibitor of κBα and the inhibition of p38 mitogen‑activated protein kinase (MAPK). These results suggested that ELJ may suppress LPS‑induced inflammatory responses in murine macrophages in vitro, through the negative regulation of p38 MAPK and nuclear factor (NF)‑κB. Therefore, ELJ may have potential as a novel candidate for the development of therapeutic strategies aimed at alleviating inflammation.

Axonal growth cones extend during neural development in response to precise distributions of extracellular cues. Deleted in colorectal cancer (DCC), a receptor for the chemotropic guidance cue netrin-1, directs F-actin reorganization, and is essential for mammalian neural development. To elucidate how the extracellular distribution of netrin-1 influences the distribution of DCC and F-actin within axonal growth cones, we patterned nanoarrays of substrate bound netrin-1 using lift-off nanocontact printing. The distribution of DCC and F-actin in embryonic rat cortical neuron growth cones was then imaged using total internal reflection fluorescence (TIRF) microscopy. Fluorescence fluctuation analysis via image cross-correlation spectroscopy (ICCS) was applied to extract the molecular density and aggregation state of DCC and F-actin, identifying the fraction of DCC and F-actin colocalizing with the patterned netrin-1 substrate. ICCS measurement of spatially segmented images based on the substrate nanodot patterns revealed distinct molecular distributions of F-actin and DCC in regions directly overlying the nanodots compared to over the reference surface surrounding the nanodots. Quantifiable variations between the populations of DCC and F-actin on and off the nanodots reveal specific responses to the printed protein substrate. We report that nanodots of substrate-bound netrin-1 locally recruit and aggregate DCC and direct F-actin organization. These effects were blocked by tetanus toxin, consistent with netrin-1 locally recruiting DCC to the plasma membrane via a VAMP2-dependent mechanism. Our findings demonstrate the utility of segmented ICCS image analysis, combined with precisely patterned immobilized ligands, to reveal local receptor distribution and signaling within specialized subcellular compartments.

Glycogen accumulation in the central nervous system of patients with classical infantile onset Pompe disease (IOPD) has been a consistent finding on the few post-mortems performed. While delays in myelination and a possible reduction in processing speed have previously been noted, it has only been recently that the potential for clinically significant progressive white matter disease has been noted. The limited reports thus far published infer that in some IOPD patients, this manifests as intellectual decline in the second decade of life. We present a CRIM negative patient, immunomodulated with rituximab and methotrexate at birth, who despite an initial good clinical response to ERT, at the age of just under 4 years, presented with evolving spasticity in the lower limbs. The investigation of which revealed progressive central nervous system involvement. Given both the earlier onset of the symptoms and consanguineous familial pedigree, extensive biochemical and genetic investigation was undertaken to ensure no alternative pathology was elucidated. In light of these findings, we review the radiology and post-mortems of previous cases and discuss the potential mechanisms that may underlie this presentation.

<h4>Introduction</h4>Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a key role in the cholesterol metabolism and is synthesized by the liver. It interacts with the LDL-receptor to promote its degradation. The model of end-stage liver disease (MELD) score is a well-established tool to estimate the risk of mortality in patients with end-stage chronic liver disease. The study aims to assess the associations between PCSK9, hypocholesterinemia, liver synthesis, cholestasis, MELD score and mortality in patients with end-stage liver disease.<h4>Methods</h4>Serum samples were obtained from 74 patients with severe liver disease. The study participants were aged between 23 and 70 y (mean: 55.8 y; 47 males [63.5%], 27 females [36.5%]). Samples were selected from those with a wide range of MELD scores (7 to 40). Patients that underwent liver transplantation (17 / 74) were censored at the time of transplantation for mortality analysis.<h4>Results</h4>PCSK9 values ranged from 31.47 ng/mL to 261.64 ng/mL. The median value was 106.39 ng/ml. PCSK9 was negatively correlated with markers of liver function and cholestasis (INR, bilirubin). Over a 90-d follow-up, 15 of 57 (26,3%) patients died within the 90-d follow-up without receiving liver transplantation. Thirteen of 31 (42%) patients with PCSK9 levels below the median died compared to 2/26 (8%) patients with higher PCSK9 levels (p = 0.006). In this cohort, there were no significant correlations between PCSK9, cholesterol, its precursors and several phytosterols.<h4>Conclusions</h4>Low PCSK9 serum concentrations were associated with higher mortality in patients with end-stage liver disease. The mean PCSK9 levels in the study population were much lower than those found in normal or healthy populations. Further studies are required to acquire a more detailed understanding of the role of PCSK9 in liver-related mortality.

<h4>Background</h4>In human IVF, embryos cultured with a lower O₂ tension (5%) can give rise to higher success rates when compared with normoxic conditions (20%). However, the mechanisms behind the beneficial effects of reduced oxygen tension in embryogenesis remain unclear. The aim of this study was to evaluate the expression of oxygen related and antioxidant genes and mitochondrial function in mouse embryo cultured under hypoxic and normoxic conditions, to investigate the beneficial effect of low oxygen tension in preimplantation embryogenesis.<h4>Methods</h4>Two-cell ICR mouse embryos were cultured to blastocysts under different oxygen tension (3% and 20%). The gene expression of oxygen-related proteins (hypoxia-inducible factor, HIF), HIF targets (vascular endothelial growth factor, VEGF; glucose transporter 3, GLUT-3) and antioxidants (manganese superoxide dismutase, MnSOD; peroxiredoxin 5, PRDX5) were assessed using quantitative RT-PCR and implantation-related protein (Leukemia Inhibitory Factor Receptor, LIFR) was validated by immunofluorescence. Apoptosis, mitochondrial membrane potential (MtMP) and ROS levels were measured by TUNEL, JC-1 and DCFDA assays, respectively.<h4>Results</h4>Blastocyst development rate (92.3% vs. 79.4%) and hatch rate (80% vs. 70.4%) were both higher in embryos cultured in 3% O₂ than in 20% O₂. The transcription levels of MnSOD, PRDX5, VEGF and GLUT-3 also significantly increased in 3% O₂ compared with 20% O₂ (P < 0.05). Immunofluorescence showed that the intensity of staining for HIF-2α, MnSOD and LIFR were higher in 3% O₂. Blastocysts cultured under 3% O₂ exhibited significantly higher MtMP compared with 20% O₂. ROS and Apoptosis levels were significantly higher in the 20% O₂ group than in the 3% O₂ group (P < 0.05).<h4>Conclusions</h4>Low O₂ tension may improve embryo viability by increasing expression of antioxidant enzymes and glucose transporter activities. It provides an environment conducive to viability by upregulation of LIFR/VEGF and increased MtMP which could enhance implantation potential and reduce apoptosis in mouse blastocyst. These effects may be initiated and regulated by HIF-2α, a key mediator in a hypoxic environment.

Duplication and segregation of chromosomes involves dynamic reorganization of their internal structure by conserved architectural proteins, including the structural maintenance of chromosomes (SMC) complexes cohesin and condensin. Despite active investigation of the roles of these factors, a genome-wide view of dynamic chromosome architecture at both small and large scale during cell division is still missing. Here, we report the first comprehensive 4D analysis of the higher-order organization of the <i>Saccharomyces cerevisiae</i> genome throughout the cell cycle and investigate the roles of SMC complexes in controlling structural transitions. During replication, cohesion establishment promotes numerous long-range intra-chromosomal contacts and correlates with the individualization of chromosomes, which culminates at metaphase. In anaphase, mitotic chromosomes are abruptly reorganized depending on mechanical forces exerted by the mitotic spindle. Formation of a condensin-dependent loop bridging the centromere cluster with the rDNA loci suggests that condensin-mediated forces may also directly facilitate segregation. This work therefore comprehensively recapitulates cell cycle-dependent chromosome dynamics in a unicellular eukaryote, but also unveils new features of chromosome structural reorganization during highly conserved stages of cell division.

Cucurbit[7]uril (CB[7]), a representative member of the host family cucurbit[n]uril, can host-guest interact with many guest molecules such as adamantane, viologen and naphthalene derivatives. This host-guest interaction provides an easy strategy in gene vector assembling. Furthermore, CB[7] can self-assemble on gold nanospheres (AuNSs). Herein, the combination of CB[7] and AuNSs provides both advantages of host-guest interaction and photo-thermal effect of AuNSs. In this study, polyethyleneimine (PEI) and polyethylene glycol (PEG) were separately interacted with CB[7] via host-guest interaction. Then by assembling on AuNSs, PEI and PEG were combined together to condense DNA into polyplexes as well as enhance circulation stability of the polyplexes. These gene vectors were found to have high cellular uptake efficiency and low cytotoxicity. Furthermore, the well distributed AuNSs in the polyplexes could transform light into heat under light exposure because of the photo-thermal effect. This was found to effectively promote the entry of gene into cytoplasm and highly enhanced gene transfection efficiency.

Flowering time is considered one of the most important agronomic traits in maize (Zea mays L.), and previous studies have indicated that this trait is correlated with genome size. We observed a significant difference in genome size between tropical and temperate inbred lines and a moderate positive correlation between genome size and 180-bp knob abundance determined by high-throughput sequencing in maize inbred lines in this study. We assembled the reads that were mapped to 180-bp knob sequences and found that the top ten abundant 180-bp knob sequences are highly variable. Moreover, our results indicate that genome size is associated with the flowering time of both male and female flowers, in both tropical and temperate inbred lines and under both tropical and temperate environments. To identify loci associated with genome size, we performed a genome-wide association study. The analysis identified three genomic regions associated with genome size, of which two were novel while the third one is located close to the known knobs K8L1 and K8L2. Overall, our results indicate that selection for breeding materials with earlier flowering times can be assisted by choosing germplasms with smaller genome sizes and that genome size can be determined based on the abundance of 180-bp knobs.

Huntington's disease (HD) is a neurodegenerative disorder whose major symptoms include progressive motor and cognitive dysfunction. Cognitive decline is a critical quality of life concern for HD patients and families. The enzyme histone deacetylase 3 (HDAC3) appears to be important in HD pathology by negatively regulating genes involved in cognitive functions. Furthermore, HDAC3 has been implicated in the aberrant transcriptional patterns that help cause disease symptoms in HD mice. HDAC3 also helps fuel CAG repeat expansions in human cells, suggesting that HDAC3 may power striatal expansions in the HTT gene thought to drive disease progression. This multifaceted role suggests that early HDAC3 inhibition offers an attractive mechanism to prevent HD cognitive decline and to suppress striatal expansions. This hypothesis was investigated by treating Hdh^Q111 knock-in mice with the HDAC3-selective inhibitor RGFP966. Chronic early treatment prevented long-term memory impairments and normalized specific memory-related gene expression in hippocampus. Additionally, RGFP966 prevented corticostriatal-dependent motor learning deficits, significantly suppressed striatal CAG repeat expansions, partially rescued striatal protein marker expression and reduced accumulation of mutant huntingtin oligomeric forms. These novel results highlight RGFP966 as an appealing multiple-benefit therapy in HD that concurrently prevents cognitive decline and suppresses striatal CAG repeat expansions.

A new and powerful approach, called imaging-wide association study (IWAS), is proposed to integrate imaging endophenotypes with GWAS to boost statistical power and enhance biological interpretation for GWAS discoveries. IWAS extends the promising transcriptome-wide association study (TWAS) from using gene expression endophenotypes to using imaging and other endophenotypes with a much wider range of possible applications. As illustration, we use gray-matter volumes of several brain regions of interest (ROIs) drawn from the ADNI-1 structural MRI data as imaging endophenotypes, which are then applied to the individual-level GWAS data of ADNI-GO/2 and a large meta-analyzed GWAS summary statistics dataset (based on about 74,000 individuals), uncovering some novel genes significantly associated with Alzheimer's disease (AD). We also compare the performance of IWAS with TWAS, showing much larger numbers of significant AD-associated genes discovered by IWAS, presumably due to the stronger link between brain atrophy and AD than that between gene expression of normal individuals and the risk for AD. The proposed IWAS is general and can be applied to other imaging endophenotypes, and GWAS individual-level or summary association data.

The members of the Tear Film Subcommittee reviewed the role of the tear film in dry eye disease (DED). The Subcommittee reviewed biophysical and biochemical aspects of tears and how these change in DED. Clinically, DED is characterized by loss of tear volume, more rapid breakup of the tear film and increased evaporation of tears from the ocular surface. The tear film is composed of many substances including lipids, proteins, mucins and electrolytes. All of these contribute to the integrity of the tear film but exactly how they interact is still an area of active research. Tear film osmolarity increases in DED. Changes to other components such as proteins and mucins can be used as biomarkers for DED. The Subcommittee recommended areas for future research to advance our understanding of the tear film and how this changes with DED. The final report was written after review by all Subcommittee members and the entire TFOS DEWS II membership.

<i>Vibrio vulnificus</i> biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTX_Vv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA1₃), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing <i>in vitro, ex vivo</i>, and <i>in vivo</i> assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apoptosis, respectively. Finally, we tested the hypothesis that RtxA1₃ contributes to human death caused by this zoonotic serovar by triggering an early cytokine storm in blood. To this end, we used a Bt2-SerE strain (R99) together with its <i>rtxA1</i>_<i>3</i> deficient mutant, and a Bt1 strain (YJ016) producing RtxA1₁ (the most studied MARTX_Vv) together with its <i>rtxA1</i>_<i>1</i> deficient mutant, as controls. Our results showed that RtxA1₃ was essential for virulence, as R99ΔΔ<i>rtxA1</i>_<i>3</i> was completely avirulent in our murine model of infection, and that R99, but not strain YJ016, induced an early, strong and dysregulated immune response involving the up-regulation of a high number of genes. This dysregulated immune response was directly linked to RtxA1₃. Based on these results and those obtained <i>ex vivo</i> (human blood), we propose a model of infection for the zoonotic serovar of <i>V. vulnificus</i>, in which RtxA1₃ would act as a sepsis-inducing toxin.

Culture of endothelial cells (ECs) embedded in 3D scaffolds of denatured collagen has shown tremendous therapeutic potential in clinical trials of tissue repair. It is postulated that these matrix-embedded ECs (MEECs) attain a differential phenotype similar to early progenitor forms, which cannot be attained in 2D culture. MEECs are compared to 2D-ECs and endothelial progenitor cells (EPCs) by secretome, phenotype, and genetic fingerprint, and are found to be altered from 2D-ECs on all levels, adopting an EPC-like phenotype. This manifests in elevation of CD34 expression-a progenitor cell marker-and protein secretion and gene expression pro-files that are similar to EPCs. Even more striking is that EPCs in 2D lose their phenotype, evident by the loss of CD34 expression, but are able to regain expression over time when embedded in the same 3D matrices, suggesting that future in vitro EPC work should use ME-EPCs to recapitulate in vivo phenotype. These findings elucidate the relationship between EPCs and the substratum-dependent regulation imparted by ECs which is critical to understand in order to optimize MEEC therapy and propel it into the clinic.

Basophils (BA) play an important role in the promotion of aberrant T helper type 2 (Th2) immune responses in asthma. It is not only the effective cell, but also modulates the initiation of Th2 immune responses. We earlier demonstrated that Notch signalling regulates the biological function of BAin vitro. However, whether this pathway plays the same role in vivo is not clear. The purpose of the present study was to investigate the effect of Notch signalling on BA function in the regulation of allergic airway inflammation in a murine model of asthma. Bone marrow BA were prepared by bone marrow cell culture in the presence of recombinant interleukin-3 (rIL-3; 300 pg/ml) for 7 days, followed by isolation of the CD49b⁺ microbeads. The recombination signal binding protein J (RBP-J^-/- ) BA were co-cultured with T cells, and the supernatant and the T-cell subtypes were examined. The results indicated disruption of the capacity of BA for antigen presentation alongside an up-regulation of the immunoregulatory function. This was possibly due to the low expression of OX40L in the RBP-J^-/- BA. Basophils were adoptively transferred to ovalbumin-sensitized recipient mice, to establish an asthma model. Lung pathology, cytokine profiles of brobchoalveolar fluid, airway hyperactivity and the absolute number of Th1/Th2 cells in lungs were determined. Overall, our results indicate that the RBP-J-mediated Notch signalling is critical for BA-dependent immunoregulation. Deficiency of RBP-J influences the immunoregulatory functions of BA, which include activation of T cells and their differentiation into T helper cell subtypes. The Notch signalling pathway is a potential therapeutic target for BA-based immunotherapy against asthma.

Biotinylation of amines is widely used to conjugate biomolecules, but either the resulting label is non-removable or its removal leaves a tag on the molecule of interest, thus affecting downstream processes. We present here a set of reagents (RevAmines) that allow traceless, reversible biotinylation under biologically compatible, mild conditions. Release following avidin-based capture is achieved through the cleavage of a (2-(alkylsulfonyl)ethyl) carbamate linker under mild conditions (200 mm ammonium bicarbonate, pH 8, 16-24 h, room temperature) that regenerates the unmodified amine. The capture and release of biotinylated proteins and peptides from neutravidin, fluorescent labelling through reversible biotinylation at the cell surface and the selective enrichment of proteins from bacterial periplasm are demonstrated. The tags are easily prepared, stable and offer the potential for future application in proteomics, activity-based protein profiling, affinity chromatography and bio-molecule tagging and purification.

Burning incense to worship deities is a popular religious ritual in large parts of Asia, and is a popular custom affecting more than 1.5 billion adherents. Due to incomplete combustion, burning incense has been well recognized to generate airborne hazards to human health. However, the correlation between burning incense and lung cancer in epidemiological studies remains controversy. Therefore, we speculated that some unknown materials in incense smoke are involved in the initiation or progression of lung cancer. Based on this hypothesis, we identified a major compound auramine O (AuO) from the water-soluble fraction of incense burned condensate using mass spectrometry. AuO is commonly used in incense manufacture as a colorant. Due to thermostable, AuO released from burned incenses becomes an unexpected air pollutant. AuO is classified as a Group 2B chemical by the International Agency of Research on Cancer (IARC), however, the damage of AuO to the respiratory system remains elusive. Our study revealed that AuO has no apparent effect on malignant transformation; but, it dramatically promotes lung cancer malignancy. AuO accumulates in the nucleus and induces the autophagy activity in lung tumor cells. AuO significantly enhances migration and invasive abilities and the in vitro and in vivo stemness features of lung tumor cells through activating the expression of aldehyde dehydrogenase family 1 member A1 (ALDH1A1), and ALDH1A1 knockdown attenuates AuO-induced autophagy activity and blocks AuO-induced lung tumor malignancy. In conclusion, we found that AuO, an ingredient of incense smoke, significantly increases the metastatic abilities and stemness characters of lung tumor cells through the activation of ALDH1A1, which is known to be associated with poor outcome and progression of lung cancer. For public health, reducing or avoiding the use of AuO in incense is recommended.

Expansions of polyglutamine (polyQ) tracts in different proteins cause 9 neurodegenerative conditions, such as Huntington disease and various ataxias. However, many normal mammalian proteins contain shorter polyQ tracts. As these are frequently conserved in multiple species, it is likely that some of these polyQ tracts have important but unknown biological functions. Here we review our recent study showing that the polyQ domain of the deubiquitinase ATXN3/ataxin-3 enables its interaction with BECN1/beclin 1, a key macroautophagy/autophagy initiator. ATXN3 regulates autophagy by deubiquitinating BECN1 and protecting it from proteasomal degradation. Interestingly, expanded polyQ tracts in other polyglutamine disease proteins compete with the shorter ATXN3 polyQ stretch and interfere with the ATXN3-BECN1 interaction. This competition results in decreased BECN1 levels and impaired starvation-induced autophagy, which phenocopies the loss of autophagic function mediated by ATXN3. Our findings describe a new autophagy-protective mechanism that may be altered in multiple neurodegenerative diseases.

Finding suitable alternative protein sources for diets of carnivorous fish species remains a major concern for sustainable aquaculture. Through genetic selection, we created a strain of rainbow trout that outperforms parental lines in utilizing an all-plant protein diet and does not develop enteritis in the distal intestine, as is typical with salmonids on long-term plant protein-based feeds. By incorporating this strain into functional analyses, we set out to determine which genes are critical to plant protein utilization in the absence of gut inflammation. After a 12-week feeding trial with our selected strain and a control trout strain fed either a fishmeal-based diet or an all-plant protein diet, high-throughput RNA sequencing was completed on both liver and muscle tissues. Differential gene expression analyses, weighted correlation network analyses and further functional characterization were performed. A strain-by-diet design revealed differential expression ranging from a few dozen to over one thousand genes among the various comparisons and tissues. Major gene ontology groups identified between comparisons included those encompassing central, intermediary and foreign molecule metabolism, associated biosynthetic pathways as well as immunity. A systems approach indicated that genes involved in purine metabolism were highly perturbed. Systems analysis among the tissues tested further suggests the interplay between selection for growth, dietary utilization and protein tolerance may also have implications for nonspecific immunity. By combining data from differential gene expression and co-expression networks using selected trout, along with ontology and pathway analyses, a set of 63 candidate genes for plant diet tolerance was found. Risk loci in human inflammatory bowel diseases were also found in our datasets, indicating rainbow trout selected for plant-diet tolerance may have added utility as a potential biomedical model.

Progression of pathology in neurodegenerative diseases is hypothesized to be a non-cell-autonomous process that may be mediated by the productive spreading of prion-like protein aggregates from a "donor cell" that is the source of misfolded aggregates to an "acceptor cell" in which misfolding is propagated by conversion of the normal protein. Although the proteins involved in the various diseases are unrelated, common pathways appear to be used for their intercellular propagation and spreading. Here, we summarize recent evidence of the molecular mechanisms relevant for the intercellular trafficking of protein aggregates involved in prion, Alzheimer's, Huntington's, and Parkinson's diseases. We focus in particular on the common roles that lysosomes and tunneling nanotubes play in the formation and spreading of prion-like assemblies.

Diffusion-weighted imaging quantified using the mono-exponential model has shown great promise for monitoring treatment response in prostate cancer bone metastases. The aim of this prospective study is to evaluate whether non-mono-exponential diffusion models better describe the water diffusion properties and may improve treatment response assessment. Diffusion-weighted imaging data of 12 treatment-naïve patients with 34 metastases acquired before and at one, two, and three months after initiation of antiandrogen treatment are analysed using the mono-exponential, the intravoxel incoherent motion, the stretched exponential, and the statistical model. Repeatability of the fitted parameters and changes under therapy are quantified. Model preference is assessed and correlation coefficients across times are calculated to delineate the relationship between the prostate-specific antigen levels and the diffusion parameters as well as between the diffusion parameters within each model. There is a clear preference for non-mono-exponential diffusion models at all time points. Particularly the stretched exponential is favoured in approximately 60% of the lesions. Its parameters increase significantly in response to treatment and are highly repeatable. Thus, the stretched exponential may be utilized as a potential optimal model for monitoring treatment response. Compared with the mono-exponential model, it may provide complementary information on tissue properties and improve response assessment.

Mutations in the X-linked gene Protocadherin-19 (Pcdh19) cause female-limited epilepsy and mental retardation in humans. Although Pcdh19 is known to be a homophilic cell-cell adhesion molecule, how its mutations bring about female-specific disorders remains elusive. Here, we report the effects of Pcdh19 knockout in mice on their development and behavior. Pcdh19 was expressed in various brain regions including the cerebral cortex and hippocampus. Although Pcdh19-positive cells were evenly distributed in layer V of wild-type cortices, their distribution became a mosaic in Pcdh19 heterozygous female cortices. In cortical and hippocampal neurons, Pcdh19 was localized along their dendrites, showing occasional accumulation on synapses. Pcdh19 mutants, however, displayed no detectable abnormalities in dendrite and spine morphology of layer V neurons. Nevertheless, Pcdh19 hemizygous males and heterozygous females showed impaired behaviors including activity defects under stress conditions. Notably, only heterozygous females exhibited decreased fear responses. In addition, Pcdh19 overexpression in wild-type cortices led to ectopic clustering of Pcdh19-positive neurons. These results suggest that Pcdh19 is required for behavioral control in mice, but its genetic loss differentially affects the male and female behavior, as seen in human, and they also support the hypothesis that the mosaic expression of Pcdh19 in brains perturbs neuronal interactions.

Epstein-Barr virus (EBV) was identified as the first human virus to be associated with a human malignancy, Burkitt's lymphoma (BL), a pediatric cancer endemic in sub-Saharan Africa. The exact mechanism of how EBV contributes to the process of lymphomagenesis is not fully understood. Recent studies have highlighted a genetic difference between endemic (EBV+) and sporadic (EBV-) BL, with the endemic variant showing a lower somatic mutation load, which suggests the involvement of an alternative virally-driven process of transformation in the pathogenesis of endemic BL. We tested the hypothesis that a global change in DNA methylation may be induced by infection with EBV, possibly thereby accounting for the lower mutation load observed in endemic BL. Our comparative analysis of the methylation profiles of a panel of BL derived cell lines, naturally infected or not with EBV, revealed that the presence of the virus is associated with a specific pattern of DNA methylation resulting in altered expression of cellular genes with a known or potential role in lymphomagenesis. These included ID3, a gene often found to be mutated in sporadic BL. In summary this study provides evidence that EBV may contribute to the pathogenesis of BL through an epigenetic mechanism.

The dissemination of carbapenem resistance in Escherichia coli has major implications for the management of common infections. bla _KPC, encoding a transmissible carbapenemase (KPC), has historically largely been associated with Klebsiella pneumoniae, a predominant plasmid (pKpQIL), and a specific transposable element (Tn4401, ~10 kb). Here we characterize the genetic features of bla _KPC emergence in global E. coli, 2008-2013, using both long- and short-read whole-genome sequencing. Amongst 43/45 successfully sequenced bla _KPC-E. coli strains, we identified substantial strain diversity (n = 21 sequence types, 18% of annotated genes in the core genome); substantial plasmid diversity (≥9 replicon types); and substantial bla _KPC-associated, mobile genetic element (MGE) diversity (50% not within complete Tn4401 elements). We also found evidence of inter-species, regional and international plasmid spread. In several cases bla _KPC was found on high copy number, small Col-like plasmids, previously associated with horizontal transmission of resistance genes in the absence of antimicrobial selection pressures. E. coli is a common human pathogen, but also a commensal in multiple environmental and animal reservoirs, and easily transmissible. The association of bla _KPC with a range of MGEs previously linked to the successful spread of widely endemic resistance mechanisms (e.g. bla _TEM, bla _CTX-M) suggests that it may become similarly prevalent.

A leading strategy in tissue engineering is the design of biomimetic scaffolds that stimulate the body's repair mechanisms through the recruitment of endogenous stem cells to sites of injury. Approaches that employ the use of chemoattractant gradients to guide tissue regeneration without external cell sources are favored over traditional cell-based therapies that have limited potential for clinical translation. Following this concept, bioactive scaffolds can be engineered to provide a temporally and spatially controlled release of biological cues, with the possibility to mimic the complex signaling patterns of endogenous tissue regeneration. Another effective way to regulate stem cell activity is to leverage the inherent chemotactic properties of extracellular matrix (ECM)-based materials to build versatile cell-instructive platforms. This review introduces the concept of endogenous stem cell recruitment, and provides a comprehensive overview of the strategies available to achieve effective cardiovascular and bone tissue regeneration.

Periodontitis is an infectious and inflammatory disease associated with significant loss of alveolar crest and soft tissue attached to the teeth. Chitosan and hydroxyapatite are biomaterials used for bone tissue repair because of their biodegradability and biocompatibility in nature. The present study evaluated the effects of chitosan (CH) in combination with hydroxyapatite (HAP) to promote alveolar bone growth. A chitosan implant mixed with hydroxyapatite was implanted into the affected area of 9 patients suffering chronic periodontitis. Patients were evaluated through X-ray images and a millimetric slide over a one year period. The application of CH/HAP produced an average alveolar bone growth of 5.77 mm (±1.87 mm). At the onset of the study, the dental pocket exhibited a depth level (DPDL) of 8.66 mm and decreased to 3.55 mm one year after the implant. Tooth mobility grade was 2.44 mm at the onset and 0.8 mm at the end of the study with a significant difference of <i>p</i> < 0.001. Moreover, the bone density in the affected areas was similar to the density of the bone adjacent to it. This result was confirmed with the software implant viewer from Anne Solutions Company. In conclusion, the CH/HAP implant promoted alveolar bone growth in periodontitis patients.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by motor dysfunction, cognitive deficits and psychosis. HD is caused by mutations in the <i>Huntingtin</i> (<i>HTT</i>) gene, resulting in the expansion of polyglutamine (polyQ) repeats in the HTT protein. Mutant HTT is prone to aggregation, and the accumulation of polyQ-expanded fibrils as well as intermediate oligomers formed during the aggregation process contribute to neurodegeneration. Distinct protein homeostasis (proteostasis) nodes such as chaperone-mediated folding and proteolytic systems regulate the aggregation and degradation of HTT. Moreover, polyQ-expanded HTT fibrils and oligomers can lead to a global collapse in neuronal proteostasis, a process that contributes to neurodegeneration. The ability to maintain proteostasis of HTT declines during the aging process. Conversely, mechanisms that preserve proteostasis delay the onset of HD. Here we will review the link between proteostasis, aging and HD-related changes.

Drosophila and extant species are the best-studied telomerase exception. In this organism, telomere elongation is coupled with targeted retrotransposition of Healing Transposon (HeT-A) and Telomere Associated Retrotransposon (TART) with sporadic additions of Telomere Associated and HeT-A Related (TAHRE), all three specialized non-Long Terminal Repeat (non-LTR) retrotransposons. These three very special retroelements transpose in head to tail arrays, always in the same orientation at the end of the chromosomes but never in interior locations. Apparently, retrotransposon and telomerase telomeres might seem very different, but a detailed view of their mechanisms reveals similarities explaining how the loss of telomerase in a Drosophila ancestor could successfully have been replaced by the telomere retrotransposons. In this review, we will discover that although HeT-A, TART, and TAHRE are still the only examples to date where their targeted transposition is perfectly tamed into the telomere biology of Drosophila, there are other examples of retrotransposons that manage to successfully integrate inside and at the end of telomeres. Because the aim of this special issue is viral integration at telomeres, understanding the base of the telomerase exceptions will help to obtain clues on similar strategies that mobile elements and viruses could have acquired in order to ensure their survival in the host genome.

The brain serotonin (5-hydroxytryptamine; 5-HT) system has been extensively studied for its role in normal physiology and behavior, as well as, neuropsychiatric disorders. The broad influence of 5-HT on brain function, is in part due to the vast connectivity pattern of 5-HT-producing neurons throughout the CNS. 5-HT neurons are born and terminally specified midway through embryogenesis, then enter a protracted period of maturation, where they functionally integrate into CNS circuitry and then are maintained throughout life. The transcriptional regulatory networks controlling progenitor cell generation and terminal specification of 5-HT neurons are relatively well-understood, yet the factors controlling 5-HT neuron maturation are only recently coming to light. In this review, we first provide an update on the regulatory network controlling 5-HT neuron development, then delve deeper into the properties and regulatory strategies governing 5-HT neuron maturation. In particular, we discuss the role of the 5-HT neuron terminal selector transcription factor (TF) Pet-1 as a key regulator of 5-HT neuron maturation. Pet-1 was originally shown to positively regulate genes needed for 5-HT synthesis, reuptake and vesicular transport, hence 5-HT neuron-type transmitter identity. It has now been shown to regulate, both positively and negatively, many other categories of genes in 5-HT neurons including ion channels, GPCRs, transporters, neuropeptides, and other transcription factors. Its function as a terminal selector results in the maturation of 5-HT neuron excitability, firing characteristics, and synaptic modulation by several neurotransmitters. Furthermore, there is a temporal requirement for Pet-1 in the control of postmitotic gene expression trajectories thus indicating a direct role in 5-HT neuron maturation. Proper regulation of the maturation of cellular identity is critical for normal neuronal functioning and perturbations in the gene regulatory networks controlling these processes may result in long-lasting changes in brain function in adulthood. Further study of 5-HT neuron gene regulatory networks is likely to provide additional insight into how neurons acquire their mature identities and how terminal selector-type TFs function in postmitotic vertebrate neurons.

Dopamine neurons of the substantia nigra compacta (SNc) and ventral tegmental area (VTA) are critical components of the neuronal machinery to control emotion and movement in mammals. The slow and gradual death of these neurons as seen in Parkinson's disease has triggered a large investment in research toward unraveling the molecular determinants that are used to generate these neurons and to get an insight in their apparent selective vulnerability. Here, I set out to summarize the current view on the molecular distinctions that exist within this mesodiencephalic dopamine (mdDA) system and elaborate on the molecular programming that is responsible for creating such diversity.

<h4>Purpose</h4>To investigate the effect of rho kinase inhibitors on oxidative stress in trabecular meshwork (TM) cells.<h4>Methods</h4>TM cells were isolated from the eyes of cynomolgus monkeys. Y-27632 and menadione were used to inhibit rho kinase and induce production of reactive oxygen species (ROS), respectively. The cynomolgus monkey array and 12,613 probes were used in DNA microarray analysis, and the affected genes were categorized using gene ontology analysis. The mRNA levels of the target genes were confirmed by real-time RT-PCR. Intracellular oxidative stress was detected using a fluorescent reagent sensitive to ROS. Cell viability was assessed by the WST-8 assay.<h4>Results</h4>Gene ontology analysis revealed upregulation of genes involved in antioxidant activity, and upregulation of catalase was confirmed by real-time RT-PCR after 30 min treatment with Y-27632. Production of ROS was increased by menadione, and the effect was partly suppressed by pretreatment with Y-27632. At a lower dose of menadione, Y-27632 stimulated TM cells and significantly increased their viability following menadione treatment compared to control cells.<h4>Conclusion</h4>Using microarray analysis, Y-27632 was shown to upregulate antioxidative genes including catalase and partially reduce ROS production and cell death by oxidative stress caused by menadione.

<h4>Introduction</h4>Previous studies have examined the correlation between hyperandrogenemia and non-alcoholic fatty liver disease (NAFLD) in women and showed contradictory results. Therefore, we aimed to evaluate the relationship between testosterone level and Fatty Liver Index (FLI), as a surrogate marker for NAFLD, in a cohort of postmenopausal women.<h4>Material and methods</h4>A total of 150 postmenopausal women were included in this cross-sectional study. Anthropometric and biochemical parameters, as well as blood pressure, were obtained. Non-alcoholic fatty liver disease is assessed by FLI, an algorithm based on body mass index, waist circumference, triglycerides and γ-glutamyl transferase, as a simple and accurate predictor of hepatic steatosis. Women were divided into three groups (FLI < 30, <i>n</i> = 80; 30 ≤ FLI < 60, <i>n</i> = 44; FLI ≥ 60, <i>n</i> = 26). Homeostasis model assessment of insulin resistance (HOMA-IR) as a surrogate marker of insulin resistance was calculated.<h4>Results</h4>Multiple linear regression analysis revealed that the best model consisted of 4 parameters (e.g., bioavailable testosterone (β = 0.288, <i>p</i> = 0.001), log HOMA-IR (β = 0.227, <i>p</i> = 0.005), log high-sensitivity C-reactive protein (β = 0.322, <i>p</i> < 0.001), and retinol-binding protein 4 (β = 0.226, <i>p</i> < 0.001)). Adjusted <i>R</i>² for the best model was 0.550, which means that as much as 55.0% of variation in FLI could be explained with this model.<h4>Conclusions</h4>Bioavailable testosterone is independently associated with FLI in postmenopausal women.

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is an aggressive subtype of prostate cancer (PCa) that arises as a consequence of rigorous androgen receptor (AR) pathway inhibition (ARPI) therapies. While the PI3K/AKT pathway has been investigated as a co-therapeutic target with ARPI for advanced PCa, whether this strategy can prevent tumor progression to t-NEPC remains unknown. Here, we report that PI3K/AKT inhibition alone reduces RE-1 silencing transcription factor (REST) protein expression and induces multiple NE markers in PCa cells. The loss of REST by PI3K/AKT inhibition is through protein degradation mediated by the E3-ubiquitin ligase β-TRCP and REST phosphorylations at the S1024, S1027, and S1030 sites. Since AR inhibition can also deplete REST, the combinational inhibition of PI3K/AKT and AR further aggravated REST protein reduction. We profiled the transcriptomes of AKT and AR inhibitions in the LNCaP cells. The Gene Set Enrichment Analysis (GSEA) showed that these transcriptomes are highly correlated with the REST-regulated gene signature. Co-targeting AKT and AR resulted in a higher correlation comparing to those of single treatment. Comparing these transcriptomes to the t-NEPC gene signature in patients by GSEA, we observed that adding AKT inhibition to AR blockade enhanced the expression of neurogenesis-related genes and resulted in a stronger and broader upregulation of REST-regulated genes specific to t-NEPC. These results indicate that AKT pathway inhibition can induce neuroendocrine differentiation of PCa cells <i>via</i> REST protein degradation. It delineates a potential risk for the AR and PI3K/AKT co-targeting strategy as it may further facilitate t-NEPC development.

The mechanism of surface-initiated atom transfer polymerization (SI-ATRP) of methacrylates in confined volumes is systematically investigated by finely tuning the distance between a grafting surface and an inert plane by means of nanosized patterns and micrometer thick foils. The polymers were synthesized from monolayers of photocleavable initiators, which allow the analysis of detached brushes by size-exclusion chromatography (SEC). Compared to brushes synthesized under "open" polymerization mixtures, nearly a 4-fold increase in brush molar mass was recorded when SI-ATRP was performed within highly confined reaction volumes. Correlating the SI-ATRP of methyl methacrylate (MMA), with and without "sacrificial" initiator, to that of lauryl methacrylate (LMA) and analyzing the brush growth rates within differently confined volumes, we demonstrate faster grafting kinetics with increasing confinement due to the progressive hindering of Cu^II-based deactivators from the brush propagating front. This effect is especially noticeable when viscous polymerization mixtures are generated and enables the synthesis of several hundred nanometer thick brushes within relatively short polymerization times. The faster rates of confined SI-ATRP can be additionally used to fabricate, in one pot, precisely structured brush gradients, when volume confinement is continuously varied across a single substrate by spatially tuning the vertical distance between the grafting and the confining surfaces.

Osteoblasts communicate both with normal cells in the bone marrow and with tumor cells that metastasized to bone. Here we show that osteoblasts release exosomes, we termed osteosomes, which may be a novel mechanism by which osteoblasts communicate with cells in their environment. We have isolated exosomes from undifferentiated/proliferating (D0 osteosomes) and differentiated/mineralizing (D24 osteosomes) primary mouse calvarial osteoblasts. The D0 and D24 osteosomes were found to be vesicles of 130-140 nm by dynamic light scattering analysis. Proteomics profiling using tandem mass spectrometry (LC-MS/MS) identified 206 proteins in D0 osteosomes and 336 in D24 osteosomes. The proteins in osteosomes are mainly derived from the cytoplasm (∼47%) and plasma membrane (∼31%). About 69% of proteins in osteosomes are also found in Vesiclepedia, and these canonical exosomal proteins include tetraspanins and Rab family proteins. We found that there are differences in both protein content and levels in exosomes isolated from undifferentiated and differentiated osteoblasts. Among the proteins that are unique to osteosomes, 169 proteins are present in both D0 and D24 osteosomes, 37 are unique to D0, and 167 are unique to D24. Among those 169 proteins present in both D0 and D24 osteosomes, 10 proteins are likely present at higher levels in D24 than D0 osteosomes based on emPAI ratios of >5. These results suggest that osteosomes released from different cellular state of osteoblasts may mediate distinct functions. Using live-cell imaging, we measured the uptake of PKH26-labeled osteosomes into C4-2B4 and PC3-mm2 prostate cancer cells. In addition, we showed that cadherin-11, a cell adhesion molecule, plays a role in the uptake of osteosomes into PC3-mm2 cells as osteosome uptake was delayed by neutralizing antibody against cadherin-11. Together, our studies suggest that osteosomes could have a unique role in the bone microenvironment under both physiological and pathological conditions.

<h4>Background and objectives</h4>Father loss during childhood has negative health and behavioral consequences, but the biological consequences are unknown. Our goal was to examine how father loss (because of separation and/or divorce, death, or incarceration) is associated with cellular function as estimated by telomere length.<h4>Methods</h4>Data come from the 9-year follow-up of the Fragile Families and Child Wellbeing Study, a birth cohort study of children in 20 large American cities (<i>N</i> = 2420). Principal measures are as follows: salivary telomere length (sTL), mother reports of father loss, and polymorphisms in genes related to serotonergic and dopaminergic signaling.<h4>Results</h4>At 9 years of age, children with father loss have significantly shorter telomeres (14% reduction). Paternal death has the largest association (16%), followed by incarceration (10%), and separation and/or divorce (6%). Changes in income partially mediate these associations (95% mediation for separation and/or divorce, 30% for incarceration, and 25% for death). Effects are 40% greater for boys and 90% greater for children with the most reactive alleles of the serotonin transporter genes when compared with those with the least reactive alleles. No differences were found by age at father loss or a child's race/ethnicity.<h4>Conclusions</h4>Father loss has a significant association with children's sTL, with the death of a father showing the largest effect. Income loss explains most of the association between child sTL and separation and/or divorce but much less of the association with incarceration or death. This underscores the important role of fathers in the care and development of children and supplements evidence of the strong negative effects of parental incarceration.

Notch signalling is a causal determinant of cancer and efforts have been made to develop targeted therapies to inhibit the so-called canonical pathway. Here we describe an unexpected pro-apoptotic role of Notch3 in regulating tumour angiogenesis independently of the Notch canonical pathway. The Notch3 ligand Jagged-1 is upregulated in a fraction of human cancer and our data support the view that Jagged-1, produced by cancer cells, is inhibiting the apoptosis induced by the aberrant Notch3 expression in tumour vasculature. We thus present Notch3 as a dependence receptor inducing endothelial cell death while this pro-apoptotic activity is blocked by Jagged-1. Along this line, using Notch3 mutant mice, we demonstrate that tumour growth and angiogenesis are increased when Notch3 is silenced in the stroma. Consequently, we show that the well-documented anti-tumour effect mediated by γ-secretase inhibition is at least in part dependent on the apoptosis triggered by Notch3 in endothelial cells.

Thiazide diuretics, commonly used antihypertensives, may cause QT interval (QT) prolongation, a risk factor for highly fatal and difficult to predict ventricular arrhythmias. We examined whether common single-nucleotide polymorphisms (SNPs) modified the association between thiazide use and QT or its component parts (QRS interval, JT interval) by performing ancestry-specific, trans-ethnic and cross-phenotype genome-wide analyses of European (66%), African American (15%) and Hispanic (19%) populations (N=78 199), leveraging longitudinal data, incorporating corrected standard errors to account for underestimation of interaction estimate variances and evaluating evidence for pathway enrichment. Although no loci achieved genome-wide significance (P<5 × 10^-8), we found suggestive evidence (P<5 × 10^-6) for SNPs modifying the thiazide-QT association at 22 loci, including ion transport loci (for example, NELL1, KCNQ3). The biologic plausibility of our suggestive results and simulations demonstrating modest power to detect interaction effects at genome-wide significant levels indicate that larger studies and innovative statistical methods are warranted in future efforts evaluating thiazide-SNP interactions.

The abuse of anabolic androgenic steroids (AAS) has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG) axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH). In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM). These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE) and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

Tuberculosis (TB) is a deadly infectious disease, which kills millions of people every year. The causative pathogen, Mycobacterium tuberculosis (MTB), is estimated to have infected up to a third of the world's population; however, only approximately 10% of infected healthy individuals progress to active TB. Despite evidence for heritability, it is not currently possible to predict who may develop TB. To explore approaches to classify susceptibility to TB, we infected with MTB dendritic cells (DCs) from putatively resistant individuals diagnosed with latent TB, and from susceptible individuals that had recovered from active TB. We measured gene expression levels in infected and non-infected cells and found hundreds of differentially expressed genes between susceptible and resistant individuals in the non-infected cells. We further found that genetic polymorphisms nearby the differentially expressed genes between susceptible and resistant individuals are more likely to be associated with TB susceptibility in published GWAS data. Lastly, we trained a classifier based on the gene expression levels in the non-infected cells, and demonstrated reasonable performance on our data and an independent data set. Overall, our promising results from this small study suggest that training a classifier on a larger cohort may enable us to accurately predict TB susceptibility.

Cardiac damage associated with iron overload is the most common cause of morbidity and mortality in patients with hereditary hemochromatosis, but the precise mechanisms leading to disease progression are largely unexplored. Here we investigated the effects of iron overload and age on cardiac hypertrophy using 1-, 5- and 12-month old Hfe-deficient mice, an animal model of hemochromatosis in humans. Cardiac iron levels increased progressively with age, which was exacerbated in Hfe-deficient mice. The heart/body weight ratios were greater in Hfe-deficient mice at 5- and 12-month old, compared with their age-matched wild-type controls. Cardiac hypertrophy in 12-month old Hfe-deficient mice was consistent with decreased alpha myosin and increased beta myosin heavy chains, suggesting an alpha-to-beta conversion with age. This was accompanied by cardiac fibrosis and up-regulation of NFAT-c2, reflecting increased calcineurin/NFAT signaling in myocyte hypertrophy. Moreover, there was an age-dependent increase in the cardiac isoprostane levels in Hfe-deficient mice, indicating elevated oxidative stress. Also, rats fed high-iron diet demonstrated increased heart-to-body weight ratios, alpha myosin heavy chain and cardiac isoprostane levels, suggesting that iron overload promotes oxidative stress and cardiac hypertrophy. Our findings provide a molecular basis for the progression of age-dependent cardiac stress exacerbated by iron overload hemochromatosis.

Diabetes mellitus type 2 predisposes patients to various microvascular complications. In the current experiment, the potent role of diabetes mellitus was investigated on the content of VEGFR-1, -2, Tie-1 and -2, and Akt in human endothelial progenitor cells. The gene expression profile of mTOR and Hedgehog signaling pathways were measured by PCR array. The possible crosstalk between RTKs, mTOR and Hedgehog signaling was also studied by bioinformatic analysis. Endothelial progenitor cells were incubated with serum from normal and diabetic for 7days. Compared to non-treated cells, diabetic serum-induced cell apoptosis (~2-fold) and prohibited cell migration toward bFGF (p<0.001). ELISA analysis showed that diabetes exposed cells had increased abundance of Tie-1, -2 and VEGFR-2 and reduced amount of VEGFR-1 (p<0.0001) in diabetic cells. Western blotting showed a marked reduction in the protein level of Akt after cells exposure to serum from diabetic subjects (p<0.0001). PCR array revealed a significant stimulation of both mTOR and Hedgehog signaling pathways in diabetic cells (p<0.05). According to data from bioinformatic datasets, we showed VEGFR-1, -2 and Tie-2, but not Tie-1, are master regulators of angiogenesis. There is a crosstalk between RTKs and mTOR signaling by involving P62, GABARAPL1, and HTT genes. It seems that physical interaction and co-expression of Akt decreased the level of VEGFR-1 in diabetic cells. Regarding data from the present experiment, diabetic serum contributed to uncontrolled induction of both mTOR and Hedgehog signaling in endothelial progenitor cells. Diabetes mellitus induces mTOR pathway by involving receptor tyrosine kinases while Hedgehog stimulation is independent of these receptors.

Autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) is a very rare neurological disorder featured with late onset, slowly progressive central nervous system demyelination. Duplication or over expression of the lamin B1 (LMNB1) gene causes ADLD. In this study, we undertook a comprehensive clinical evaluation and genetic detection for a Chinese family with ADLD. The proband is a 52-year old man manifested with autonomic abnormalities, pyramidal tract dysfunction. MRI brain scan identified bilateral symmetric white matter (WM) hyper-intensities in periventricular and semi-oval WM, cerebral peduncles and middle cerebellar peduncles. The proband has a positive autosomal dominant family history with similar clinical manifestations with a trend of genetic anticipation. In order to understand the genetic cause of the disease in this family, target exome capture based next generation sequencing has been done, but no causative variants or possibly pathogenic variants has been identified. However, Multiplex ligand-dependent probe amplification (MLPA) showed whole duplication of <i>LMNB1</i> gene which is co-segregated with the disease phenotype in this family. This is the first genetically confirmed <i>LMNB1</i> associated ADLD pedigree from China.

The use of non-<i>Saccharomyces</i> yeast for wine making is becoming a common trend in many innovative wineries. The application is normally aimed at increasing aromas, glycerol, reducing acidity, and other improvements. This manuscript focuses on the reproduction of the native microbiota from the vineyard in the inoculum. Thus, native selected yeasts (<i>Hanseniaspora uvarum, Metschnikowia pulcherrima, Torulaspora delbrueckii, Starmerella bacillaris</i> species and three different strains of <i>Saccharomyces cerevisiae</i>) were inoculated sequentially, or only <i>S. cerevisiae</i> (three native strains together or one commercial) was used. Inoculations were performed both in laboratory conditions with synthetic must (400 mL) as well as in industrial conditions (2000 kg of grapes) in red winemaking in two different varieties, Grenache and Carignan. The results showed that all the inoculated <i>S. cerevisiae</i> strains were found at the end of the vinifications, and when non-<i>Saccharomyces</i> yeasts were inoculated, they were found in appreciable populations at mid-fermentation. The final wines produced could be clearly differentiated by sensory analysis and were of similar quality, in terms of sensory analysis panelists' appreciation.

The genetic factors associated with susceptibility or resistance to viral infections are likely to involve a sophisticated array of immune response. These genetic elements may modulate other biological factors that account for significant influence on the gene expression and/or protein function in the host. Among them, the role of the major histocompatibility complex in viral pathogenesis in particular human immunodeficiency virus (HIV) and hepatitis C virus (HCV), is very well documented. We, recently, added a novel insight into the field by identifying the molecular mechanism associated with the protective role of human leukocyte antigen (HLA)-B27/B57 CD8⁺ T cells in the context of HIV-1 infection and why these alleles act as a double-edged sword protecting against viral infections but predisposing the host to autoimmune diseases. The focus of this review will be reexamining the role of classical and non-classical HLA alleles, including class Ia (HLA-A, -B, -C), class Ib (HLA-E, -F, -G, -H), and class II (HLA-DR, -DQ, -DM, and -DP) in immune regulation and viral pathogenesis (e.g., HIV and HCV). To our knowledge, this is the very first review of its kind to comprehensively analyze the role of these molecules in immune regulation associated with chronic viral infections.

Cigarette smoking results in the accumulation of iron both systemically and locally, in the lung thereby causing imbalance in iron homeostasis. This disruption in iron homeostasis can be associated with oxidative stress and consequent tissue injury. Therefore, in this study, we tested the association between iron homeostasis and airway obstruction by examining a large cohort of smokers and non-smokers for relationships between 1) serum ferritin and iron concentrations and transferrin saturation and 2) forced vital capacity (FVC), forced expiratory volume in 1 s (FEV₁), and their ratio (FEV₁/FVC). Data from the National Health and Examination Survey III were analyzed. The study population included persons aged 20 years and above with their following data recorded: race, gender, serum ferritin and iron concentrations, and transferrin saturation; the final sample number was 7,251. In the total population, Pearson correlation coefficients between 1) serum ferritin and iron concentrations and transferrin saturation and 2) FVC and FEV₁ were significantly positive; whereas those between 1) serum ferritin concentrations and transferrin saturation and 2) FEV₁/FVC were significantly negative. With separate analyses, serum ferritin concentrations demonstrated positive associations with FVC and FEV₁ but an inverse relationship with FEV₁/FVC in smokers and non-smokers. Serum ferritin levels increased with worsening airway obstruction among smokers, and its highest concentrations were found among those with the lowest values of FEV₁/FVC ratio (<60%). Comparable to cigarette smokers, serum ferritin concentrations among non-smokers were greatest in those with the lowest FEV₁/FVC ratio. Furthermore, elevated levels of serum iron and saturation of transferrin also corresponded with decreased FEV₁/FVC ratio among non-smokers. Thus, we conclude that indices of iron homeostasis are associated with airway obstruction in both smokers and non-smokers.

This article reports the oral manifestations of an unusual presentation of Marfan syndrome (MFS) and provides an evidence to the importance of recognizing the oral features in confirming the diagnosis of MFS. Dentists have a vital role in confirming the diagnosis of developmental disorders that involve the craniofacial compendium.

Lung adenocarcinoma (LUAD) is the most common histological subtype of non-small cell lung cancer, but novel biomarkers for early diagnosis are lacking. Extensive effort has been exerted to identify miRNA biomarkers in LUAD. Unfortunately, high inter-lab variability and small sample sizes have produced inconsistent conclusions in this field. To resolve the above-mentioned limitations, we performed a comprehensive analysis based on LUAD miRNome profiling studies using the robust rank aggregation (RRA) method. Moreover, miRNA-gene interaction network, pathway enrichment analysis and Kaplan-Meier survival curves were used to investigate the clinical values and biological functions of the identified miRNAs. A total of six common differentially expressed miRNAs (DEMs) were identified in LUAD. An independent cohort further confirmed that four miRNAs (miR-21-5p, miR-210-3p, miR-182-5p and miR-183-5p) were up-regulated and two miRNAs (miR-126-3p and miR-218-5p) were down-regulated in LUAD tissues. Pathway enrichment analysis also suggested that the above-listed six DEMs may affect LUAD progression via the estrogen signaling pathway. Survival analysis based on the TCGA dataset revealed the potential prognostic values of six DEMs in patients with LUAD (<i>P</i>-value<0.01). In conclusion, we identified a panel of six miRNAs from LUAD using miRNome profiling studies. Our results provide evidence for the use of these six DEMs as novel diagnostic and prognostic biomarkers for LUAD patients.

<h4>Background</h4>Metastatic colorectal cancer (CRC) is associated with highly variable clinical outcome and response to therapy. The recently identified consensus molecular subtypes (CMS1-4) have prognostic and therapeutic implications in primary CRC, but whether these subtypes are valid for metastatic disease is unclear. We performed multi-level analyses of resectable CRC liver metastases (CLM) to identify molecular characteristics of metastatic disease and evaluate the clinical relevance.<h4>Methods</h4>In this ancillary study to the Oslo-CoMet trial, CLM and tumor-adjacent liver tissue from 46 patients were analyzed by profiling mutations (targeted sequencing), genome-wide copy number alteration (CNAs), and gene expression.<h4>Results</h4>Somatic mutations and CNAs detected in CLM were similar to reported primary CRC profiles, while CNA profiles of eight metastatic pairs suggested intra-patient divergence. A CMS classifier tool applied to gene expression data, revealed the cohort to be highly enriched for CMS2. Hierarchical clustering of genes with highly variable expression identified two subgroups separated by high or low expression of 55 genes with immune-related and metabolic functions. Importantly, induction of genes and pathways associated with immunogenic cell death (ICD) was identified in metastases exposed to neoadjuvant chemotherapy (NACT).<h4>Conclusions</h4>The uniform classification of CLM by CMS subtyping may indicate that novel class discovery approaches need to be explored to uncover clinically useful stratification of CLM. Detected gene expression signatures support the role of metabolism and chemotherapy in shaping the immune microenvironment of CLM. Furthermore, the results point to rational exploration of immune modulating strategies in CLM, particularly by exploiting NACT-induced ICD.

The present study was designed to explore the molecular mechanism at the early stage of hepatocarcinoma (HCC) and identify the candidate genes and pathways changed significantly. We downloaded the gene expression file dataset GSE6764 from GEO, adopted the Robust Multi-array Average (RMA) algorithm to preprocess the raw file. 797 differentially expressed genes (DEGs) were screened out based on the SAM method using R language. Ingenuity Pathway Analysis (IPA) was used to perform canonical pathway analysis in order to calculate the most significantly changed pathways and predict the upstream regulators. In order to confirm the results from the DEGs which based on the individual gene level, the gene set enrichment analysis (GSEA) was done from the gene set level and the leading edge analysis was performed to find out the most appeared genes in several gene sets. The PPI network was built using GeneMANIA and the key genes were calculated using cytoHubba plugin based on cytoscape 3.4.0. We found that the Cell Cycle: G2/M DNA damage checkpoint regulation is the top-ranked pathways at the early stage of HCC by IPA. The high expression of several genes including CCNB1, CDC25B, XPO1, GMPS, KPNA2 and MELK is correlated with high risk, poor prognosis and shorter overall survival time in HCC patients by use of Kaplan-Meier Survival analysis. Taken together, our study showed that the G2/M checkpoint plays a vital role at the early HCC and the genes participate in the process may serve as biomarkers for the diagnosis and prognosis.

Off-target cardiotoxicity has been a significant impediment to the development of new drugs. Traditional platforms for screening for cardiotoxicity are both overly sensitive and limited in their ability to predict cardiotoxicity that is often only uncovered after years of clinical use. A major impediment has been the lack of a human cardiomyocyte cell line. The recent discovery that adult somatic human cells (white blood cells or skin fibroblasts) can be reprogrammed into pluripotent stem cells (hiPSCs) and then differentiated into beating cardiomyocytes (hiPSC-CMs) provides an exciting new platform for drug cardiotoxicity and efficacy testing. One major advantage of using patient-derived hiPSC-CMs for drug testing is their ability to recapitulate population genetic variations (single nucleotide polymorphisms) that influence drug toxicity, providing a powerful new tool in the field of pharmacogenomics and personalized medicine.

Systemic lupus erythematosus (SLE) is an autoimmune disease with marked gender and ethnic disparities. We report a large transancestral association study of SLE using Immunochip genotype data from 27,574 individuals of European (EA), African (AA) and Hispanic Amerindian (HA) ancestry. We identify 58 distinct non-HLA regions in EA, 9 in AA and 16 in HA (∼50% of these regions have multiple independent associations); these include 24 novel SLE regions (P<5 × 10^-8), refined association signals in established regions, extended associations to additional ancestries, and a disentangled complex HLA multigenic effect. The risk allele count (genetic load) exhibits an accelerating pattern of SLE risk, leading us to posit a cumulative hit hypothesis for autoimmune disease. Comparing results across the three ancestries identifies both ancestry-dependent and ancestry-independent contributions to SLE risk. Our results are consistent with the unique and complex histories of the populations sampled, and collectively help clarify the genetic architecture and ethnic disparities in SLE.

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a 'piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (K_d=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

Personal data for 108 individuals were collected during a 9-month period, including whole genome sequences; clinical tests, metabolomes, proteomes, and microbiomes at three time points; and daily activity tracking. Using all of these data, we generated a correlation network that revealed communities of related analytes associated with physiology and disease. Connectivity within analyte communities enabled the identification of known and candidate biomarkers (e.g., gamma-glutamyltyrosine was densely interconnected with clinical analytes for cardiometabolic disease). We calculated polygenic scores from genome-wide association studies (GWAS) for 127 traits and diseases, and used these to discover molecular correlates of polygenic risk (e.g., genetic risk for inflammatory bowel disease was negatively correlated with plasma cystine). Finally, behavioral coaching informed by personal data helped participants to improve clinical biomarkers. Our results show that measurement of personal data clouds over time can improve our understanding of health and disease, including early transitions to disease states.

Endothelial cells and leptin receptor⁺ (LepR⁺) stromal cells are critical sources of haematopoietic stem cell (HSC) niche factors, including stem cell factor (SCF), in bone marrow. After irradiation or chemotherapy, these cells are depleted while adipocytes become abundant. We discovered that bone marrow adipocytes synthesize SCF. They arise from Adipoq-Cre/ER⁺ progenitors, which represent ∼5% of LepR⁺ cells, and proliferate after irradiation. Scf deletion using Adipoq-Cre/ER inhibited haematopoietic regeneration after irradiation or 5-fluorouracil treatment, depleting HSCs and reducing mouse survival. Scf from LepR⁺ cells, but not endothelial, haematopoietic or osteoblastic cells, also promoted regeneration. In non-irradiated mice, Scf deletion using Adipoq-Cre/ER did not affect HSC frequency in long bones, which have few adipocytes, but depleted HSCs in tail vertebrae, which have abundant adipocytes. A-ZIP/F1 'fatless' mice exhibited delayed haematopoietic regeneration in long bones but not in tail vertebrae, where adipocytes inhibited vascularization. Adipocytes are a niche component that promotes haematopoietic regeneration.

UK Biobank is among the world's largest repositories for phenotypic and genotypic information in individuals of European ancestry. We performed a genome-wide association study in UK Biobank testing ∼9 million DNA sequence variants for association with coronary artery disease (4,831 cases and 115,455 controls) and carried out meta-analysis with previously published results. We identified 15 new loci, bringing the total number of loci associated with coronary artery disease to 95 at the time of analysis. Phenome-wide association scanning showed that CCDC92 likely affects coronary artery disease through insulin resistance pathways, whereas experimental analysis suggests that ARHGEF26 influences the transendothelial migration of leukocytes.

Huntington's Disease (HD) is an autosomal dominant progressive neurodegenerative disorder characterized by cognitive, behavioral and motor dysfunctions. HD is caused by a CAG repeat expansion in exon 1 of the HD gene that is translated into an expanded polyglutamine tract in the encoded protein, huntingtin (HTT). While the most significant neuropathology of HD occurs in the striatum, other brain regions are also affected and play an important role in HD pathology. To date there is no cure for HD, and recently strategies aiming at silencing HTT expression have been initiated as possible therapeutics for HD. However, the essential functions of HTT in the adult brain are currently unknown and hence the consequence of sustained suppression of HTT expression is unpredictable and can potentially be deleterious. Using the Cre-loxP system of recombination, we conditionally inactivated the mouse HD gene homologue at 3, 6 and 9 months of age. Here we show that elimination of Htt expression in the adult mouse results in behavioral deficits, progressive neuropathological changes including bilateral thalamic calcification, and altered brain iron homeostasis.

Kaposi's sarcoma (KS) is a highly prevalent cancer in AIDS patients, especially in sub-Saharan Africa. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of KS and other cancers like Primary Effusion Lymphoma (PEL). In KS and PEL, all tumors harbor latent KSHV episomes and express latency-associated viral proteins and microRNAs (miRNAs). The exact molecular mechanisms by which latent KSHV drives tumorigenesis are not completely understood. Recent developments have highlighted the importance of aberrant long non-coding RNA (lncRNA) expression in cancer. Deregulation of lncRNAs by miRNAs is a newly described phenomenon. We hypothesized that KSHV-encoded miRNAs deregulate human lncRNAs to drive tumorigenesis. We performed lncRNA expression profiling of endothelial cells infected with wt and miRNA-deleted KSHV and identified 126 lncRNAs as putative viral miRNA targets. Here we show that KSHV deregulates host lncRNAs in both a miRNA-dependent fashion by direct interaction and in a miRNA-independent fashion through latency-associated proteins. Several lncRNAs that were previously implicated in cancer, including MEG3, ANRIL and UCA1, are deregulated by KSHV. Our results also demonstrate that KSHV-mediated UCA1 deregulation contributes to increased proliferation and migration of endothelial cells.

<h4>Background</h4>In addition to conventional chemotherapeutics, nucleic acid-based therapeutics like antisense oligodeoxynucleotides (AS-ODN) represent a novel approach for the treatment of bladder cancer (BCa). An efficient delivery of AS-ODN to the urothelium and then into cancer cells might be achieved by the local application of multi-walled carbon nanotubes (MWCNT). In the present study, pristine MWCNT and MWCNT functionalized with hydrophilic moieties were synthesized and then investigated regarding their physicochemical characteristics, dispersibility, biocompatibility, cellular uptake and mucoadhesive properties. Finally, their binding capacity for AS-ODN via hybridization to carrier strand oligodeoxynucleotides (CS-ODN), which were either non-covalently adsorbed or covalently bound to the different MWCNT types, was evaluated.<h4>Results</h4>Pristine MWCNT were successfully functionalized with hydrophilic moieties (MWCNT-OH, -COOH, -NH₂, -SH), which led to an improved dispersibility and an enhanced dispersion stability. A viability assay revealed that MWCNT-OH, MWCNT-NH₂ and MWCNT-SH were most biocompatible. All MWCNT were internalized by BCa cells, whereupon the highest uptake was observed for MWCNT-OH with 40% of the cells showing an engulfment. Furthermore, all types of MWCNT could adhere to the urothelium of explanted mouse bladders, but the amount of the covered urothelial area was with 2-7% rather low. As indicated by fluorescence measurements, it was possible to attach CS-ODN by adsorption and covalent binding to functionalized MWCNT. Adsorption of CS-ODN to pristine MWCNT, MWCNT-COOH and MWCNT-NH₂ as well as covalent coupling to MWCNT-NH₂ and MWCNT-SH resulted in the best binding capacity and stability. Subsequently, therapeutic AS-ODN could be hybridized to and reversibly released from the CS-ODN coupled via both strategies to the functionalized MWCNT. The release of AS-ODN at experimental conditions (80 °C, buffer) was most effective from CS-ODN adsorbed to MWCNT-OH and MWCNT-NH₂ as well as from CS-ODN covalently attached to MWCNT-COOH, MWCNT-NH₂ and MWCNT-SH. Furthermore, we could exemplarily demonstrate that AS-ODN could be released following hybridization to CS-ODN adsorbed to MWCNT-OH at physiological settings (37 °C, urine).<h4>Conclusions</h4>In conclusion, functionalized MWCNT might be used as nanotransporters in antisense therapy for the local treatment of BCa.

<h4>Objectives</h4>Smoking is associated with adverse health outcomes among drug users, including those in treatment. To date, however, there has been little evidence about smoking patterns among people receiving opioid-dependence treatment in developing countries. We examined self-reported nicotine dependence and associated factors in a large sample of opioid-dependent patients receiving methadone maintenance treatment (MMT) in northern Vietnam.<h4>Setting</h4>Five clinics in Hanoi (urban area) and Nam Dinh (rural area).<h4>Participants</h4>Patients receiving MMT in the settings during the study period.<h4>Primary and secondary outcome measures</h4>We collected data about smoking patterns, levels of nicotine dependence and other covariates such as socioeconomic status, health status, alcohol use and drug use. The Fagerström test was used to measure nicotine dependence (FTND). Logistic regression and Tobit regression were employed to examine relationships between the smoking rate, nicotine dependence and potentially associated variables.<h4>Results</h4>Among 1016 drug users undergoing MMT (98.7% male), 87.2% were current smokers. The mean FTND score was 4.5 (SD 2.4). Longer duration of MMT (OR 0.98, 95% CI 0.96 to 0.99) and being HIV-positive (OR 0.46, 95% CI 0.24 to 0.88) were associated with lower likelihood of smoking. Being employed, older age at first drug injection and having long duration of MMT were inversely related with FTND scores. Higher age and continuing drug and alcohol use were significantly associated with higher FTND scores.<h4>Conclusion</h4>Smoking prevalence is high among methadone maintenance drug users. Enhanced smoking cessation support should be integrated into MMT programmes in order to reduce risk factors for cigarette smoking and improve the health and well-being of people recovering from opiate dependence.

<h4>Introduction</h4>Chronic breathlessness is highly prevalent and distressing to patients and families. No medication is registered for its symptomatic reduction. The strongest evidence is for regular, low-dose, extended- release (ER) oral morphine. A recent large phase III study suggests the subgroup most likely to benefit have chronic obstructive pulmonary disease (COPD) and modified Medical Research Council breathlessness scores of 3 or 4. This protocol is for an adequately powered, parallel-arm, placebo-controlled, multisite, factorial, block-randomised study evaluating regular ER morphine for chronic breathlessness in people with COPD.<h4>Methods and analysis</h4>The primary question is what effect regular ER morphine has on worst breathlessness, measured daily on a 0-10 numerical rating scale. Uniquely, the coprimary outcome will use a FitBit to measure habitual physical activity. Secondary questions include safety and, whether upward titration after initial benefit delivers greater net symptom reduction. Substudies include longitudinal driving simulation, sleep, caregiver, health economic and pharmacogenetic studies. Seventeen centres will recruit 171 participants from respiratory and palliative care. The study has five phases including three randomisation phases to increasing doses of ER morphine. All participants will receive placebo or active laxatives as appropriate. Appropriate statistical analysis of primary and secondary outcomes will be used.<h4>Ethics and dissemination</h4>Ethics approval has been obtained. Results of the study will be submitted for publication in peer-reviewed journals, findings presented at relevant conferences and potentially used to inform registration of ER morphine for chronic breathlessness.<h4>Trial registration number</h4>NCT02720822; Pre-results.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily that act as ligand-dependent transcription factors. Here we identified the ten-eleven translocation protein 3 (TET3) as a TR interacting protein increasing cell sensitivity to T3. The interaction between TET3 and TRs is independent of TET3 catalytic activity and specifically allows the stabilization of TRs on chromatin. We provide evidence that TET3 is required for TR stability, efficient binding of target genes, and transcriptional activation. Interestingly, the differential ability of different TRα1 mutants to interact with TET3 might explain their differential dominant activity in patients carrying TR germline mutations. So this study evidences a mode of action for TET3 as a nonclassical coregulator of TRs, modulating its stability and access to chromatin, rather than its intrinsic transcriptional activity. This regulatory function might be more general toward nuclear receptors. Indeed, TET3 interacts with different members of the superfamily and also enhances their association to chromatin.

Toll-like receptors (TLRs) are innate immune receptors for sensing microbial molecules and damage-associated molecular patterns released from host cells. Double-stranded RNA and the synthetic analog polyinosinic:polycytidylic acid (poly(I:C)) bind and activate TLR3. This stimulation leads to recruitment of the adaptor molecule TRIF (Toll/IL-1 resistance (TIR) domain-containing adapter-inducing interferon β) and activation of the transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3), classically inducing IFNβ production. Here we report that, unlike non-metastatic intestinal epithelial cells (IECs), metastatic IECs express TLR3 and that TLR3 promotes invasiveness of these cells. In response to poly(I:C) addition, the metastatic IECs also induced the chemokine CXCL10 in a TLR3-, TRIF-, and IRF3-dependent manner but failed to produce IFNβ. This was in contrast to healthy and non-metastatic IECs, which did not respond to poly(I:C) stimulation. Endolysosomal acidification and the endosomal transporter protein UNC93B1 was required for poly(I:C)-induced CXCL10 production. However, TLR3-induced CXCL10 was triggered by immobilized poly(I:C), was only modestly affected by inhibition of endocytosis, and could be blocked with an anti-TLR3 antibody, indicating that TLR3 can still signal from the cell surface of these cells. Furthermore, plasma membrane fractions from metastatic IECs contained both full-length and cleaved TLR3, demonstrating surface expression of both forms of TLR3. Our results imply that metastatic IECs express surface TLR3, allowing it to sense extracellular stimuli that trigger chemokine responses and promote invasiveness in these cells. We conclude that altered TLR3 expression and localization may have implications for cancer progression.

The z-disc is a structural component at the lateral borders of the sarcomere and is important for mechanical stability and contractility of both cardiac and skeletal muscles. Of note, the sarcomeric z-disc also represents a nodal point in cardiomyocyte function and signaling. Mutations of numerous z-disc proteins are associated with cardiomyopathies and muscle diseases. To identify additional z-disc proteins that might contribute to cardiac disease, we employed an <i>in silico</i> screen for cardiac-enriched cDNAs. This screen yielded a previously uncharacterized protein named <u>c</u>ardiac-<u>e</u>nriched <u>F</u>HL2-<u>i</u>nteracting <u>p</u>rotein (CEFIP), which exhibited a heart- and skeletal muscle-specific expression profile. Importantly, CEFIP was located at the z-disc and was up-regulated in several models of cardiomyopathy. We also found that CEFIP overexpression induced the fetal gene program and cardiomyocyte hypertrophy. Yeast two-hybrid screens revealed that CEFIP interacts with the calcineurin-binding protein four and a half LIM domains 2 (FHL2). Because FHL2 binds calcineurin, a phosphatase controlling hypertrophic signaling, we examined the effects of CEFIP on the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. These experiments revealed that CEFIP overexpression further enhances calcineurin-dependent hypertrophic signal transduction, and its knockdown repressed hypertrophy and calcineurin/NFAT activity. In summary, we report on a previously uncharacterized protein CEFIP that modulates calcineurin/NFAT signaling in cardiomyocytes, a finding with possible implications for the pathogenesis of cardiomyopathy.

Deciphering gene regulatory networks requires identification of gene expression modules. We describe a novel bottom-up approach to identify gene modules regulated by cis-regulatory motifs from a human gene co-expression network. Target genes of a cis-regulatory motif were identified from the network via the motif's enrichment or biased distribution towards transcription start sites in the promoters of co-expressed genes. A gene sub-network containing the target genes was extracted and used to derive gene modules. The analysis revealed known and novel gene modules regulated by the NF-Y motif. The binding of NF-Y proteins to these modules' gene promoters were verified using ENCODE ChIP-Seq data. The analyses also identified 8,048 Sp1 motif target genes, interestingly many of which were not detected by ENCODE ChIP-Seq. These target genes assemble into house-keeping, tissues-specific developmental, and immune response modules. Integration of Sp1 modules with genomic and epigenomic data indicates epigenetic control of Sp1 targets' expression in a cell/tissue specific manner. Finally, known and novel target genes and modules regulated by the YY1, RFX1, IRF1, and 34 other motifs were also identified. The study described here provides a valuable resource to understand transcriptional regulation of various human developmental, disease, or immunity pathways.

HOXA11 antisense RNA (HOXA11-AS) has been shown to be involved in tumorigenesis and development of different cancers. However, the role of HOXA11-AS in non-small cell lung cancer (NSCLC) remains unclear. In this study, we firstly explored and confirmed the expression of HOXA11-AS in NSCLC tissues and cells. Cytometry, CCK-8, cell scratch, migration, Matrigel invasion and flow cytometry assays were performed to determine the biological impact of HOXA11-AS in vitro. Furthermore, a chick embryo chorioallantoic membrane (CAM) model of NSCLC was constructed to explore the effect of HOXA11-AS on tumorigenicity and angiogenesis in vivo. Additionally, bioinformatics analyses were performed to investigate the prospective pathways of HOXA11-AS co-expressed genes. As results, HOXA11-AS was markedly highly expressed in NSCLC tissues and cells. Furthermore, the proliferation, migration, invasion, tumorigenic and angiogenic ability of NSCLC cells were all inhibited and apoptosis was induced after HOXA11-AS knock-down. HOXA11-AS RNAi also led to cell cycle arrest on G0/G1 or G2/M phase. In addition, the non-small cell lung cancer pathway might be involved in regulating the co-expressed genes of HOXA11-AS in NSCLC. These results indicate that HOXA11-AS plays pivotal roles in NSCLC and it can become a novel therapeutic direction for treating NSCLC.

The R5 subfamily of receptor-type protein tyrosine phosphatases (RPTPs) comprises PTPRZ and PTPRG. A recent study on primary human glioblastomas suggested a close association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles of PTPRZ activity in glioma stem cells have remained unclear. In the present study, we found that sphere-forming cells from the rat C6 and human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B, the short receptor isoform of PTPRZ. Stable PTPRZ knockdown altered the expression levels of stem cell transcription factors such as SOX2, OLIG2, and POU3F2 and decreased the sphere-forming abilities of these cells. Suppressive effects on the cancer stem-like properties of the cells were also observed following the knockdown of PTPRG. Here, we identified NAZ2329, a cell-permeable small molecule that allosterically inhibits both PTPRZ and PTPRG. NAZ2329 reduced the expression of SOX2 in C6 and U251 cells and abrogated the sphere-forming abilities of these cells. Tumor growth in the C6 xenograft mouse model was significantly slower with the co-treatment of NAZ2329 with temozolomide, an alkylating agent, than with the individual treatments. These results indicate that pharmacological inhibition of R5 RPTPs is a promising strategy for the treatment of malignant gliomas.

Condensin complexes play crucial roles in chromosome condensation that is a fundamental process to establish the "rod-like" shape of chromosome structure in mitosis. Failure of the chromosome assembly causes chromosome segregation errors and subsequent genomic instability. However, a molecular mechanism that controls condensin function for the chromosomal organization has not been fully understood. Here, we show that the abundance of CAP-H2, one of the condensin II subunits, is fluctuated during the cell cycle in accordance with Plk1 kinase activity. Inhibition of Plk1 leads to Cdc20-mediated degradation of CAP-H2 in mitosis. Plk1 phosphorylation of CAP-H2 at Ser288 is required for the accumulation of CAP-H2 and accurate chromosomal condensation during prophase. These findings suggest that Plk1 phosphorylation regulates condensin II function by modulating CAP-H2 expression levels to facilitate proper mitotic chromosome organization.

<h4>Background</h4>Survival and longevity of xenotransplants depend on immune function and ability to integrate energy metabolism between cells from different species. However, mechanisms for interspecies cross talk in energy metabolism are not well understood. White adipose tissue stores energy and is capable of mobilization and dissipation of energy as heat (thermogenesis) by adipocytes expressing uncoupling protein 1 (Ucp1). Both pathways are under the control of vitamin A metabolizing enzymes. Deficient retinoic acid production in aldehyde dehydrogenase 1 A1 (Aldh1a1) knockout adipocytes (KO) inhibits adipogenesis and increases thermogenesis. Here we test the role Aldh1a1 in regulation of lipid metabolism in xenocultures.<h4>Methods</h4>Murine wide-type (WT) and KO pre-adipocytes were encapsulated into a poly-L-lysine polymer that allows exchange of humoral factors <32kD via nanopores. Encapsulated murine adipocytes were co-incubated with primary differentiated canine adipocytes. Then, expression of adipogenic and thermogenic genes in differentiated canine adipocytes was detected by real-time polymerase chain reaction (PCR). The regulatory factors in WT and KO cells were identified by comparison of secretome using proteomics and in transcriptome by gene microarray.<h4>Results</h4>Co-culture of encapsulated mouse KO vs WT adipocytes increased expression of peroxisome proliferator-activated receptor gamma (Pparg), but reduced expression of its target genes fatty acid binding protein 4 (Fabp4), and adipose triglyceride lipase (Atgl) in canine adipocytes, suggesting inhibition of PPARγ activation. Co-culture with KO adipocytes also induced expression of Ucp1 in canine adipocytes compared to expression in WT adipocytes. Cumulatively, murine KO compared to WT adipocytes decreased lipid accumulation in canine adipocytes. Comparative proteomics revealed significantly higher levels of vitamin A carriers, retinol binding protein 4 (RBP4), and lipokalin 2 (LCN2) in KO vs WT adipocytes.<h4>Conclusions</h4>Our data demonstrate the functional exchange of regulatory factors between adipocytes from different species for regulation of energy balance. RBP4 and LCN2 appear to be involved in the transport of retinoids for regulation of lipid accumulation and thermogenesis in xenocultures. While the rarity of thermogenic adipocytes in humans and dogs precludes their use for autologous transplantation, our study demonstrates that xenotransplantation of engineered cells could be a potential solution for the reduction in obesity in dogs and a strategy for translation to patients.

In the brain, the Homer protein family modulates excitatory signal transduction and receptor plasticity through interactions with other proteins in dendritic spines. Homer proteins are implicated in a variety of psychiatric disorders such as schizophrenia and addiction. Since long Homers serve as scaffolding proteins, identifying their interacting partners is an important first step in understanding their biological function and could help to guide the design of new therapeutic strategies. The present study set out to document Homer2-interacting proteins in the mouse brain using a co-immunoprecipitation-based mass spectrometry approach where Homer2 knockout samples were used to filter out non-specific interactors. We found that in the mouse brain, Homer2 interacts with a limited subset of its previously reported interacting partners (3 out of 31). Importantly, we detected an additional 15 novel Homer2-interacting proteins, most of which are part of the N-methyl-D-aspartate receptor signaling pathway. These results corroborate the central role Homer2 plays in glutamatergic transmission and expand the network of proteins potentially contributing to the behavioral abnormalities associated with altered Homer2 expression.<h4>Significance</h4>Long Homer proteins are scaffolding proteins that regulate signal transduction in neurons. Identifying their interacting partners is key to understanding their function. We used co-immunoprecipitation in combination with mass spectrometry to establish the first comprehensive list of Homer2-interacting partners in the mouse brain. The specificity of interactions was evaluated using Homer2 knockout brain tissue as a negative control. The set of proteins that we identified minimally overlaps with previously reported interacting partners of Homer2; however, we identified novel interactors that are part of a signaling cascade activated by glutamatergic transmission, which improves our mechanistic understanding of the role of Homer2 in behavior.

Gastric cancer (GC) is a complex disease with heterogeneous genetic mechanisms. Genomic mutational profiling of gastric cancer not only expands our knowledge about cancer progression at a fundamental genetic level, but also could provide guidance on new treatment decisions, currently based on tumor histology. The fact that precise medicine-based treatment is successful in a subset of tumors indicates the need for better identification of clinically related molecular tumor phenotypes, especially with regard to those driver mutations on tumor suppressor genes (TSGs) and oncogenes (ONGs). We surveyed 313 TSGs and 160 ONGs associated with 48 protein coding and 19 miRNA genes with both TSG and ONG roles. Using public cancer mutational profiles, we confirmed the dual roles of <i>CDKN1A</i> and <i>CDKN1B</i>. In addition to the widely recognized alterations, we identified another 82 frequently mutated genes in public gastric cancer cohort. In summary, these driver mutation profiles of individual GC will form the basis of personalized treatment of gastric cancer, leading to substantial therapeutic improvements.

A straightforward enantiomerically pure synthesis of the heterocyclic core of the D-series GE2270 is reported. The synthetic strategy combines the Hantzsch thiazole's building condensation with a cross-coupling reaction including direct C-H hetarylation to build and connect step-by-step thiazolyl moieties to the 5-bromopicolinate as readily available starting material.

Complex DNA sequences are difficult to detect and profile, but are important contributors to human health and disease. Existing hybridization probes lack the capability to selectively bind and enrich hypervariable, long or repetitive sequences. Here, we present a generalized strategy for constructing modular hybridization probes (M-Probes) that overcomes these challenges. We demonstrate that M-Probes can tolerate sequence variations of up to 7 nt at prescribed positions while maintaining single nucleotide sensitivity at other positions. M-Probes are also shown to be capable of sequence-selectively binding a continuous DNA sequence of more than 500 nt. Furthermore, we show that M-Probes can detect genes with triplet repeats exceeding a programmed threshold. As a demonstration of this technology, we have developed a hybrid capture method to determine the exact triplet repeat expansion number in the Huntington's gene of genomic DNA using quantitative PCR.

No abstract available.

Sepsis is an inflammatory response to pathogens (such as Gram‑positive and Gram‑negative bacteria), which has high morbidity and mortality in critically ill patients. The present study aimed to identify the key genes in Gram‑positive and Gram‑negative sepsis. GSE6535 was downloaded from Gene Expression Omnibus, containing 17 control samples, 18 Gram‑positive samples and 25 Gram‑negative samples. Subsequently, the limma package in R was used to screen the differentially expressed genes (DEGs). Hierarchical clustering was conducted for the specific DEGs in Gram‑negative and Gram‑negative samples using cluster software and the TreeView software. To analyze the correlation of samples at the gene level, a similarity network was constructed using Cytoscape software. Functional and pathway enrichment analyses were conducted for the DEGs using DAVID. Finally, stochastic perturbation was used to determine the significantly differential functions between Gram‑positive and Gram‑negative samples. A total of 340 and 485 DEGs were obtained in Gram‑positive and Gram‑negative samples, respectively. Hierarchical clustering revealed that there were significant differences between control and sepsis samples. In Gram‑positive and Gram‑negative samples, myeloid cell leukemia sequence 1 was associated with apoptosis and programmed cell death. Additionally, NADH:ubiquinone oxidoreductase subunit S4 was associated with mitochondrial respiratory chain complex I assembly. Stochastic perturbation analysis revealed that NADH:ubiquinone oxidoreductase subunit B2 (NDUFB2), NDUFB8 and ubiquinol‑cytochrome c reductase hinge protein (UQCRH) were associated with cellular respiration in Gram‑negative samples, whereas large tumor suppressor kinase 2 (LATS2) was associated with G1/S transition of the mitotic cell cycle in Gram‑positive samples. NDUFB2, NDUFB8 and UQCRH may be biomarkers for Gram‑negative sepsis, whereas LATS2 may be a biomarker for Gram‑positive sepsis. These findings may promote the therapies of sepsis caused by Gram‑positive and Gram‑negative bacteria.

No abstract available.

Resveratrol, SIRT1 activator, inhibits carcinogenesis predominantly performed in transgenic animal models, orthotopic cancers of nude mice or different cancer cell lines, but its effects during process of spontaneous tumors using vertebrate models remain untested. Spontaneous liver neoplasm is an age-related disease and is inhibited by resveratrol in the annual fish <i>Nothobranchius guentheri</i>, which indicates that the fish can act as an excellent model to study spontaneous tumorigenesis. Totally, 175 fish were fed with resveratrol and another 175 fish for controls. Treated fish were fed with resveratrol (25 μg/fish/day) from sexual maturity (4-month-old) until they were sacrificed at 6-, 9- and 12-month-old. Immunoblot, immunohistochemistry and co-immunoprecipitation were employed to investigate the underlying mechanisms that resveratrol inhibited age-dependent spontaneous tumorigenesis in the fish. Results showed that resveratrol increased protein level of SIRT1 and alleviated age-associated tumorigenesis in liver. With SIRT1 up-regulation, resveratrol reduced proliferation by deacetylating K-Ras and inactivating K-Ras/PI3K/AKT pathway; and promoted apoptosis through deacetylation and dephosphorylation of FoxOs, up-regulation of DLC1 and interaction between SIRT1 and DLC1, and dephosphorylation of DLC1 in spontaneous neoplasms. We established a novel short-lived fish model for understanding the molecular mechanisms of drugs on age-dependent spontaneous tumorigenesis.

Numerous genetic studies have been conducted regarding the occurrence of colorectal cancer (CRC) and the prognosis using microarrays. However, adequate investigations into the diagnostic application of microarrays have yet to be performed. The simplicity and accuracy of diagnosis and prognosis tracking are important requirements for its processes, and the use of blood cells for diagnosis is considered to be suitable to meet these requirements. The patients involved in the study were 28 preoperative patients with CRC and 6 healthy individuals who served as controls. RNA was extracted from the blood cells of the patients and analyzed using a sense/antisense RNA custom microarray. In the patients with CRC, the expression levels of 20 sense RNA and 20 antisense RNA species were identified as being significantly altered compared with that of the healthy volunteers (P<0.05; fold-change, >2.0). Cluster analysis of these RNA species revealed that the top 10 antisense RNAs significantly clustered patients with cancer and healthy individuals separately. Patients with stage I or II CRC exhibited significant changes in the expression levels of 33 sense and 39 antisense RNA species, as compared with healthy volunteers (P<0.01; fold-change >2.0). Cluster analysis demonstrated that patients with stage I or II CRC and healthy volunteers formed separate clusters only among the top 20 antisense RNA species. A tracking study of expression levels of haloacid dehalogenase-like hydrolase domain-containing 1 (HDHD1) antisense RNA was performed and a significant difference was identified between the CRC and healthy groups revealing that the levels at one week and three months following surgical removal of the cancerous tissue, decreased to almost same levels of the healthy individuals. The results of the current study indicate that HDHD1 antisense RNA may serve as a potential biomarker for the prognosis of CRC.

Ciita was discovered for its role in regulating transcription of major histocompatibility complex class II (MHCII) genes. Subsequently, CIITA was predicted to control many other genes based on reporter and ChIP-seq analysis but few such predictions have been verified in vivo using Ciita^-/- mice. Testing these predictions for classical dendritic cells (cDCs) has been particularly difficult, since Ciita^-/- mice lack MHCII expression required to identify cDCs. However, recent identification of the cDC-specific transcription factor Zbtb46 allows the identification of cDCs independently of MHCII expression. We crossed Zbtb46^gfp mice onto the Ciita^-/- background and found that all cDC lineages developed in vivo in the absence of Ciita. We then compared the complete transcriptional profile of wild-type and Ciita^-/- cDCs to define the physiological footprint of CIITA for both immature and activated cDCs. We find that CIITA exerts a highly restricted control over only the MHCII, H2-DO and H2-DM genes, in DC1 and DC2 cDC subsets, but not over other proposed targets, including Ii. These findings emphasize the caveats needed in interpreting transcription factor binding sites identified by in-vitro reporter analysis, or by ChIP-seq, which may not necessarily indicate their functional activity in vivo.

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in myriad dominant and recessive disease phenotypes. Glycyl-tRNA synthetase (GARS) is a bifunctional ARS that charges tRNA^Gly in the cytoplasm and mitochondria. GARS variants have been associated with dominant Charcot-Marie-Tooth disease but have not been convincingly implicated in recessive phenotypes. Here, we describe a patient from the NIH Undiagnosed Diseases Program with a multisystem, developmental phenotype. Whole-exome sequence analysis revealed that the patient is compound heterozygous for one frameshift (p.Glu83Ilefs*6) and one missense (p.Arg310Gln) GARS variant. Using in vitro and in vivo functional studies, we show that both GARS variants cause a loss-of-function effect: the frameshift variant results in depleted protein levels and the missense variant reduces GARS tRNA charging activity. In support of GARS variant pathogenicity, our patient shows striking phenotypic overlap with other patients having ARS-related recessive diseases, including features associated with variants in both cytoplasmic and mitochondrial ARSs; this observation is consistent with the essential function of GARS in both cellular locations. In summary, our clinical, genetic, and functional analyses expand the phenotypic spectrum associated with GARS variants.

<h4>Background</h4>Staufen2 (STAU2) is an RNA-binding protein involved in the post-transcriptional regulation of gene expression. This protein was shown to be required for organ formation and cell differentiation. Although STAU2 functions have been reported in neuronal cells, its role in dividing cells remains deeply uncharacterized. Especially, its regulation during the cell cycle is completely unknown.<h4>Results</h4>In this study, we showed that STAU2 isoforms display a mitosis-specific slow migration pattern on SDS-gels in all tested transformed and untransformed cell lines. Deeper analyses in hTert-RPE1 and HeLa cells further indicated that the slow migration pattern of STAU2 isoforms is due to phosphorylation. Time course studies showed that STAU2 phosphorylation occurs before prometaphase and terminates as cells exit mitosis. Interestingly, STAU2 isoforms were phosphorylated on several amino acid residues in the C-terminal half via the cyclin-dependent kinase 1 (Cdk1), an enzyme known to play crucial roles during mitosis. Introduction of phospho-mimetic or phospho-null mutations in STAU2 did not impair its RNA-binding capacity, its stability, its interaction with protein co-factors or its sub-cellular localization, suggesting that STAU2 phosphorylation in mitosis does not regulate these functions. Similarly, STAU2 phosphorylation is not likely to be crucial for cell cycle progression since expression of phosphorylation mutants in hTert-RPE1 cells did not impair cell proliferation.<h4>Conclusions</h4>Altogether, these results indicate that STAU2 isoforms are phosphorylated during mitosis and that the phosphorylation process involves Cdk1. The meaning of this post-translational modification is still elusive.

MicroRNAs (miRNAs) are crucial regulators of gene expression in tumorigenesis and are of great interest to researchers, but miRNA profiles are often inconsistent between studies. The aim of this study was to confirm candidate miRNA biomarkers for esophageal cancer from integrated-miRNA expression profiling data and TCGA (The Cancer Genome Atlas) data in tissues. Here, we identify five significant miRNAs by a comprehensive analysis in esophageal cancer, and two of them (hsa-miR-100-5p and hsa-miR-133b) show better prognoses with significant difference for both 3-year and 5-year survival. Additionally, they participate in esophageal cancer occurrence and development according to KEGG and Panther enrichment analyses. Therefore, these five miRNAs may serve as miRNA biomarkers in esophageal cancer. Analysis of differential expression for target genes of these miRNAs may also provide new therapeutic alternatives in esophageal cancer.

<b>Purpose:</b> Carcinoembryonic antigen (CEA) is a candidate target for cellular immunotherapy of pancreatic cancer. In this study, we have characterized the antigen-specific function of autologous cytotoxic T lymphocytes (CTL) specific for the HLA-A2-restricted peptide, pCEA691-699, isolated from the peripheral T-cell repertoire of pancreatic cancer patients and sought to determine if <i>ex vivo</i> PD-L1 and TIM-3 blockade could enhance CTL function.<b>Experimental Design:</b> CD8⁺ T-cell lines were generated from peripheral blood mononuclear cells of 18 HLA-A2⁺ patients with pancreatic cancer and from 15 healthy controls. <i>In vitro</i> peptide-specific responses were evaluated by flow cytometry after staining for intracellular cytokine production and carboxy fluorescein succinimydyl ester cytotoxicity assays using pancreatic cancer cell lines as targets.<b>Results:</b> Cytokine-secreting functional CEA691-specific CTL lines were successfully generated from 10 of 18 pancreatic cancer patients, with two CTL lines able to recognize and kill both CEA691 peptide-loaded T2 cells and CEA⁺ HLA-A2⁺ pancreatic cancer cell lines. In the presence of <i>ex vivo</i> PD-L1 blockade, functional CEA691-specific CD8⁺ T-cell responses, including IFNγ secretion and proliferation, were enhanced, and this effect was more pronounced on Ag-specific T cells isolated from tumor draining lymph nodes.<b>Conclusions:</b> These data demonstrate that CEA691-specific CTL can be readily expanded from the self-restricted T-cell repertoire of pancreatic cancer patients and that their function can be enhanced by PD-L1 blockade. <i>Clin Cancer Res; 23(20); 6178-89. ©2017 AACR</i>.

<b>Purpose:</b> Sarcomatoid renal cell carcinoma (SRCC) ranks among the most aggressive clinicopathologic phenotypes of RCC. However, the paucity of high-quality, genome-wide molecular examinations of SRCC has hindered our understanding of this entity.<b>Experimental Design:</b> We interrogated the mutational, copy number, and transcriptional characteristics of SRCC and compared these data with those of nonsarcomatoid RCC (RCC). We evaluated whole-exome sequencing, single-nucleotide polymorphism, and RNA sequencing data from patients with SRCC (<i>n</i> = 65) and RCC (<i>n</i> = 598) across different parent RCC subtypes, including clear-cell RCC, papillary RCC, and chromophobe RCC subtypes.<b>Results:</b> SRCC was molecularly discrete from RCC and clustered according to its parent RCC subtype, though with upregulation of TGFβ signaling across all subtypes. The epithelioid (E-) and spindled (S-) histologic components of SRCC did not show differences in mutational load among cancer-related genes despite a higher mutational burden in S-. Notably, sarcomatoid clear-cell RCC (SccRCC) showed significantly fewer deletions at 3p21-25, a lower rate of two-hit loss for <i>VHL</i> and <i>PBRM1</i>, and more mutations in <i>PTEN</i>, <i>TP53</i>, and <i>RELN</i> compared with ccRCC. A two-hit loss involving <i>VHL</i> predicted for ccRCC and a better prognosis, whereas mutations in <i>PTEN</i>, <i>TP53</i>, or <i>RELN</i> predicted for SccRCC and worse prognosis.<b>Conclusions:</b> SRCC segregates by parent subtype, and SccRCC has a fundamentally different early molecular pathogenesis, usually lacking the classic 3p21-25 deletion and showing distinctive mutational and transcriptional profiles. These features prompt a more precise molecular classification of RCC, with diagnostic, prognostic, and therapeutic implications. <i>Clin Cancer Res; 23(21); 6686-96. ©2017 AACR</i><i>See related commentary by Bergerot et al., p. 6381</i>.

Gliomas, a common type of brain tumor, are characterized by aggressive infiltration, making it difficultly to cure by surgery. Netrin-1, an extracellular guidance cue critical for neuronal axon path-finding, has been reported to play an important role in cell invasion and migration in several types of cancers. However, the role of netrin-1 in glioma remains largely unknown. Here, we provide evidence suggested that Netrin-1 has a critical role in glioma growth. We found that netrin-1 was significantly increased in glioma samples and positively correlated with cell proliferation, tumor grade and malignancy. Netrin-1 knockdown reduced cell proliferation and attenuated tumor growth in a xenograft mouse model. Further studies found that netrin-1 induced NF-κB p65^ser536 phosphorylation and c-Myc expression in vitro and in vivo. Interestingly, activation of NF-κB by netrin-1 was dependent on UNC5A receptor, because suppression of UNC5A significantly inhibited NF-κB p65^ser536 phosphorylation, c-Myc up-regulation and reduced cell proliferation. Taken together, these results suggested netrin-1 promotes glioma cell proliferation by activating NF-κB signaling via UNC5A, netrin-1 may be a potential therapeutic target for the treatment of glioma.

<h4>Background</h4>Phase 1 studies have shown potential benefit of gene replacement in RPE65-mediated inherited retinal dystrophy. This phase 3 study assessed the efficacy and safety of voretigene neparvovec in participants whose inherited retinal dystrophy would otherwise progress to complete blindness.<h4>Methods</h4>In this open-label, randomised, controlled phase 3 trial done at two sites in the USA, individuals aged 3 years or older with, in each eye, best corrected visual acuity of 20/60 or worse, or visual field less than 20 degrees in any meridian, or both, with confirmed genetic diagnosis of biallelic RPE65 mutations, sufficient viable retina, and ability to perform standardised multi-luminance mobility testing (MLMT) within the luminance range evaluated, were eligible. Participants were randomly assigned (2:1) to intervention or control using a permuted block design, stratified by age (<10 years and ≥10 years) and baseline mobility testing passing level (pass at ≥125 lux vs <125 lux). Graders assessing primary outcome were masked to treatment group. Intervention was bilateral, subretinal injection of 1·5 × 10¹¹ vector genomes of voretigene neparvovec in 0·3 mL total volume. The primary efficacy endpoint was 1-year change in MLMT performance, measuring functional vision at specified light levels. The intention-to-treat (ITT) and modified ITT populations were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, number NCT00999609, and enrolment is complete.<h4>Findings</h4>Between Nov 15, 2012, and Nov 21, 2013, 31 individuals were enrolled and randomly assigned to intervention (n=21) or control (n=10). One participant from each group withdrew after consent, before intervention, leaving an mITT population of 20 intervention and nine control participants. At 1 year, mean bilateral MLMT change score was 1·8 (SD 1·1) light levels in the intervention group versus 0·2 (1·0) in the control group (difference of 1·6, 95% CI 0·72-2·41, p=0·0013). 13 (65%) of 20 intervention participants, but no control participants, passed MLMT at the lowest luminance level tested (1 lux), demonstrating maximum possible improvement. No product-related serious adverse events or deleterious immune responses occurred. Two intervention participants, one with a pre-existing complex seizure disorder and another who experienced oral surgery complications, had serious adverse events unrelated to study participation. Most ocular events were mild in severity.<h4>Interpretation</h4>Voretigene neparvovec gene replacement improved functional vision in RPE65-mediated inherited retinal dystrophy previously medically untreatable.<h4>Funding</h4>Spark Therapeutics.

Annonaceous acetogenins (ACGs) are a large family of fatty acid derived natural products that are exclusively isolated from the <i>Annonaceae</i> species. Many members of this diverse family have a broad spectrum of biological activities, the most impressive of which is anticancer activity. However, their poor solubility and severe toxicity restrict their clinical application, and their complicated composition hinders their formulation and drug delivery. In this study, β-cyclodextrin was modified with folic acid (FA) and then combined with soybean lecithin to prepare FA-modified ACGs nanosuspensions (FA-ACGs-NSps). The obtained FA-ACGs-NSps had a high drug payload of 57.59% and average particle size of 199.5 nm, and they exhibited sustained drug release within 142 hours. In comparison with ACGs-NSps, FA-ACGs-NSps showed significantly enhanced cytotoxicity and higher cell uptake toward folate receptor-positive 4T1 cell lines. An in vivo study demonstrated that FA-ACGs-NSps more effectively accumulated in tumors and enhanced the antitumor therapeutic efficacy with less toxicity in 4T1 tumor bearing mice. Therefore, FA-ACGs-NSps may be a promising drug delivery system for ACGs to improve their therapeutic window and may be suitable for clinical application to treat folate-positive tumors.

The cross-linked pentapeptides (2<i>R</i>,7<i>R</i>)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2<i>R</i>,7<i>R</i>)-BVD-74D, (2<i>R</i>,7<i>R</i>)-<b>1</b>) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) (<b>2</b>) as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide (<b>3</b>) were previously described as neuropeptide Y Y₄ receptor (Y₄R) partial agonists. Here, we report on a series of analogues of (2<i>R</i>,7<i>R</i>)-<b>1</b> and <b>2</b> in which Arg², Leu³, or Arg⁴ were replaced by the respective aza-amino acids. The replacement of Arg² in <b>3</b> with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr-<i>N</i>^ω-[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (<b>35</b>), which was used as a precursor for a d-amino acid scan. The target compounds were investigated for Y₄R functional activity in assays with complementary readouts: aequorin Ca²⁺ and β-arrestin 1 or β-arrestin 2 assays. In contrast to the parent compounds, which are Y₄R agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide Y₄R antagonists.

<h4>Background</h4>There is increasing evidence supporting the relevance of aberrant splicing in multiple disorders. In antithrombin deficiency only 22 intronic mutations affecting splicing sites (7% of <i>SERPINC1</i> mutations) are considered as splicing mutations.<h4>Methods</h4><i>SERPINC1</i> was analyzed by Sanger sequencing and MLPA in 141 unrelated cases with antithrombin deficiency. Plasma antithrombin was studied by functional and western blot assays, purified by FPLC and characterized by proteomic analysis. <i>In silico</i> predictions on splicing was done with the Human Splicing Finder software.<h4>Results</h4>We detected 89 different <i>SERPINC1</i> defects, 13 with potential effect on splicing. Ten cases presented 9 mutations disturbing splicing sites, 5 new. Three gross or small gene defects also disturbed a correct splicing. Interestingly, the first duplication of a single exon ever described (c.1154-13_1218+115dup), caused mild deficiency (75%). A deeper intronic mutation (c.1154-14G>A), identified in three unrelated patients with traces of disulphide dimers of antithrombin in plasma, created a cryptic splicing site that might generate a variant with 4 additional in frame residues according to <i>in silico</i> predictions. This aberrant splicing was confirmed by proteomic analysis of the dimer purified from plasma.<h4>Conclusions</h4>A high proportion of cases with antithrombin deficiency (up to 13%) may be explained by an aberrant splicing. Up to 15% of mutations in <i>SERPINC1</i>: splicing site variations, gross gene defects and deep intronic mutations, may affect a correct splicing with three potential consequences type I, type II, and even moderate antithrombin deficiency.

<h4>Significance</h4>Peroxiredoxins (Prxs) are thiol peroxidases with multiple functions in the antioxidant defense and redox signaling network of the cell. Our progressing understanding assigns both local and global significance to plant Prxs, which are grouped in four Prx types. In plants they are localized to the cytosol, mitochondrion, plastid, and nucleus. Antioxidant defense is fundamentally connected to redox signaling, cellular communication, and acclimation. The thiol-disulfide network is central part of the stress sensing and processing response and integrates information input with redox regulation. Recent Advances: Prxs function both as redox sensory system within the network and redox-dependent interactors. The processes directly or indirectly targeted by Prxs include gene expression, post-transcriptional reactions, including translation, post-translational regulation, and switching or tuning of metabolic pathways, and other cell activities. The most advanced knowledge is available for the chloroplast 2-CysPrx wherein recently a solid interactome has been defined. An in silico analysis of protein structure and coexpression reinforces new insights into the 2-CysPrx functionality.<h4>Critical issues</h4>Up to now, Prxs often have been investigated for local properties of enzyme activity. In vitro and ex vivo work with mutants will reveal the ability of Prxs to interfere with multiple cellular components, including crosstalk with Ca²⁺-linked signaling pathways, hormone signaling, and protein homeostasis.<h4>Future directions</h4>Complementation of the Prxs knockout lines with variants that mimic specific states, namely devoid of peroxidase activity, lacking the oligomerization ability, resembling the hyperoxidized decamer, or with truncated C-terminus, should allow dissecting the roles as thiol peroxidase, oxidant, interaction partner, and chaperone. Antioxid. Redox Signal. 28, 609-624.

Tendons and ligaments provide connections between muscle and bone or bone and bone to enable locomotion. Damage to tendons and ligaments caused by acute or chronic injury or associated with aging and arthritis is a prevalent cause of disability. Improvements in approaches for the treatment of these conditions depend on a better understanding of tendon and ligament development, cell biology, and pathophysiology. This review focuses on recent advances in the discovery of transcription factors that control ligament and tendon cell differentiation, how cell and extracellular matrix homeostasis are altered in disease, and how this new insight can lead to novel therapeutic approaches. © 2017 American Society for Bone and Mineral Research.

Chromosomal rearrangements of the human MLL/KMT2A gene are associated with infant, pediatric, adult and therapy-induced acute leukemias. Here we present the data obtained from 2345 acute leukemia patients. Genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) were determined and 11 novel TPGs were identified. Thus, a total of 135 different MLL rearrangements have been identified so far, of which 94 TPGs are now characterized at the molecular level. In all, 35 out of these 94 TPGs occur recurrently, but only 9 specific gene fusions account for more than 90% of all illegitimate recombinations of the MLL gene. We observed an age-dependent breakpoint shift with breakpoints localizing within MLL intron 11 associated with acute lymphoblastic leukemia and younger patients, while breakpoints in MLL intron 9 predominate in AML or older patients. The molecular characterization of MLL breakpoints suggests different etiologies in the different age groups and allows the correlation of functional domains of the MLL gene with clinical outcome. This study provides a comprehensive analysis of the MLL recombinome in acute leukemia and demonstrates that the establishment of patient-specific chromosomal fusion sites allows the design of specific PCR primers for minimal residual disease analyses for all patients.

Globally, lung cancer results in more deaths worldwide than any other cancer, indicating a need for better treatments. Members of the eicosanoid metabolism pathway represent promising therapeutic targets, as several enzymes involved in the generation of these lipids are dysregulated in many cancers and their inhibition reduces lung cancer growth in mouse models. However, genetic variation of enzymes involved in eicosanoid metabolism has not been adequately examined for association with lung cancer. The goal of this study was to determine whether germline genetic variation altering eicosanoid producing enzyme function and/or expression are associated with differences in lung cancer survival. We examined the association of genetic variation with mortality within eicosanoid metabolism genes in 395 non-small-cell lung cancer (NSCLC) cases from the Southern Community Cohort Study (SCCS). A total of 108 SNPs, both common and rare, in 19 genes, were examined for association. No common or rare variants were associated with lung cancer survival across the entire study population. However, rare variants in ALOX15B (arachidonate 15-lipoxygenase, type B) and the common variant rs12529 in AKR1C3 (prostaglandin F synthase) were associated with NSCLC mortality in women and African Americans, respectively. Rare variants in ALOX15B were associated with greater mortality in women (HR = 2.10, 95% CI = 1.25-3.54, p-value = 0.005). The major allele of rs12529 in AKCR1C3 associated with improved survival in African Americans (HR = 0.74, 95% CI = 0.59-0.92, p-value = 0.008). The lack of genetic associations among all NSCLC cases and the association among women only for rare variants in ALOX15B may, in part, explain the better NSCLC survival observed among women. These results raise the possibility that some subgroups within the NSCLC population may benefit from drugs targeting eicosanoid metabolism.

We have reported previously that Short Interspersed Degenerate Retroposons of the SIDER2 subfamily, largely located within 3'UTRs of Leishmania transcripts, promote rapid turnover of mRNAs through endonucleolytic cleavage within the highly conserved second tandem 79-nt hallmark sequence (79-nt SII). Here, we used site-directed mutagenesis and in silico RNA structural studies to delineate the cis-acting requirements within 79-nt SII for cleavage and mRNA degradation. The putative cleavage site(s) and other nucleotides predicted to alter the RNA secondary structure of 79-nt SII were either deleted or mutated and their effect on mRNA turnover was monitored using a gene reporter system. We found that short deletions of 8-nt spanning the two predicted cleavage sites block degradation of SIDER2-containing transcripts, leading to mRNA accumulation. Furthermore, single or double substitutions of the dinucleotides targeted for cleavage as well as mutations altering the predicted RNA secondary structure encompassing both cleavage sites also prevent mRNA degradation, confirming that these dinucleotides are the bona fide cleavage sites. In line with these results, we show that stage-regulated SIDER2 inactivation correlates with the absence of endonucleolytic cleavage. Overall, these data demonstrate that both cleavage sites within the conserved 79-nt SII as well as RNA folding in this region are essential for SIDER2-mediated mRNA decay, and further support that SIDER2-harboring transcripts are targeted for degradation by endonucleolytic cleavage.

Small isoprenoid diphosphates, such as (<i>E</i>)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9Vδ2 T cells. We demonstrate by small-angle X-ray scattering (SAXS) that HMBPP binding to the internal domain of BTN3A1 induces a conformational change in the position of the B30.2 domain relative to the juxtamembrane (JM) region. To better understand the molecular details of this conformational rearrangement, NMR spectroscopy was used to discover that the JM region interacts with HMBPP, specifically at the diphosphate. The spectral location of the affected amide peaks, partial NMR assignments, and JM mutants (ST²⁹⁶AA or T³⁰⁴A) investigated, confirm that the backbone amide of at least one Thr (Thr³⁰⁴), adjacent to conserved Ser, comes close to the HMBPP diphosphate, whereas double mutation of nonconserved residues (Ser/Thr^296/297) may perturb the local fold. Cellular mutation of either of the identified Thr residues reduces the activation of Vγ9Vδ2 T cells by HMBPP, zoledronate, and POM₂-C-HMBP, but not by a partial agonist BTN3 antibody. Taken together, our results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation.-Nguyen, K., Li, J., Puthenveetil, R., Lin, X., Poe, M. M., Hsiao, C.-H. C., Vinogradova, O., Wiemer, A. J. The butyrophilin 3A1 intracellular domain undergoes a conformational change involving the juxtamembrane region.

Silks are protein-based natural structured materials with an unusual combination of high strength and elongation. Their unique microstructural features composed of hard β-sheet crystals aligned within a soft amorphous region lead to the robust properties of silks. Herein we report a large enhancement in the intrinsic properties of silk through the transformation of the basic building blocks into a poly-hexagonal carbon structure by a simple heat treatment with axial stretching. The carbon clusters originating from the β-sheet retain the preferred orientation along the fibre axis, resulting in a long-range-ordered graphitic structure by increasing heat-treatment temperatures and leading improvements in mechanical properties with a maximum strength and modulus up to ∼2.6 and ∼470 GPa, respectively, almost four and thirty times surpassing those of raw silk. Moreover, the formation of sp ² carbon configurations induce a significant change in the electrical properties (e.g. an electrical conductivity up to 4.37 × 10³ S cm^-1).The mechanical properties of silk are determined by tight stacks of sheet-like peptide crystals distributed in amorphous regions. Here, the authors heat and stretch silk fibres to align these crystal into a long range ordered carbon structure and dramatically enhance the silk strength.

Huntington's disease is characterized by a complex and heterogeneous pathogenic profile. Studies have shown that disturbance in lipid homeostasis may represent a critical determinant in the progression of several neurodegenerative disorders. The recognition of perturbed lipid metabolism is only recently becoming evident in HD. In order to provide more insight into the nature of such a perturbation and into the effect its modulation may have in HD pathology, we investigated the metabolism of Sphingosine-1-phosphate (S1P), one of the most important bioactive lipids, in both animal models and patient samples. Here, we demonstrated that S1P metabolism is significantly disrupted in HD even at early stage of the disease and importantly, we revealed that such a dysfunction represents a common denominator among multiple disease models ranging from cells to humans through mouse models. Interestingly, the in vitro anti-apoptotic and the pro-survival actions seen after modulation of S1P-metabolizing enzymes allows this axis to emerge as a new druggable target and unfolds its promising therapeutic potential for the development of more effective and targeted interventions against this incurable condition.

The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFII ^F/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2⁺ neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.

<h4>Background</h4>The association of type 2 diabetes mellitus (T2DM) with the <i>KCNJ11, CDKAL1, SLC30A8, CDKN2B,</i> and <i>FTO</i> genes in the Russian population has not been well studied. In this study, we analysed the population frequencies of polymorphic markers of these genes.<h4>Methods</h4>The study included 862 patients with T2DM and 443 control subjects of Russian origin. All subjects were genotyped for 10 single nucleotide polymorphisms (SNPs) of the genes using real-time PCR (TaqMan assays). HOMA-IR and HOMA-<i>β</i> were used to measure insulin resistance and <i>β</i>-cell secretory function, respectively.<h4>Results</h4>The analysis of the frequency distribution of polymorphic markers for genes <i>KCNJ11, CDKAL1, SLC30A8</i> and <i>CDKN2B</i> showed statistically significant associations with T2DM in the Russian population. The association between the <i>FTO</i> gene and T2DM was not statistically significant. The polymorphic markers <i>rs5219</i> of the <i>KCNJ11</i> gene, <i>rs13266634</i> of the <i>SLC30A8</i> gene, <i>rs10811661</i> of the <i>CDKN2B</i> gene and <i>rs9465871</i>, <i>rs7756992</i> and <i>rs10946398</i> of the <i>CDKAL1</i> gene showed a significant association with impaired glucose metabolism or impaired <i>β</i>-cell function.<h4>Conclusion</h4>In the Russian population, genes, which affect insulin synthesis and secretion in the <i>β</i>-cells of the pancreas, play a central role in the development of T2DM.

The associations between proteins and diseases are crucial information for investigating pathological mechanisms. However, the number of known and reliable protein-disease associations is quite small. In this study, an analysis framework to infer associations between proteins and diseases was developed based on a large data set of a human protein-protein interaction network integrating an effective network search, namely, the reverse <i>k</i>-nearest neighbor (R<i>k</i>NN) search. The R<i>k</i>NN search was used to identify an impact of a protein on other proteins. Then, associations between proteins and diseases were inferred statistically. The method using the R<i>k</i>NN search yielded a much higher precision than a random selection, standard nearest neighbor search, or when applying the method to a random protein-protein interaction network. All protein-disease pair candidates were verified by a literature search. Supporting evidence for 596 pairs was identified. In addition, cluster analysis of these candidates revealed 10 promising groups of diseases to be further investigated experimentally. This method can be used to identify novel associations to better understand complex relationships between proteins and diseases.

Epilepsy is a common episodic neurological disorder or condition characterized by recurrent epileptic seizures, and genetics seems to play a key role in its etiology. Early linkage studies have localized multiple loci that may harbor susceptibility genes to epilepsy, and mutational analyses have detected a number of mutations involved in both ion channel and nonion channel genes in patients with idiopathic epilepsy. Genome-wide studies of epilepsy have found copy number variants at 2q24.2-q24.3, 7q11.22, 15q11.2-q13.3, and 16p13.11-p13.2, some of which disrupt multiple genes, such as <i>NRXN1</i>, <i>AUTS2</i>, <i>NLGN1</i>, <i>CNTNAP2</i>, <i>GRIN2A</i>, <i>PRRT2</i>, <i>NIPA2</i>, and <i>BMP5</i>, implicated for neurodevelopmental disorders, including intellectual disability and autism. Unfortunately, only a few common genetic variants have been associated with epilepsy. Recent exome-sequencing studies have found some genetic mutations, most of which are located in nonion channel genes such as the <i>LGI1</i>, <i>PRRT2</i>, <i>EFHC1</i>, <i>PRICKLE</i>, <i>RBFOX1</i>, and <i>DEPDC5</i> and in probands with rare forms of familial epilepsy, and some of these genes are involved with the neurodevelopment. Since epigenetics plays a role in neuronal function from embryogenesis and early brain development to tissue-specific gene expression, epigenetic regulation may contribute to the genetic mechanism of neurodevelopment through which a gene and the environment interacting with each other affect the development of epilepsy. This review focused on the analytic tools used to identify epilepsy and then provided a summary of recent linkage and association findings, indicating the existence of novel genes on several chromosomes for further understanding of the biology of epilepsy.

The carcinogenic potential of phenobarbital (PB) was assessed in a mouse line carrying a mutant <i>Mmh</i> allele of the <i>Mmh/Ogg1</i> gene encoding the enzyme oxoguanine DNA glycosylase (Ogg1) responsible for the repair of 8-hydroxy-2'-deoxyguanosine (8-OHdG). <i>Mmh</i> homozygous mutant (<i>Ogg1</i>^-/-) and wild-type (<i>Ogg1</i>^+/+) male and female, 10-week-old, mice were treated with 500 ppm PB in diet for 78 weeks. Hepatocellular carcinomas (HCCs) were found in PB-treated <i>Ogg1</i>^-/- mice, while <i>Ogg1</i>^+/+ animals developed only hepatocellular adenomas (HCAs) at the same rate. This was coordinated with PB-induced significant elevation of 8-OHdG formation in DNA and cell proliferation in adjacent liver of <i>Ogg1</i>^-/- mice. Proteome analysis predicted activation of transcriptional factor Nrf2 in the livers and HCAs of PB-administered <i>Ogg1</i>^+/+ mice; however, its activation was insufficient or absent in the livers and HCCs of <i>Ogg1</i>^-/- mice, respectively. Significant elevation of phase I and II metabolizing enzymes was demonstrated in both <i>Ogg1</i>^-/- and <i>Ogg1</i>^+/+ animals. Treatment of <i>Ogg1</i>^-/- mice with PB resulted in significant elevation of cell proliferation in the liver. These results indicate that PB induced progression from HCA to HCC in <i>Ogg1</i>^-/- mice, due to persistent accumulation of DNA oxidative base modifications and suppression of Nrf2-mediated oxidative stress response, resulting in significant elevation of cell proliferation.

The vaccinia virus B1 kinase is highly conserved among poxviruses and is essential for the viral life cycle. B1 exhibits a remarkable degree of similarity to vaccinia virus-related kinases (VRKs), a family of cellular kinases, suggesting that the viral enzyme has evolved to mimic VRK activity. Indeed, B1 and VRKs have been demonstrated to target a shared substrate, the DNA binding protein BAF, elucidating a signaling pathway important for both mitosis and the antiviral response. In this study, we further characterize the role of B1 during vaccinia infection to gain novel insights into its regulation and integration with cellular signaling pathways. We begin by describing the construction and characterization of the first B1 deletion virus (vvΔB1) produced using a complementing cell line expressing the viral kinase. Examination of vvΔB1 revealed that B1 is critical for the production of infectious virions in various cell types and is sufficient for BAF phosphorylation. Interestingly, the severity of the defect in DNA replication following the loss of B1 varied between cell types, leading us to posit that cellular VRKs partly complement for the absence of B1 in some cell lines. Using cell lines devoid of either VRK1 or VRK2, we tested this hypothesis and discovered that VRK2 expression facilitates DNA replication and allows later stages of the viral life cycle to proceed in the absence of B1. Finally, we present evidence that the impact of VRK2 on vaccinia virus is largely independent of BAF phosphorylation. These data support a model in which B1 and VRK2 share additional substrates important for the replication of cytoplasmic poxviruses.<b>IMPORTANCE</b> Viral mimicry of cellular signaling modulators provides clear evidence that the pathogen targets an important host pathway during infection. Poxviruses employ numerous viral homologs of cellular proteins, the study of which have yielded insights into signaling pathways used by both virus and cells alike. The vaccinia virus B1 protein is a homolog of cellular vaccinia virus-related kinases (VRKs) and is needed for viral DNA replication and likely other stages of the viral life cycle. However, much remains to be learned about how B1 and VRKs overlap functionally. This study utilizes new tools, including a B1 deletion virus and VRK knockout cells, to further characterize the functional links between the viral and cellular enzymes. As a result, we have discovered that B1 and VRK2 target a common set of substrates vital to productive infection of this large cytoplasmic DNA virus.

Proteins exist as conformational ensembles, exchanging between substates to perform their function. Advances in experimental techniques yield unprecedented access to structural snapshots of their conformational landscape. However, computationally modeling how proteins use collective motions to transition between substates is challenging owing to a rugged landscape and large energy barriers. Here, we present a new, robotics-inspired motion planning procedure called dCC-RRT that navigates the rugged landscape between substates by introducing dynamic, interatomic constraints to modulate frustration. The constraints balance non-native contacts and flexibility, and instantaneously redirect the motion towards sterically favorable conformations. On a test set of eight proteins determined in two conformations separated by, on average, 7.5 Å root mean square deviation (RMSD), our pathways reduced the Cα atom RMSD to the goal conformation by 78%, outperforming peer methods. We then applied dCC-RRT to examine how collective, small-scale motions of four side-chains in the active site of cyclophilin A propagate through the protein. dCC-RRT uncovered a spatially contiguous network of residues linked by steric interactions and collective motion connecting the active site to a recently proposed, non-canonical capsid binding site 25 Å away, rationalizing NMR and multi-temperature crystallography experiments. In all, dCC-RRT can reveal detailed, all-atom molecular mechanisms for small and large amplitude motions. Source code and binaries are freely available at https://github.com/ExcitedStates/KGS/.

Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that <u>tri</u>partite <u>m</u>otif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased <u>s</u>oluble <u>N</u>-ethylmaleimide <u>a</u>ttachment protein <u>re</u>ceptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons.

In addition to mutations or aberrant expression in the protein-coding genes, mutations and misregulation of noncoding RNAs, in particular long noncoding RNAs (lncRNA), appear to play major roles in cancer. Genome-wide association studies of tumor samples have identified a large number of lncRNAs associated with various types of cancer. Alterations in lncRNA expression and their mutations promote tumorigenesis and metastasis. LncRNAs may exhibit tumor-suppressive and -promoting (oncogenic) functions. Because of their genome-wide expression patterns in a variety of tissues and their tissue-specific expression characteristics, lncRNAs hold strong promise as novel biomarkers and therapeutic targets for cancer. In this article, we have reviewed the emerging functions and association of lncRNAs in different types of cancer and discussed their potential implications in cancer diagnosis and therapy. <i>Cancer Res; 77(15); 3965-81. ©2017 AACR</i>.

Huntington's disease (HD) is a progressive and fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin gene. Although HD is monogenic, its molecular manifestation appears highly complex and involves multiple cellular processes. The recent application of high throughput platforms such as microarrays and mass-spectrometry has indicated multiple pathogenic routes. The massive data generated by these techniques together with the complexity of the pathogenesis, however, pose considerable challenges to researchers. Network-based methods can provide valuable tools to consolidate newly generated data with existing knowledge, and to decipher the interwoven molecular mechanisms underlying HD. To facilitate research on HD in a network-oriented manner, we have developed HDNetDB, a database that integrates molecular interactions with many HD-relevant datasets. It allows users to obtain, visualize and prioritize molecular interaction networks using HD-relevant gene expression, phenotypic and other types of data obtained from human samples or model organisms. We illustrated several HDNetDB functionalities through a case study and identified proteins that constitute potential cross-talk between HD and the unfolded protein response (UPR). HDNetDB is publicly accessible at http://hdnetdb.sysbiolab.eu .

Aberrant microRNA expression contributes to breast cancer progression and endocrine resistance. We reported that although tamoxifen stimulated miR-29b-1/a transcription in tamoxifen (TAM)-resistant breast cancer cells, ectopic expression of miR-29b-1/a did not drive TAM-resistance in MCF-7 breast cancer cells. However, miR-29b-1/a overexpression significantly repressed TAM-resistant LCC9 cell proliferation, suggesting that miR-29b-1/a is not mediating TAM resistance but acts as a tumor suppressor in TAM-resistant cells. The target genes mediating this tumor suppressor activity were unknown. Here, we identify miR-29b-1 and miR-29a target transcripts in both MCF-7 and LCC9 cells. We find that miR-29b-1 and miR-29a regulate common and unique transcripts in each cell line. The cell-specific and common downregulated genes were characterized using the MetaCore Gene Ontology (GO) enrichment analysis algorithm. LCC9-sepecific miR-29b-1/a-regulated GO processes include oxidative phosphorylation, ATP metabolism, and apoptosis. Extracellular flux analysis of cells transfected with anti- or pre- miR-29a confirmed that miR-29a inhibits mitochondrial bioenergetics in LCC9 cells. qPCR,luciferase reporter assays, and western blot also verified the ATP synthase subunit genes ATP5G1 and ATPIF1 as bone fide miR29b-1/a targets. Our results suggest that miR-29 repression of TAM-resistant breast cancer cell proliferation is mediated in part through repression of genes important in mitochondrial bioenergetics.

Eicosanoids may play a role in ischemic stroke. However, the associations of variants in cyclooxygenase (COX) pathway genes and interaction among these variants with carotid plaque vulnerability are not fully understood. In present study, twelve variants in COX pathway genes were examined using matrix-assisted laser desorption ionization time-of-flight mass spectrometry method in 396 patients with ischemic stroke and 291 controls. Platelet aggregation, platelet-leukocyte aggregates, and urine 11-dehydrothromboxane B2 (11-dTxB2) were also measured. According to the results of carotid high-resolution B-mode ultrasound, the patients were stratified into the following groups [i.e., non-carotid plaque and carotid plaque. The carotid plaque was further classified into subgroups of echolucent plaque (ELP) and echogenic plaque (EGP)]. Additionally, gene-gene interactions were analyzed to assess whether there was any interactive role for assessed variants in affecting carotid plaque vulnerability, platelet activation and 11-dTxB2 levels. There were no significant differences in the frequencies of genotypes of the twelve variants between patients and controls. Among 396 patients, 294 cases (74.2%) had carotid plaques (106 had ELP, 188 had EGP). Frequency of PTGS2 rs20417CC, TXAS1 rs2267679TT, TXAS1 rs41708TT, PTGIS rs5602CC, and TXA2R rs1131882TT genotype was significantly higher in patients with plaque compared with patients without plaque, or in patients with ELP compared with patients with EGP. 11-dTxB2 levels, platelet aggregation and platelet-leukocyte aggregates were significantly higher in patients with ELP compared with patients without plaque or with EGP. Multivariate logistic regression analysis revealed that PTGS2 rs20417CC, TXA2R rs1131882TT, and high-risk interaction among variants in PTGS2 rs20417, TXA2R rs1131882 and TXAS1 rs41708 were independently associated with the risk of ELP after adjusting for confounding variables. The variants in COX pathway genes and the high-risk interactions among variants in PTGS2 rs20417, TXA2R rs1131882 and TXAS1 rs41708 were associated with high 11-dTxB2 and platelet activation, and independently associated with the risk of carotid plaque vulnerability. These variants might be potential markers for plaque instability.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a single-stranded positive-sense RNA virus that can cause devastating reproductive failure and respiratory tract lesions, which has led to serious damage to the swine industry worldwide. Our previous studies have indicated that Tongcheng (TC) pigs, a Chinese local breed, have stronger resistance or tolerance to PRRSV infection than Large White (LW) pigs. This study aims to investigate their host transcriptome differences in porcine alveolar macrophages (PAMs) at 7 days post challenge. Transcriptome profiling of PAMs from PRRSV infected and control pigs of these two breeds were performed using RNA-sequencing. For both breeds, there were 1257 common differentially expressed genes (DEGs) in response to PRRSV infection, involving hepatic fibrosis/hepatic stellate cell activation, phospholipase C, and granulocyte adhesion and diapedesis pathways. For TC pig, 549 specific DEGs were identified, including VAV2, BCL2 and BAX, which were enriched in activation of leukocyte extravasation and suppression of apoptosis. While, 898 specific DEGs were identified in LW pigs, including GNAQ, GNB5, GNG2, CALM4 and RHOQ, which were involved in suppression of Gαq and PI3K-AKT signaling. This study provides an insight into the transcriptomic comparison of resistant and susceptible pigs to PRRSV infection. TC pigs may promote the extravasation and migration of leukocytes to defend against PRRSV infections and suppress apoptosis of the infected macrophages to increase antigen presentation, thereby reducing the lung lesions.

The objective of this study was to determine the effects of feeding protein-iron complex (PIC) on productive performance and indicators of iron metabolism, hematology parameters, antioxidant and immune status during first 35 days of a calf's life. Preparation of the complex involved enzymatic hydrolysis of milk casein (serine protease from <i>Yarrowia lipolytica</i> yeast). Iron chloride was then added to the hydrolyzate and lyophilizate. Calves were divided into treated groups: LFe (low iron dose) 10 g/day calf of protein-iron complex, HFe (height iron dose) 20 g/day calf, and control group. Dietary supplements containing the lower dose of concentrate had a significant positive effect on iron metabolism, while the higher dose of concentrate resulted in increase of total iron binding capacity (TIBC), saturation of transferrin and decrease of and unsaturated iron binding capacity (UIBC), which suggest iron overload. Additionally, treatment with the lower dose of iron remarkably increased the antioxidant parameters, mainly total antioxidant (TAS) and glutathione peroxidase activity (GPx). Higher doses of PIC were related to lower total antioxidant status. IgG, IgM, insulin, glucose, TNFα and IGF-1 concentration did not change significantly in either group after supplementation. In practice, the use of protein-iron complex concentrate requires taking into account the iron content in milk replacers and other feedstuffs.

There are anatomical and functional differences between human dental pulp (DP) and periodontal ligament (PDL). However, the molecular biological differences and function of these tissues are poorly understood. In the present study, we employed a cDNA microarray array to screen for differentially expressed genes (DEGs) between human DP and PDL tissues, and used the online software WebGestalt to perform the functional analysis of the DEGs. In addition, the STRING database and KEGG pathway analysis were applied for interaction network and pathway analysis of the DEGs. DP and PDL samples were obtained from permanent premolars (n=16) extracted for orthodontic purposes. The results of the microarray assay were confirmed by RT-qPCR. The DEGs were found to be significantly associated with the extracellular matrix and focal adhesion. A total of 10 genes were selected to confirm the results. The mRNA levels of integrin alpha 4 (ITGA4), integrin alpha 8 (ITGA8), neurexin 1 (NRXN1) and contactin 1 (CNTN1) were significantly higher in the DP than in the PDL tissues. However, the levels of collagen type XI alpha 1 (COL11A1), aggrecan (ACAN), collagen type VI alpha 1 (COL6A1), chondroadherin (CHAD), laminin gamma 2 (LAMC2) and laminin alpha 3 (LAMA3) were higher in the PDL than in the DP samples. The gene expression profiles provide novel insight into the characterization of DP and PDL tissues, and contribute to our understanding of the potential molecular mechanisms of dental tissue mineralization and regeneration.

Allergic rhinitis (AR) is a common inflammatory disease of the upper airway. Recent evidence suggests that gene‑gene interactions between tumor necrosis factor receptor superfamily 4 (TNFSF4) and B cell lymphocyte kinase (BLK) may have a synergistic effect on T and B cells in determining immunologic aberration, via the nuclear factor‑κB pathway. The present study was performed to evaluate the potential association between specific single nucleotide polymorphisms (SNPs) in the TNFSF4 and BKL genes with susceptibility to AR in Chinese subjects. A population‑based case‑control study was performed in 600 Chinese AR patients and 700 controls. Blood was drawn for DNA extraction, and 9 SNPs (6 in TNFSF4 and 3 in BKL genes) were selected and genotyped. The TNFSF4 SNPs rs1234314 and rs1234315, and the BLK SNPs rs13277113 and rs1600249 were observed to occur in different frequencies between the AR patients and the controls. The CC (rs1234314, rs1234315) and AA (rs1600249, rs13277113) genotypes provided protective effects against AR, whereas the AG (rs13277113) genotype presented a risk factor for AR. The haplotypes ACC in the rs1234313‑rs1234314‑rs1234315 block and GA in the rs2254546‑rs13277113 block significantly decreased the risk of AR, whereas the GGT and AG haplotypes served protective roles. SNP interaction analysis further indicated that there may be synergistic effects among the selected sets of polymorphisms. The present study suggests a novel association between specific TNFSF4 and BLK gene polymorphisms and AR risk, highlighting their potential utility as genetic biomarkers for AR susceptibility in a Chinese Han population.