Gene Literature Dashboard

<h4>Objectives</h4>This study compares the expression levels of tumor necrosis factor ligand superfamily member 4 (TNFSF4) and TNF-R-associated factor 2 (TRAF2) mRNAs in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE) against healthy controls. The association of SLE disease activity index (SLEDAI) and clinical features of SLE with altered expression levels of TNFSF4 and TRAF2 mRNAs were also evaluated.<h4>Design</h4>We used real-time reverse transcription polymerase chain reaction to measure TNFSF4 and TRAF2 mRNAs expression levels in peripheral blood mononuclear cells of 57 SLE patients and 57 healthy controls.<h4>Results</h4>The expression level of TNFSF4 mRNA was significantly higher in SLE patients than in the control group. Overexpression of TNFSF4 was correlated with arthritis, atherosclerosis and lupus nephritis. TRAF2 mRNA was underexpressed in PBMCs of SLE patients, and its lower expression was associated with atherosclerosis and lupus nephritis. The altered expression levels of TNFSF4 and TRAF2 mRNAs was significantly correlated with SLEDAI.<h4>Conclusion</h4>Our results suggest that changes in the expression levels of TNFSF4 and TRAF2 mRNAs may significantly correlate with the pathogenesis of SLE, the disease activity and different clinical features of lupus, particularly lupus nephritis, atherosclerosis and arthritis.

<h4>Background</h4>The psychobiological model of personality by Cloninger and colleagues originally hypothesized that interindividual variability in the temperament dimension 'harm avoidance' (HA) is explained by differences in the activity of the brain serotonin system. We assessed brain serotonin transporter (5-HTT) density in vivo with positron emission tomography (PET) in healthy individuals with high or low HA scores using an 'oversampling' study design. Method Subjects consistently in either upper or lower quartiles for the HA trait were selected from a population-based cohort in Finland (n = 2075) with pre-existing Temperament and Character Inventory (TCI) scores. A total of 22 subjects free of psychiatric and somatic disorders were included in the matched high- and low-HA groups. The main outcome measure was regional 5-HTT binding potential (BPND) in high- and low-HA groups estimated with PET and [11C]N,N-dimethyl-2-(2-amino-4-methylphenylthio)benzylamine ([11C]MADAM). In secondary analyses, 5-HTT BPND was correlated with other TCI dimensions.<h4>Results</h4>5-HTT BPND did not differ between high- and low-HA groups in the midbrain or any other brain region. This result remained the same even after adjusting for other relevant TCI dimensions. Higher 5-HTT BPND in the raphe nucleus predicted higher scores in 'self-directedness'.<h4>Conclusions</h4>This study does not support an association between the temperament dimension HA and serotonin transporter density in healthy subjects. However, we found a link between high serotonin transporter density and high 'self-directedness' (ability to adapt and control one's behaviour to fit situations in accord with chosen goals and values). We suggest that biological factors are more important in explaining variability in character than previously thought.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aβ42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Hereditary hemochromatosis is characterized by tissue iron loading and associated organ damage. However, the phenotype can be highly variable. The relationship between iron loading of different organs and the temporal nature of its deposition is still not well understood. We examined the progression of tissue iron loading in three mouse models to advance our understanding of the natural history of iron deposition in hereditary hemochromatosis. Wild-type, Hfe(-/-), Tfr2(-/-), and Hfe(-/-)/Tfr2(-/-) mice were analyzed at 3, 5, 10, 26, and 52 weeks, respectively. Hepatic, splenic, cardiac, and pancreatic iron concentrations were determined. Expression of both iron-regulatory and fibrosis genes was determined by quantitative real-time PCR in livers and hearts of 52-week-old mice. In all models, hepatic iron increased rapidly, plateauing before 10 weeks at different levels, depending on the genotype. Iron deposition in the pancreas and heart occurred after maximal iron loading of the liver was reached and was most marked in the Hfe(-/-)/Tfr2(-/-) mice. Although a significant positive correlation was identified between pancreatic and cardiac iron in all models at 52 weeks, there was no correlation between hepatic and either pancreatic or cardiac iron. There is variability in the timing and extent of tissue iron loading within a genotype, suggesting that hepatic iron levels in hereditary hemochromatosis may not accurately predict the iron content of other organs.

Signal transduction pathways activated by Toll-like Receptors and the IL-1 family of cytokines are fundamental to mounting an innate immune response and thus to clearing pathogens and promoting wound healing. Whilst mechanistic understanding of the regulation of innate signalling pathways has advanced considerably in recent years, there are still a number of critical controllers to be discovered. In order to characterise novel regulators of macrophage inflammation, we have carried out an extensive, cDNA-based forward genetic screen and identified 34 novel activators, based on their ability to induce the expression of cxcl2. Many are physiologically expressed in macrophages, although the majority of genes uncovered in our screen have not previously been linked to innate immunity. We show that expression of particular activators has profound but distinct impacts on LPS-induced inflammatory gene expression, including switch-type, amplifier and sensitiser behaviours. Furthermore, the novel genes identified here interact with the canonical inflammatory signalling network via specific mechanisms, as demonstrated by the use of dominant negative forms of IL1/TLR signalling mediators.

Mesotelencephalic pathways in the adult central nervous system have been studied in great detail because of their implication in major physiological functions as well as in psychiatric, neurological, and neurodegenerative diseases. However, the ontogeny of these pathways and the molecular mechanisms that guide dopaminergic axons during embryogenesis have been only recently studied. This line of research is of crucial interest for the repair of lesioned circuits in adulthood following neurodegenerative diseases or common traumatic injuries. For instance, in the adult, the anatomic and functional repair of the nigrostriatal pathway following dopaminergic embryonic neuron transplantation suggests that specific guidance cues exist which govern embryonic fibers outgrowth, and suggests that axons from transplanted embryonic cells are able to respond to theses cues, which then guide them to their final targets. In this review, we first synthesize the work that has been performed in the last few years on developing mesotelencephalic pathways, and summarize the current knowledge on the identity of cellular and molecular signals thought to be involved in establishing mesotelencephalic dopaminergic neuronal connectivity during embryogenesis in the central nervous system of rodents. Then, we review the modulation of expression of these molecular signals in the lesioned adult brain and discuss their potential role in remodeling the mesotelencephalic dopaminergic circuitry, with a particular focus on Parkinson's disease (PD). Identifying guidance molecules involved in the connection of grafted cells may be useful for cellular therapy in Parkinsonian patients, as these molecules may help direct axons from grafted cells along the long distance they have to travel from the substantia nigra to the striatum.

<h4>Aims</h4>Serotonin transporter knockout (5-HTT KO) mice exhibit elevated basal extracellular serotonin, increased depressive-like behaviors and increased rapid eye movement sleep. Because abnormalities of circadian rhythms are associated with mood disorders, we tested the hypothesis that 5-HTT KO mice would have altered circadian rhythmicity.<h4>Main methods</h4>Homecage locomotor activity was recorded in wild-type (WT) and KO mice under a standard 12:12 light-dark cycle. After 4weeks of recording, mice received a one-hour light pulse at circadian time (CT) 14 and then were kept under constant darkness for 3weeks.<h4>Key findings</h4>There were no significant differences in amplitude, period, acrophase or total home cage locomotor activity between WT and KO mice during the 12:12 light-dark cycle or during constant darkness. The mean phase delay to a CT 14 light pulse was significantly attenuated in KO compared to WT mice.<h4>Significance</h4>Acute increases in serotonin have been reported to attenuate photic phase shifts. The current study demonstrates that this effect is maintained in the face of a lifelong absence of 5-HTT.

Activating mutations in the receptor tyrosine kinase KIT, most notably KIT D816V, are commonly observed in patients with systemic mastocytosis. Thus, inhibition of KIT has been a major focus for treatment of this disorder. Here we investigated a novel approach to such inhibition. Utilizing rational drug design, we targeted the switch pocket (SP) of KIT, which regulates its catalytic conformation. Two SP inhibitors thus identified, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. Autophosphorylation of both wild-type and, where also examined, KIT D816V activation was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34(+)-derived human mast cells activated by stem cell factor (SCF). Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Moreover, the SP inhibitors more selectively blocked SCF potentiation of FcɛRI-mediated degranulation. Overall, SP inhibitors represent an innovative mechanism of KIT inhibition whose dual suppression of KIT D816V neoplastic mast cell proliferation and SCF-enhanced mast cell activation may provide significant therapeutic benefits.

<h4>Background</h4>Anticoagulation, the use of sequential compression devices on the lower extremities perioperatively, and early ambulation are thought to reduce the incidence of venous thromboembolism (VTE) postoperatively and are recommended to reduce VTE risk. However, the evidence on which this recommendation has been based is not particularly strong. We have demonstrated that even a large, multicenter cohort with carefully collected prospective data is inadequate to provide sufficient evidence to support, or refute, this recommendation.<h4>Methods</h4>Longitudinal Assessment of Bariatric Surgery participants from 10 centers in the United States who underwent their first bariatric surgery between March 2005 and December 2007 constituted the study group. We examined the ability to address the question of whether anticoagulation therapy, in addition to sequential compression, reduces the 30-day incidence of VTE or death sufficiently to recommend the use of prophylactic anticoagulation, a therapy that is not without risk.<h4>Results</h4>Of 4416 patients, 396 (9.0%) received sequential compression alone, and 4020 also received anticoagulation therapy. The incidence of VTE within 30 days of surgery was low (.25% among those receiving sequential compression alone and .47% when anticoagulation therapy was added), and the 30-day incidence of death was also low (.25% versus .34%, respectively, P = .76, for sequential compression alone versus sequential compression plus anticoagulation therapy). Estimates of the number of cases required to address the question of whether a difference exists in the outcome related to VTE chemoprophylaxis or whether the outcome rates are equivalent have ranged from 13,680 to ≥ 35,760 patients, depending on whether superiority or equivalence was being analyzed.<h4>Conclusion</h4>Sufficient evidence from a clinical trial study to determine whether prophylactic anticoagulation added to compression devices further prevents VTE is not available, and such a trial is likely to be impractical. The data presented are insufficient to make a final recommendation concerning prophylactic treatment to prevent VTE in the 30 days after bariatric surgery.

Human Vγ9Vδ2 T cells are well known for their rapid and potent response to infection and tumorigenesis when in the presence of endogenous or exogenous phosphoisoprenoids. However, the molecular mechanisms behind the activation of this γδ T cell population remains unclear. Evidence pointing to a role for the CD277/butyrophilin-3 (BTN3A) molecules in this response led us to investigate the structures of these molecules and their modifications upon binding to an agonist antibody (20.1) that mimics phosphoisoprenoid-mediated Vγ9Vδ2 activation and an antagonist antibody (103.2) that inhibits this reactivity. We find that the three BTN3A isoforms: BTN3A1, BTN3A2, and BTN3A3, have high structural homology to the B7 superfamily of proteins and exist as V-shaped homodimers in solution, associating through the membrane proximal C-type Ig domain. The 20.1 and 103.2 antibodies bind to separate epitopes on the BTN3A Ig-V domain with high affinity but likely with different valencies based on their binding orientation. These structures directly complement functional studies of this system that demonstrate that BTN3A1 is necessary for Vγ9Vδ2 activation and begin to unravel the extracellular events that occur during stimulation through the Vγ9Vδ2 T cell receptor.

Ca(V)β subunits are formed by a Src homology 3 domain and a guanylate kinase-like (GK) domain connected through a variable HOOK domain. Complete deletion of the Src homology 3 domain (75 residues) as well as deletion of the HOOK domain (47 residues) did not alter plasma membrane density of Ca(V)2.3 nor its typical activation gating. In contrast, six-residue deletions in the GK domain disrupted cell surface trafficking and functional expression of Ca(V)2.3. Mutations of residues known to carry nanomolar affinity binding in the GK domain of Ca(V)β (P175A, P179A, M195A, M196A, K198A, S295A, R302G, R307A, E339G, N340G, and A345G) did not significantly alter cell surface targeting or gating modulation of Ca(V)2.3. Nonetheless, mutations of a quartet of leucine residues (either single or multiple mutants) in the α3, α6, β10, and α9 regions of the GK domain were found to significantly impair cell surface density of Ca(V)2.3 channels. Furthermore, the normalized protein density of Ca(V)2.3 was nearly abolished with the quadruple Ca(V)β3 Leu mutant L200G/L303G/L337G/L342G. Altogether, our observations suggest that the four leucine residues in Ca(V)β3 form a hydrophobic pocket surrounding key residues in the α-interacting domain of Ca(V)2.3. This interaction appears to play an essential role in conferring Ca(V)β-induced modulation of the protein density of Ca(V)α1 subunits in Ca(V)2 channels.

A new poly(aminoester) (EPAE-FA) containing folic acid and amino groups in the backbone and side chain was synthesized. EPAE-FA self-assembled readily with the plasmid DNA (pCMV-βgal) in HEPES buffer and was characterized by dynamic light scattering, zeta potential, fluorescence images, and XTT cell viability assays. To evaluate the transfection effect of graft ratio of FA on the EPAE system, EPAE-FA polymers with two different graft ratios (EPAE-FA12k and EPAE-FA14k) were also prepared. This study found that all EPAE-FA polymers were able to bind plasmid DNA and yielded positively charged complexes with nano-sized particles (< 200 nm). To assess the transfection efficiency mediated by EPAE and EPAE-FA polymers, we performed in vitro transfection activity assays using FR-negative (COS-7) and FR-positive (HeLa) cells. The EPAE-FA12k/DNA and EPAE-FA14k/DNA complexes were able to transfect HeLa cell in vitro with higher transfection efficiency than PEI25k/DNA at the similar weight ratio. These results demonstrated that the introduction of FA into EPAE system had a significant effect on transferring ability for FR-positive cells (HeLa). Examination of the cytotoxicity of PEI25k and EPAE-FA system revealed that EPAE-FA system had lower cytotoxicity. In this paper, EPAE-FA seemed to be a novel cationic poly(aminoester) for gene delivery and an interesting candidate for further study.

<h4>Background and aims</h4>Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge.<h4>Methods and results</h4>Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration.<h4>Conclusions</h4>The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Hepatitis B or C infections are the main causes of HCC with hepatitis C being the major risk factor for liver cancer in the developed countries. Recently, complications with bacteria of the genus Helicobacter have been associated with HCV-induced HCC. To further understand the mechanisms leading to the development of HCC in the presence of HCV and/or Helicobacter spp., investigation of the differential protein expression in Huh7 cells harbouring HCV-replicon, and replicon cured-Huh7 cells cocultured with H. bilis was done employing two-dimensional gel electrophoresis and mass spectrometry. In the transfected-Huh7 cells exposed to sublethal inoculum densities of H. bilis, 53 different proteins were identified comprising of 28 upregulated and 16 downregulated proteins including 9 potential protein isoforms; in the cured Huh7 cells, 45 different proteins were identified including 33 upregulated, 8 downregulated and, 9 potential protein isoforms. H. bilis affected the modulation of proteins involved in different pathways of Huh7-derived cells physiology including proteins involved in the progression from dysplasia to neoplasm. The result also indicated that the response of the Huh7-derived cells to the presence of H. bilis depended on whether or not HCV replicon was present.

Obesity and related complications are major health burdens. Almost 700 million adults are currently obese globally and the prevalence is predicted to rise towards 2030. The sudden change of lifestyle with physical inactivity and excessive calorie intake undoubtedly have a major part of the epidemic development; however, some individuals seem to be more prone to be affected by an unhealthy lifestyle than others. Hence, genetic predisposition also has an essential role in determining disease susceptibility and response to lifestyle factors. Since the introduction of genome-wide association studies (GWAS), the success of identifying obesity susceptibility variants have increased, and a total of 32 variants have been identified associating genome-wide significantly with body mass index (BMI) and 18 with measures of fat distribution during four overall obesity GWAS waves. However, the immediate success of the GWAS approach has eased off, but the proportion of explained variance for BMI by the identified obesity variants remains low. This review suggests and discusses new initiatives to take GWAS of obesity to the next level, including gene-environment interactions as modulating/masking factors, low-frequent or rare variants and ways to address such analyses, and finally reflections about the applicability of epigenetic modifications when elucidating the genetic background of obesity.

We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like C>T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.

The mechanisms that regulate the T(H)9 subset of helper T cells and diseases mediated by T(H)9 cells remain poorly defined. Here we found that the costimulatory receptor OX40 was a powerful inducer of T(H)9 cells in vitro and T(H)9 cell-dependent airway inflammation in vivo. In polarizing conditions based on transforming growth factor-β (TGF-β), ligation of OX40 inhibited the production of induced regulatory T cells and the T(H)17 subset of helper T cells and diverted CD4(+)Foxp3(-) T cells to a T(H)9 phenotype. Mechanistically, OX40 activated the ubiquitin ligase TRAF6, which triggered induction of the kinase NIK in CD4(+) T cells and the noncanonical transcription factor NF-κB pathway; this subsequently led to the generation of T(H)9 cells. Thus, our study identifies a previously unknown mechanism for the induction of T(H)9 cells and may have important clinical implications in allergic inflammation.

<h4>Aims</h4>To determine venesection patterns in hereditary haemochromatosis (HC) patients in Christchurch, New Zealand, their contribution to the blood supply, and reasons for deferral.<h4>Methods</h4>Review of clinical records of 412 HC patients venesected by the NZ Blood Service at least once during 2009.<h4>Results</h4>Of 275 males and 137 females, 384 had been tested for HFE gene mutations--76% were C282Y homozygotes, 12.8%, C282Y/H63D compound heterozygous, 8.6%, either H63D homozygotes, C282Y heterozygotes or H63D heterozygotes. Small numbers had no detectable mutations, were not iron overloaded but had been venesected for isolated hyperferritiniaemia. 53% were donors. C282Y homozygotes required significantly more venesections than patients of other genotypes. Eligible HC patients donated 3 units/donor/year compared to 1.63/person/year by healthy donors (p<0.001). HC patients contributed 3.4% of whole blood collections in 2009. There were 212 permanent or temporary donation deferrals--common reasons were abnormal liver functions, chronic or malignant disease, or immigration from vCJD risk countries.<h4>Conclusions</h4>HC donors donate at nearly twice the rate of healthy donors but contribute only a small amount to the blood pool. Revision of selection criteria may increase this contribution without compromising blood safety.

Hepatic angiomyolipoma is rare and may pose differential diagnostic difficulty, particularly if encountered in core needle biopsy. We studied 6 cases from 5 males and one female (median age, 48.6 yrs). All presented with non-specific symptoms or an incidentally discovered tumor mass. Two patients had a remote history of chemotherapy for hematological neoplasms (acute lymphoblastic leukemia and Hodgkin lymphoma respectively) and another had clear cell renal cell carcinoma and anaplastic pancreatic carcinoma diagnosed at autopsy without definable syndrome. None of the patients had evidence of the tuberous sclerosis complex or renal or other extra-renal angiomyolipoma. Three tumors were resected completely and three have been only biopsied and followed up. None of the resected cases recurred at a mean follow-up of 35 months. Histologically, tumors were classified as classical triphasic (1), lipomatous (2), epithelioid/oncocytoid (1), epithelioid trabecular (1) and myelolipoma-like (1). The adjacent liver parenchyma was normal in 3 cases, showed pigment cirrhosis in one case and mild fatty change in another case. One case had clinically diagnosed but histologically unverified cirrhosis. The initial diagnostic impression/frozen section was misleading in 5 of the cases and included vascular lesion, focal fatty change, myelolipoma, hepatocellular tumor and oncocytic neoplasm. All tumors expressed HMB45 and variably desmin. One epithelioid lesion expressed HMB45 and TFE3, but lacked desmin expression. In conclusion, hepatic angiomyolipomas are increasingly recognized as incidental findings during surveillance for cirrhosis or investigations for unrelated conditions. Awareness of their diverse morphological spectrum in liver biopsy is necessary to avoid misdiagnosis as hepatocellular carcinoma, metastatic melanoma or other malignant neoplasms.

Neural development in metazoans is characterized by the establishment of initial process tracts by pioneer axons and the subsequent extension of follower axons along these pioneer processes. Mechanisms governing the fidelity of follower extension along pioneered routes are largely unknown. In C. elegans, formation of the right angle-shaped lumbar commissure connecting the lumbar and preanal ganglia is an example of pioneer/follower dynamics. We find that the dystroglycan ortholog DGN-1 mediates the fidelity of follower lumbar commissure axon extension along the pioneer axon route. In dgn-1 mutants, the axon of the pioneer PVQ neuron faithfully establishes the lumbar commissure, but axons of follower lumbar neurons, such as PVC, frequently bypass the lumbar commissure and extend along an oblique trajectory directly toward the preanal ganglion. In contrast, disruption of the UNC-6/netrin guidance pathway principally perturbs PVQ ventral guidance to pioneer the lumbar commissure. Loss of DGN-1 in unc-6 mutants has a quantitatively similar effect on follower axon guidance regardless of PVQ axon route, indicating that DGN-1 does not mediate follower/pioneer adhesion. Instead, DGN-1 appears to block premature responsiveness of follower axons to a preanal ganglion-directed guidance cue, which mediates ventral-to-anterior reorientation of lumbar commissure axons. Deletion analysis shows that only the most N-terminal DGN-1 domain is required for these activities. These studies suggest that dystroglycan modulation of growth cone responsiveness to conflicting guidance cues is important for restricting follower axon extension to the tracts laid down by pioneers.

Recent studies have described Huntington's disease (HD) patients with atypical onset of ataxia. Symptoms in these patients can overlap with those of spinocerebellar ataxia (SCA). We retrospectively examined clinical data for 82 HD probands and found 7 had initially been clinically diagnosed as SCA cases. Clinical features in these patients were further investigated and the number of CAG repeats in the huntingtin (HTT) gene was determined by direct sequencing. Genetic screenings for SCAs in the 7 patients were all negative. By contrast, HTT was heterozygous in each patient. The distribution of CAG number in the 7 patients was statistically the same as that in the other 75 patients. Each of 7 HD patients had presented with atypical onset of ataxia. The mean time from onset to HTT genetic testing was 5.6 ± 5.52 years. Three of the patients developed chorea, but the others did not. Our observations confirm the clinical heterogeneity of HD in Han Chinese. Based on these findings, testing for HTT expansions should be considered for clinically diagnosed SCA patients who test negatively in genetic screening of SCA genes.

New mixed-valence iron-nickel dithiolates are described that exhibit structures similar to those of mixed-valence diiron dithiolates. The interaction of tricarbonyl salt [(dppe)Ni(pdt)Fe(CO)(3)]BF(4) ([1]BF(4), where dppe = Ph(2)PCH(2)CH(2)PPh(2) and pdt(2-) = -SCH(2)CH(2)CH(2)S-) with P-donor ligands (L) afforded the substituted derivatives [(dppe)Ni(pdt)Fe(CO)(2)L]BF(4) incorporating L = PHCy(2) ([1a]BF(4)), PPh(NEt(2))(2) ([1b]BF(4)), P(NMe(2))(3) ([1c]BF(4)), P(i-Pr)(3) ([1d]BF(4)), and PCy(3) ([1e]BF(4)). The related precursor [(dcpe)Ni(pdt)Fe(CO)(3)]BF(4) ([2]BF(4), where dcpe = Cy(2)PCH(2)CH(2)PCy(2)) gave the more electron-rich family of compounds [(dcpe)Ni(pdt)Fe(CO)(2)L]BF(4) for L = PPh(2)(2-pyridyl) ([2a]BF(4)), PPh(3) ([2b]BF(4)), and PCy(3) ([2c]BF(4)). For bulky and strongly basic monophosphorus ligands, the salts feature distorted coordination geometries at iron: crystallographic analyses of [1e]BF(4) and [2c]BF(4) showed that they adopt "rotated" Fe(I) centers, in which PCy(3) occupies a basal site and one CO ligand partially bridges the Ni and Fe centers. Like the undistorted mixed-valence derivatives, members of the new class of complexes are described as Ni(II)Fe(I) (S = ½) systems according to electron paramagnetic resonance spectroscopy, although with attenuated (31)P hyperfine interactions. Density functional theory calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the spin for [1e](+) is mostly localized in a Fe(I)-centered d(z(2)) orbital, orthogonal to the Fe-P bond. The PCy(3) complexes, rare examples of species featuring "rotated" Fe centers, both structurally and spectroscopically incorporate features from homobimetallic mixed-valence diiron dithiolates. Also, when the NiS(2)Fe core of the [NiFe]-hydrogenase active site is reproduced, the "hybrid models" incorporate key features of the two major classes of hydrogenase. Furthermore, cyclic voltammetry experiments suggest that the highly basic phosphine ligands enable a second oxidation corresponding to the couple [(dxpe)Ni(pdt)Fe(CO)(2)L](+/2+). The resulting unsaturated 32e(-) dications represent the closest approach to modeling the highly electrophilic Ni-SI(a) state. In the case of L = PPh(2) (2-pyridyl), chelation of this ligand accompanies the second oxidation.

<h4>Background & aims</h4>We aimed to characterize offers of organs to candidates awaiting liver transplantation (LT).<h4>Methods</h4>We analyzed data from the United Network for Organ Sharing registry on all US LT candidates with nonfulminant disease who were offered livers from February 1, 2005, to January 31, 2010, and ultimately received transplants. We excluded candidates with a final Model for End-stage Liver Disease score of less than 15. Livers were classified as high quality if they were from donors 18-50 years of age who were ≥ 170 cm tall, of non-black race, suffered brain death secondary to trauma, hepatitis C antibody-negative, not categorized as high risk by the Centers for Disease Control, and locally or regionally located.<h4>Results</h4>Of 33,389 candidates for LT, 20% died or were removed from the list and 64% received LT; the median (interquartile range) number of liver offers for all candidates was 5 (range, 2-12). Of those who died or were removed from the list, 84% received 1 or more liver offers. Overall, 55% of those who died or were removed from the list, and 57% of those who received LT, received 1 or more offers of a high-quality liver when they had Model for End-stage Liver Disease scores of 15 or greater (P = .005). However, the proportion of last liver offers of high quality to patients who underwent LT was twice that of patients who died or were removed from the list (28% vs 14%; P < .001). Most liver offers (68%) were refused for reasons related to donor quality.<h4>Conclusions</h4>Most candidates for LT who died or were removed from the list received 1 or more offers of a liver beforehand, and 55% received 1 or more offers of a high-quality liver. These findings indicate that a substantial proportion of wait-list mortality results in part from declined livers, rather than lack of opportunity, for transplantation. Understanding the real-time factors involved in the complex decision to accept a liver offer is vital to reducing wait-list mortality for LT candidates.

We report on a de novo 17q21.33 microdeletion, 1.8 Mb in size, detected in a patient with mild intellectual disability, growth retardation, poor weight gain, microcephaly, long face, large beaked nose, thick lower lip, micrognathia and other dysmorphic features. The deletion was detected by whole-genome genotyping BeadChip assay and involves the genomic region between 45,682,246 and 47,544,816 bp on chromosome 17. Among the 24 RefSeq genes included in this deletion are the CA10 and CACNA1G genes that are involved in brain development and neurological processes. A possible candidate gene for the prenatal and postnatal growth retardation is the CHAD gene, which product chondroadherin is a cartilage protein with cell binding properties. These three genes may be responsible for the patient's phenotype.

Trastuzumab is effective in about half of HER2-positive breast cancer patients. The PI3K/Akt signalling pathway plays an important role in the process of primary and secondary resistance to anti-HER2 targeted therapy. We evaluated the relationship between expression, activation and subcellular localization of selected Akt isoforms and response to trastuzumab-based anti-HER2 targeted therapy in patients with HER2-positive metastatic breast cancer. Seventy-four women with verified HER2-positive breast cancer were treated with trastuzumab for metastatic disease. Immunohistochemistry was used to evaluate Akt1, Akt2, pAkt Thr308 and pAkt Ser473 expression. For pAkt, cytoplasmic and nuclear fractions were assessed separately. Even though Akt isoforms were expressed in the majority of tumours, activated Akt (pAkt) was present in the cytoplasm only and not in the nucleus in >20% of tumours, and there was no pAkt at all in another 7-13% of tumours. Patients whose tumours showed strong Akt2 expression and had pAkt (pAkt-Thr308 and/or pAkt-Ser473) detectable in the cytoplasm as well as nucleus (n+c), exhibited improved time to progression (TTP) and overall survival from the initiation of trastuzumab therapy (OSt). Patients with tumours with strong Akt2 and pAkt Thr308 (n+c) had superior TTP (17.0 vs. 7.6 months, P=0.024; HR 0.52) and OSt (51.8 vs. 16.8 months, P=0.0009; HR 0.34) compared to other tumours. Similar results were found for strong Akt2 and pAkt Ser473 (n+c): TTP 13.1 vs. 7.2 months (P=0.085, HR 0.62) and OSt 50.8 vs. 17.0 months (P=0.009; HR 0.45). This study is the first to prove the significance of Akt kinase isoform, activity and compartmentalization for the prediction of response to trastuzumab-based therapy in patients with HER2-positive metastatic breast cancer.

The mechanosensory lateral line, found only in fishes and amphibians, is an important sense organ associated with aquatic life. Lateral line patterns differ among teleost, the most diverse vertebrate taxa, hypothetically in response to selective pressures from different aquatic habitats. In this article, we conduct evolutionary genomic analyses of 34 genes associated with lateral line system development in teleosts to elucidate the significance of contrasting evolutionary rates and changes in the protein coding sequences. We find that duplicated copies of these genes are preferentially retained in the teleost genomes and that episodic events of positive selection have occurred in 22 of the 30 postduplication branches. In general, teleost genes evolved at a faster rate relative to their tetrapod counterparts, and the mutation rates of 26 of the 34 genes differed among teleosts and tetrapods. We conclude that following whole genome duplication, evolutionary rates and episodic events of positive selection on the lateral line system development genes might have been one of the factors favoring the subsequent adaptive radiation of teleosts into diverse habitats. These results provide the foundation for further detailed explorations into lateral line system genes and the evolution of diverse phenotypes and adaptations.

Neutrophils kill bacteria generally through oxidative and nonoxidative mechanisms. Whereas much research has focused on the enzymes essential for neutrophil killing, little is known about the regulatory molecules responsible for such killing. In this study, we investigated the role of olfactomedin 4 (OLFM4), an olfactomedin-related glycoprotein, in neutrophil bactericidal capability and host innate immunity. Neutrophils from OLFM4⁻/⁻ mice have increased intracellular killing of Staphylococcus aureus and Escherichia coli in vitro. The OLFM4⁻/⁻ mice have enhanced in vivo bacterial clearance and are more resistant to sepsis when challenged with S. aureus or E. coli by i.p. injection. OLFM4 was found to interact with cathepsin C, a cysteine protease that plays an important role in bacterial killing and immune regulation. We demonstrated that OLFM4 inhibited cathepsin C activity in vitro and in vivo. The cathepsin C activity in neutrophils from OLFM4⁻/⁻ mice was significantly higher than that in neutrophils from wild-type littermate mice. The activities of three serine proteases (neutrophil elastase, cathepsin G, and proteinase 3), which require cathepsin C activity for processing and maturity, were also significantly higher in OLFM4⁻/⁻ neutrophils. The bacterial killing and clearance capabilities observed in OLFM4⁻/⁻ mice that were enhanced relative to wild-type mice were significantly compromised by the additional loss of cathepsin C in mice with OLFM4 and cathepsin C double deficiency. These results indicate that OLFM4 is an important negative regulator of neutrophil bactericidal activity by restricting cathepsin C activity and its downstream granule-associated serine proteases.

In Huntington's disease (HD; MIM ID #143100), a fatal neurodegenerative disorder, transcriptional dysregulation is a key pathogenic feature. Histone modifications are altered in multiple cellular and animal models of HD suggesting a potential mechanism for the observed changes in transcriptional levels. In particular, previous work has suggested an important link between decreased histone acetylation, particularly acetylated histone H3 (AcH3; H3K9K14ac), and downregulated gene expression. However, the question remains whether changes in histone modifications correlate with transcriptional abnormalities across the entire transcriptome. Using chromatin immunoprecipitation paired with microarray hybridization (ChIP-chip), we interrogated AcH3-gene interactions genome-wide in striata of 12-week old wild-type (WT) and transgenic (TG) R6/2 mice, an HD mouse model, and correlated these interactions with gene expression levels. At the level of the individual gene, we found decreases in the number of sites occupied by AcH3 in the TG striatum. In addition, the total number of genes bound by AcH3 was decreased. Surprisingly, the loss of AcH3 binding sites occurred within the coding regions of the genes rather than at the promoter region. We also found that the presence of AcH3 at any location within a gene strongly correlated with the presence of its transcript in both WT and TG striatum. In the TG striatum, treatment with histone deacetylase (HDAC) inhibitors increased global AcH3 levels with concomitant increases in transcript levels; however, AcH3 binding at select gene loci increased only slightly. This study demonstrates that histone H3 acetylation at lysine residues 9 and 14 and active gene expression are intimately tied in the rodent brain, and that this fundamental relationship remains unchanged in an HD mouse model despite genome-wide decreases in histone H3 acetylation.

Various water-soluble L-valine-, L-glutamate-, and glycine ester prodrugs of two 3-Carboranyl Thymidine Analogs (3-CTAs), designated N5 and N5-2OH, were synthesized for Boron Neutron Capture Therapy (BNCT) of brain tumors since the water solubilities of the parental compounds proved to be insufficient in preclinical studies. The amino acid ester prodrugs were prepared and stored as hydrochloride salts. The water solubilities of these amino acid ester prodrugs, evaluated in phosphate buffered saline (PBS) at pH 5, pH 6 and pH 7.4, improved 48-6600 times compared with parental N5 and N5-2OH. The stability of the amino acid ester prodrugs was evaluated in PBS at pH 7.4, Bovine serum, and Bovine cerebrospinal fluid (CSF). The rate of the hydrolysis in all three incubation media depended primarily on the amino acid promoiety and, to a lesser extend, on the site of esterification at the deoxyribose portion of the 3-CTAs. In general, 3'-amino acid ester prodrugs were less sensitive to chemical and enzymatic hydrolysis than 5'-amino acid ester prodrugs and the stabilities of the latter decreased in the following order: 5'-valine > 5'-glutamate > 5'-glycine. The rate of the hydrolysis of the 5'-amino acid ester prodrugs in Bovine CSF was overall higher than in PBS and somewhat lower than in Bovine serum. Overall, 5'-glutamate ester prodrug of N5 and the 5'-glycine ester prodrugs of N5 and N5-2OH appeared to be the most promising candidates for preclinical BNCT studies.

Tumor necrosis factor superfamily-15 (TNFSF15; also known as VEGI or TL1A) is a unique cytokine that functions in the modulation of vascular homeostasis and inflammation. TNFSF15 is expressed abundantly in established vasculature but is down-regulated at sites of neovascularization such as in cancers and wounds. TNFSF15 inhibits endothelial cell proliferation and endothelial progenitor cell differentiation. Additionally, TNFSF15 stimulates T cell activation, Th1 cytokine production, and dendritic cell maturation. Some of the functions of TNFSF15 are mediated by death receptor-3. We review the experimental evidences on TNFSF15 activities in angiogenesis, vasculogenesis, inflammation, and immune system mobilization.

<h4>Background</h4>Patients with end-stage renal disease (ESRD) have elevated cancer risk. Cancer risk increases with age, but associations of ESRD with specific malignancies are incompletely studied for older individuals.<h4>Methods</h4>We conducted a population-based case-control study (1,029,695 cancer and 99,610 controls) among the U.S. elderly using SEER-Medicare linked data. We defined ESRD as presence of dialysis claims in the 3 months prior to selection.<h4>Results</h4>Although ESRD was not associated with excess cancer risk overall (odds ratio 1.02; 95%CI 0.91-1.14), risk was specifically increased for cancers of the stomach (1.45; 1.16-1.81), small intestine (1.92; 1.27-2.92), colon (1.17; 1.00-1.36), liver (1.53; 1.16-2.01), biliary tract (1.78; 1.20-2.65), lung (1.17; 1.02-1.34), cervix (2.12; 1.39-3.23), kidney (2.42; 2.01-2.92), and for multiple myeloma (1.77; 1.40-2.24) and chronic myeloid leukemia (1.74; 1.08-2.80). The association between liver cancer and ESRD was attenuated upon adjustment for hepatitis B and C infection or diabetes mellitus. Multiple myeloma risk was highest with short ESRD duration (p < 0.0001), possibly reflecting reverse causality, while kidney cancer risk showed a borderline rise over time (p = 0.08).<h4>Conclusions</h4>Among elderly individuals with ESRD, the excess risks for some cancers may reflect immune dysfunction or a high prevalence of other risk factors, such as viral infections or diabetes mellitus. Our results underscore the need for studying biological pathways of carcinogenesis in ESRD.

<h4>Background</h4>Tubulin-binding agents (TBAs) are effective in non-small cell lung cancer (NSCLC) treatment. Both βIII- and βV-tubulins are expressed by cancer cells and may lead to resistance against TBAs.<h4>Methods</h4>Pre-treatment samples from 65 locally advanced or oligometastatic NSCLC patients, who underwent uniform induction chemotherapy with paclitaxel and platinum followed by radiochemotherapy with vinorelbine and platinum were retrospectively analysed by immunohistochemistry. Protein expression of βIII- and βV-tubulin was morphometrically quantified.<h4>Results</h4>Median pre-treatment H-score for βIII-tubulin was 110 (range: 0-290), and 160 for βV-tubulin (range: 0-290). Low βIII-tubulin expression was associated with improved overall survival (OS) (P=0.0127, hazard ratio (HR): 0.328). An association between high βV-tubulin expression and prolonged progression-free survival (PFS, median 19.2 vs 9.4 months in high vs low expressors; P=0.0315, HR: 1.899) was found. Further, high βV-tubulin expression was associated with objective response (median H-score 172.5 for CR+PR vs 120 for SD+PD patients, P=0.0104) or disease control following induction chemotherapy (170 for CR+PR+SD vs 100 for PD patients, P=0.0081), but not radiochemotherapy.<h4>Conclusion</h4>Expression of βV-tubulin was associated with treatment response and PFS following paclitaxel-based chemotherapy of locally advanced and oligometastatic NSCLC patients. Prolonged OS was associated with low levels of βIII-tubulin. Prospective evaluation of βIII/βV-tubulin expression in NSCLC is warranted.

It is unknown whether adipokines derived from adipose tissues modulate endoplasmic reticulum (ER) stress induced in obesity. Here, we show that visceral adipose tissue-derived serine protease inhibitor (vaspin) binds to cell-surface 78-kDa glucose-regulated protein (GRP78), which is recruited from ER to plasma membrane under ER stress. Vaspin transgenic mice were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis, while vaspin-deficient mice developed glucose intolerance associated with upregulation of ER stress markers. With tandem affinity tag purification using HepG2 cells, we identified GRP78 as an interacting molecule. The complex formation of vaspin, GRP78, and murine tumor cell DnaJ-like protein 1 (MTJ-1) (DnaJ homolog, subfamily C, member 1) on plasma membrane was confirmed by cell-surface labeling with biotin and immunoprecipitation in liver tissues and H-4-II-E-C3 cells. The addition of recombinant human vaspin in the cultured H-4-II-E-C3 cells also increased the phosphorylation of Akt and AMP-activated protein kinase (AMPK) in a dose-dependent manner, and anti-GRP78 antibodies completely abrogated the vaspin-induced upregulation of pAkt and pAMPK. Vaspin is a novel ligand for cell-surface GRP78/MTJ-1 complex, and its subsequent signals exert beneficial effects on ER stress-induced metabolic dysfunctions.

Expansions of the noncoding GGGGCC hexanucleotide repeat in the Chromosome 9 open reading frame 72 (C9ORF72) gene cause frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this study we aimed to determine whether the length of the normal-unexpanded-allele of the GGGGCC repeat in C9ORF72 plays a role in the presentation of disease or affects age at onset in C9ORF72 mutation carriers. We also studied whether the GGGGCC repeat length confers risk or affects age at onset in FTD and ALS patients without C9ORF72 repeat expansions. C9ORF72 genotyping was performed in 580 FTD, 995 ALS, and 160 FTD-ALS patients, and 1444 controls, leading to the identification of 211 patients with pathogenic C9ORF72 repeat expansions. No meaningful association between the repeat length of the normal alleles of the GGGGCC repeat in C9ORF72 and disease phenotype or age at onset was observed in C9ORF72 mutation carriers or nonmutation carriers.

We describe a method that combines an optimized titanium dioxide protocol and hydrophilic interaction liquid chromatography to simultaneously enrich, identify and quantify phosphopeptides and formerly N-linked sialylated glycopeptides to monitor changes associated with cell signaling during mouse brain development. We initially applied the method to enriched membrane fractions from HeLa cells, which allowed the identification of 4468 unique phosphopeptides and 1809 formerly N-linked sialylated glycopeptides. We subsequently combined the method with isobaric tagging for relative quantification to compare changes in phosphopeptide and formerly N-linked sialylated glycopeptide abundance in the developing mouse brain. A total of 7682 unique phosphopeptide sequences and 3246 unique formerly sialylated glycopeptides were identified. Moreover 669 phosphopeptides and 300 formerly N-sialylated glycopeptides differentially regulated during mouse brain development were detected. This strategy allowed us to reveal extensive changes in post-translational modifications from postnatal mice from day 0 until maturity at day 80. The results of this study confirm the role of sialylation in organ development and provide the first extensive global view of dynamic changes between N-linked sialylation and phosphorylation.

Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.

<h4>Background and objective</h4>Emerging evidence from biological and epidemiological studies has suggested that body iron stores and heme-iron intake may be related to the risk of type 2 diabetes (T2D). We aimed to examine the association of body iron stores and heme-iron intake with T2D risk by conducting a systematic review and meta-analysis of previously published studies.<h4>Research design and methods</h4>Systematic review and subsequent meta-analysis were conducted by searching MEDLINE database up to June 22, 2012 to identify studies that analyzed the association of body iron stores or dietary heme-iron intake with T2D risk. The meta-analysis was performed using the effect estimates and 95% confidence intervals (CIs) to calculate the pooled risk estimates, while the heterogeneity among studies was examined using the I(2) and Q statistic.<h4>Results</h4>The meta-analysis included 16 high-quality studies: 12 studies analyzed ferritin levels (4,366 T2D patients and 41,091 controls) and 4 measured heme-iron intake (9,246 T2D patients and 179,689 controls). The combined relative risk (RR) comparing the highest and lowest category of ferritin levels was 1.66 (95% CI: 1.15-2.39) for prospective studies, 2.29 (95% CI: 1.48-3.54) for cross-sectional studies with heterogeneity (Q = 14.84, p = 0.01, I(2) = 66.3%; Q = 44.16, p<0.001, I(2) = 88.7%). The combined RR comparing the highest and lowest category of heme-iron intake was 1.31 (95% CI: 1.21-1.43) with heterogeneity (Q = 1.39, p = 0.71, I(2) = 0%). No publication bias was found. Additional 15 studies that were of good quality, had significant results, and analyzed the association between body iron stores and T2D risk were qualitatively included in the systematic review.<h4>Conclusions</h4>The meta-analysis and systematic review suggest that increased ferritin levels and heme-iron intake are both associated with higher risk of T2D.

The inherent cytotoxicity of aberrantly folded protein aggregates contributes substantially to the pathogenesis of amyloid diseases. It was recently shown that a class of evolutionary conserved proteins, called MOAG-4/SERF, profoundly alter amyloid toxicity via an autonomous but yet unexplained mode. We show that the biological function of human SERF1a originates from its atypical ability to specifically distinguish between amyloid and nonamyloid aggregation. This inherently unstructured protein directly affected the aggregation kinetics of a broad range of amyloidogenic proteins in vitro, while being inactive against nonamyloid aggregation. A representative biophysical analysis of the SERF1a:α-synuclein (aSyn) complex revealed that the amyloid-promoting activity resulted from an early and transient interaction, which was sufficient to provoke a massive increase of soluble aSyn amyloid nucleation templates. Therefore, the autonomous amyloid-modifying activity of SERF1a observed in living organisms relies on a direct and dedicated manipulation of the early stages in the amyloid aggregation pathway.

<h4>Purpose</h4>Primary open-angle glaucoma (POAG) is one of the leading causes of irreversible blindness in the world. To make progress in understanding POAG, it is necessary to identify more POAG-causing genes.<h4>Methods</h4>Using haplotype analysis, we found that mutational region is located on chromosome 2 in two families. Furthermore, we screened 11 candidate genes on chromosome 2 by protein-protein interaction (PPI) analysis, including mutS homolog 6 (MSH6), mutS homolog 2 (MSH2), v-rel reticuloendotheliosis viral oncogene homolog (REL), endothelial PAS domain protein 1 (EPAS1), vaccinia related kinase 2 (VRK2), F-box protein 11 (FBXO11), EGF containing fibulin-like extracellular matrix protein 1 (EFEMP1), reticulon 4 (RTN4), RAB1A, member RAS oncogene family (RAB1A), ARP2 actin-related protein 2 homolog (ACTR2), and calmodulin 2 (phosphorylase kinase, delta; CALM2). These 11 genes are all predicted to be related to trabecular meshwork changes and progressive loss of retinal ganglion cells in POAG patients.<h4>Results</h4>According to our study, FBXO11 and VRK2 may interact with tumor protein p53 to regulate mitochondrial membrane permeability, mitochondrial membrane organization, and apoptosis. MSH2 is responsible for repairing DNA mismatches and RTN4 is for neuronal regeneration. Therefore, they are supposed to play a negative role in cellular process in POAG. CALM2 may be involved in retinal ganglion cell death and oxidative damage to cell communication.<h4>Conclusions</h4>The results demonstrate that the genes above may be associated with pathogenesis of POAG.

Huntington's and Parkinson's diseases are neurodegenerative disorders associated with unusual protein interactions. Although the origin and evolution of these diseases are completely different, characteristic deposits of protein aggregates (huntingtin and α-synuclein resp.), are a common feature in both diseases. After these observations, many studies are performed with both proteins. Some of them try to understand the nature and driving forces of the aggregation process; others try to find a correlation between the genetic and failure in protein function. Finally with the combination of both approaches, it was proposed that possible strategies deal with pathologic aggregation. Unfortunately, if protein aggregation is a cause or a consequence of the neurodegeneration observed in these pathologies, it is still debatable. This paper describes the process of aggregation of two proteins: huntingtin and α synuclein. The characteristics of the aggregation reaction of these proteins have been followed with novel methods both in vivo and in vitro; these studies include both the combination with other proteins and the presence of various chemical compounds. The ultimate goal of this study was to summarize recent findings on protein aggregation and its possible role as a therapeutic target in neurodegenerative diseases and their role in biomaterial science.

The serotonergic system has been hypothesized to contribute to the biological susceptibility to type 2 diabetes mellitus (T2DM) and body-mass index (BMI) categories. We investigate a possible association of 5-HTTLPR polymorphism (L and S alleles) in the promoter region of the serotonin transporter gene (SLC6A4) with the development of T2DM and/or higher BMI by analyzing a sample of 138 individuals diagnosed with T2DM and 172 unrelated controls from the Mexican general population. In the total sample genotypes were distributed according to Hardy-Weinberg equilibrium, and S allele frequency was 0.58. There was no statistical association between 5-HTTLPR polymorphism and the development of T2DM in this Mexican population sample (p = 0.12). Nevertheless, logistic regression analysis of the L allele and increased BMI disclosed an association, after adjusting for age, sex and T2DM (p = 0.02, OR 1.74, 95% CI: 1.079-2.808).

Few standardized tools are available for time-efficient screening of emotional health status across diagnostic categories, especially in primary care. We evaluated the 45-question Brief Risk-resilience Index for SCreening (BRISC) and the 15-question mini-BRISC in identifying poor emotional health and coping capacity across a range of diagnostic groups - compared with a detailed clinical assessment - in a large sample of adult outpatients. Participants 18-60 years of age (n = 1079) recruited from 12 medical research and clinical sites completed the computerized assessments. Three index scores were derived from the full BRISC and the mini-BRISC: one for risk (negativity-positivity bias) and two for coping (resilience and social capacity). Summed answers were converted to standardized z-scores. BRISC scores were compared with detailed health assessment and diagnostic interview (for current psychiatric, psychological, and neurological conditions) by clinicians at each site according to diagnostic criteria. Clinicians were blinded to BRISC scores. Clinical assessment stratified participants as having "clinical" (n = 435) or "healthy" (n = 644) diagnostic status. Receiver operating characteristic analyses showed that a z-score threshold of -1.57 on the full BRISC index of emotional health provided an optimal classification of "clinical" versus "healthy" status (sensitivity: 81.2%, specificity: 92.7%, positive predictive power: 80.2%, and negative predictive power: 93.1%). Comparable findings were revealed for the mini-BRISC. Negativity-positivity bias index scores contributed the most to prediction. The negativity-positivity index of emotional health was most sensitive to classifying major depressive disorder (100%), posttraumatic stress disorder (95.8%), and panic disorder (88.7%). The BRISC and mini-BRISC both offer a brief, clinically useful screen to identify individuals at risk of disorders characterized by poor emotion regulation, from those with good emotional health and coping.

Hepatocellular carcinoma (HCC) is diagnosed in more than half a million individuals worldwide every year. It is often invasive and metastatic, resulting in a poor prognosis. Our knowledge of the genomic alterations implicated in HCC initiation and progression is fragmentary, and few molecular alterations unique to HCC are known. We performed whole-exome sequencing for a pleomorphic cell-type HCC tissue and matched normal tissue, and uncovered seven non-synonymous somatic variants in SPATA21, PPCS, CDH12, OR1L3, PCK2, HUWE1 and PHF16. These variants were validated by PCR and sequencing, with the exception of that in PPCS. We further performed a bioinformatics analysis of the six validated variants. The results suggested that the function of the proteins of the three mutated genes, PCK2, HUWE1 and PHF16, may be changed significantly. Among these genes, PCK2, within the insulin signaling pathway, and HUWE1, within the ubiquitin-mediated proteolysis pathway, may be essential for cell proliferation. These pathways are known to be important for hepatocarcinogenesis. Hence, we suggest that PCK2 and HUWE1 are associated with carcinoma cell proliferation in HCC.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by motor, cognitive and behavioral disturbances, caused by the expansion of a CAG trinucleotide repeat in the HD gene. The CAG allele size is the major determinant of age at onset (AO) of motor symptoms, although the remaining variance in AO is highly heritable. The rs7665116 SNP in PPARGC1A, encoding the mitochondrial regulator PGC-1α, has been reported to be a significant modifier of AO in three European HD cohorts, perhaps due to affected cases from Italy. We attempted to replicate these findings in a large collection of (1,727) HD patient DNA samples of European origin. In the entire cohort, rs7665116 showed a significant effect in the dominant model (p value = 0.008) and the additive model (p value = 0.009). However, when examined by origin, cases of Southern European origin had an increased rs7665116 minor allele frequency (MAF), consistent with this being an ancestry-tagging SNP. The Southern European cases, despite similar mean CAG allele size, had a significantly older mean AO (p < 0.001), suggesting population-dependent phenotype stratification. When the generalized estimating equations models were adjusted for ancestry, the effect of the rs7665116 genotype on AO decreased dramatically. Our results do not support rs7665116 as a modifier of AO of motor symptoms, as we found evidence for a dramatic effect of phenotypic (AO) and genotypic (MAF) stratification among European cohorts that was not considered in previously reported association studies. A significantly older AO in Southern Europe may reflect population differences in genetic or environmental factors that warrant further investigation.

Genetic variation in the human serotonin system has long been studied because of its functional consequences and links to various neuropsychiatric and behavior-related disorders. Among non-human primates, the common marmosets (Callithrix jacchus) and tufted capuchins monkeys (Cebus apella) are becoming increasingly used as models to study the effects of genes, environments, and their interaction on physiology and complex behavior. In order to investigate the independent functions of and potential interactions between serotonin-related genes, anxiety and neuropsychiatric disorders, we analyzed the presence and variability of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) in marmoset and capuchin monkeys. By PCR and using heterologous primers from the human sequence, we amplified and then sequenced the corresponding 5-HTT region in marmosets and capuchins. The resulting data revealed the presence of a tandem repeat sequence similar to that described in humans, but unlike humans and other Old World primates, no variable length alleles were detected in these New World monkeys, suggesting that if serotonin transporter is involved in modulating behavior in these animals it does so through different molecular mechanisms.

Cellular differentiation and lineage commitment are considered to be robust and irreversible processes during development. Recent work has shown that mouse and human fibroblasts can be reprogrammed to a pluripotent state with a combination of four transcription factors. We hypothesized that combinatorial expression of chondrocyte-specific transcription factors could directly convert human placental cells into chondrocytes. Starting from a pool of candidate genes, we identified a combination of only five genes (5F pool)-BCL6, T (also called BRACHYURY), c-MYC, MITF, and BAF60C (also called SMARCD3)-that rapidly and efficiently convert postnatal human chorion and decidual cells into chondrocytes. The cells generated expressed multiple cartilage-specific genes, such as Collagen type II α1, LINK PROTEIN-1, and AGGRECAN, and exhibited characteristics of cartilage both in vivo and in vitro. Expression of the endogenous genes for T and MITF was initiated, implying that the cell conversion is due to not only the forced expression of the transgenes, but also to cellular reprogramming by the transgenes. This direct conversion system from noncartilage tissue to cartilaginous tissue is a substantial advance toward understanding cartilage development, cell-based therapy, and oncogenesis of chondrocytes.

Parkinson disease is characterized by the loss of dopaminergic neurons mainly in the substantia nigra. Accumulation of α-synuclein and cell loss has been also reported in many other brain regions including the hippocampus, where it might impair adult neurogenesis, contributing to nonmotor symptoms. However, the molecular mechanisms of these alterations are still unknown. In this report we show that α-synuclein-accumulating adult rat hippocampus neural progenitors present aberrant neuronal differentiation, with reduction of Notch1 expression and downstream signaling targets. We characterized a Notch1 proximal promoter that contains p53 canonical response elements. In vivo binding of p53 represses the transcription of Notch1 in neurons. Moreover, we demonstrated that α-synuclein directly binds to the DNA at Notch1 promoter vicinity and also interacts with p53 protein, facilitating or increasing Notch1 signaling repression, which interferes with maturation and survival of neural progenitors cells. This study provides a molecular basis for α-synuclein-mediated disruption of adult neurogenesis in Parkinson disease.

<h4>Background</h4>There is inconsistent evidence of interaction between stressful events and a serotonin transporter promoter polymorphism (5-HTTLPR) in depression. Recent studies have indicated that the moderating effect of 5-HTTLPR may be strongest when adverse experiences have occurred in childhood and the depressive symptoms persist over time. However, it is unknown whether this gene-environment interaction is present for recurrent depressive disorder and different forms of maltreatment. Therefore, patients with recurrent clinically diagnosed depression and controls screened for the absence of depression were utilised to examine the moderating effect of 5-HTTLPR on associations between specific forms of childhood adversity and recurrent depression.<h4>Method</h4>A sample of 227 recurrent unipolar depression cases and 228 never psychiatrically ill controls completed the Childhood Trauma Questionnaire to assess exposure to sexual, physical and emotional abuse, physical and emotional neglect in childhood. DNA extracted from blood or cheek swabs was genotyped for the short (s) and long (l) alleles of 5-HTTLPR.<h4>Results</h4>All forms of childhood maltreatment were reported as more severe by cases than controls. There was no direct association between 5-HTTLPR and depression. Significant interactions with additive and recessive 5-HTTLPR genetic models were found for overall severity of maltreatment, sexual abuse and to a lesser degree for physical neglect, but not other maltreatment types.<h4>Limitations</h4>The cross-sectional design limits causal inference. Retrospective report of childhood adversity may have reduced the accuracy of the findings.<h4>Conclusions</h4>This study provides support for the role of interplay between 5-HTTLPR and a specific early environmental risk in recurrent depressive disorder.

The purpose of this review paper is to present evidence that rat animal models of alcoholism provide an ideal platform for developing and screening medications that target alcohol abuse and dependence. The focus is on the 5 oldest international rat lines that have been selectively bred for a high alcohol-consumption phenotype. The behavioral and neurochemical phenotypes of these rat lines are reviewed and placed in the context of the clinical literature. The paper presents behavioral models for assessing the efficacy of pharmaceuticals for the treatment of alcohol abuse and dependence in rodents, with particular emphasis on rats. Drugs that have been tested for their effectiveness in reducing alcohol/ethanol consumption and/or self-administration by these rat lines and their putative site of action are summarized. The paper also presents some current and future directions for developing pharmacological treatments targeting alcohol abuse and dependence.

Kynurenic acid (KYNA), a classical ionotropic glutamate receptor antagonist is also purported to block the α7-subtype nicotinic acetylcholine receptor (α7* nAChR). Although many published studies cite this potential effect, few have studied it directly. In this study, the α7*-selective agonist, choline, was pressure-applied to interneurons in hippocampal subregions, CA1 stratum radiatum and hilus of acute brain hippocampal slices from adolescent to adult mice and adolescent rats. Stable α7* mediated whole-cell currents were measured using voltage-clamp at physiological temperatures. The effects of bath applied KYNA on spontaneous glutamatergic excitatory postsynaptic potentials (sEPSC) as well as choline-evoked α7* currents were determined. In mouse hilar interneurons, KYNA totally blocked sEPSC whole-cell currents in a rapid and reversible manner, but had no effect on choline-evoked α7* whole-cell currents. To determine if this lack of KYNA effect on α7* function was due to regional and/or species differences in α7* nAChRs, the effects of KYNA on choline-evoked α7* whole-cell currents in mouse and rat stratum radiatum interneurons were tested. KYNA had no effect on either mouse or rat stratum radiatum interneuron choline-evoked α7* whole-cell currents. Finally, to test whether the lack of effect of KYNA was due to unlikely slow kinetics of KYNA interactions with α7* nAChRs, recordings of a7*-mediated currents were made from slices that were prepared and stored in the presence of 1 mM KYNA (>90 minutes exposure). Under these conditions, KYNA had no measurable effect on α7* nAChR function. The results show that despite KYNA-mediated blockade of glutamatergic sEPSCs, two types of hippocampal interneurons that express choline-evoked α7* nAChR currents fail to show any degree of modulation by KYNA. Our results indicate that under our experimental conditions, which produced complete KYNA-mediated blockade of sEPSCs, claims of KYNA effects on choline-evoked α7* nAChR function should be made with caution.

<h4>Background</h4>Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study.<h4>Objective</h4>The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome.<h4>Methods</h4>A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations.<h4>Results</h4>Eighteen patients (9 males, 9 females) were diagnosed to have Dravet syndrome. Among them, 83% (15/18) had SCN1A mutations including truncating (7), splice site (2) and missense mutations (6). The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05). During the progression of disease, 73% (11/15) had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15) had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant.<h4>Conclusion</h4>A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense variant were identified in a patient with Dravet syndrome.

Stroke is a devastating brain injury that is a leading cause of adult disability with limited treatment options. Using a rat model of middle cerebral artery occlusion (MCAO) to induce cerebral ischemia, we profiled microRNAs (miRNAs), small non-protein coding RNAs, in the ischemic cortex. Many miRNAs were confirmed by qPCR to be robustly upregulated 24 hours following MCAO surgery including miR-155, miR-297a, miR-466f, miR-466h, and miR-1224. In addition, we treated MCAO rats with valproic acid (VPA), a mood stabilizer and histone deacetylase inhibitor. This post-insult treatment was shown to improve neurological deficits and motor performance following MCAO. To provide mechanistic insight into the potential targets and pathways that may underlie these benefits, we profiled miRNAs regulated following this VPA treatment. Two promising post-insult VPA-regulated candidates were miR-331 and miR-885-3p. miR-331 was also regulated by VPA pre-treatment in rat cortical neuronal cultures subjected to oxygen-glucose deprivation, an in vitro ischemic model. The predicted targets of these miRNAs analyzed by Ingenuity Pathway Analysis (IPA) identified networks involved in hematological system development, cell death, and nervous system development. These predicted networks were further filtered using IPA and showed significant associations with neurological diseases including movement disorders, neurodegenerative disorders, damage to cerebral cortex, and seizure disorders among others. Collectively, these data support common disease mechanisms that may be under miRNA control and provide exciting directions for further investigations aimed at elucidating the miRNA mechanisms and targets that may yield new therapies for neurological disorders.

Venous thromboembolism (VTE) is major health problem and is sometimes complicated by lethal pulmonary embolism (PE). Disturbances of the coagulation and anticoagulation systems are important risk factors for VTE. Comparative studies suggest that coagulation and innate immunity have a shared evolutionary origin. It is therefore unsurprising that the immune and coagulation systems are linked, with many molecular components being important for both systems. Systemic inflammation modulates thrombotic responses by suppressing fibrinolysis, upregulating procoagulant, and downregulating anticoagulants, and autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), and Behçet's syndrome have been linked to an increased risk of VTE. Recent reports have further shown that a majority of autoimmune and immune-mediated disorders are linked to an increased risk of venous thrombosis, PE, or VTE. For instance, a Swedish nationwide study found that the risk of PE was increased in the first year after hospitalization for 33 different autoimmune disorders. Especially high risks were noted for several autoimmune diseases such as immune thrombocytopenic purpura, polyarteritis nodosa, polymyositis/dermatomyositis, ulcerative colitis, and SLE. Another study from England, also based on hospitalization data, found that immune-mediated disorders were associated with an increased risk of VTE compared with other medical causes of hospitalization. Multiple mechanisms may operate and disease-specific factors, such as cardiolipin antibodies, have been identified. However, inflammation by itself appears to change the hemostatic balance in a thrombogenic direction. Recent epidemiological studies, together with previous experimental and clinical studies, indicate that autoimmune disorders should not only be viewed as inflammatory disorders, but also hypercoagulable disorders. Research to identify thrombotic risk factors, elucidate the mechanisms involved, and investigate prophylactic regiments is needed. The present review describes the epidemiological, clinical, and experimental evidence for the connection between VTE and autoimmune and immune-mediated disorders.

Oncogene activation leads to dramatic changes in numerous biological pathways controlling cellular division, and results in the initiation of a transcriptional program that promotes transformation. Conversely, it also triggers an irreversible cell cycle exit called cellular senescence, which allows the organism to counteract the potentially detrimental uncontrolled proliferation of damaged cells. Therefore, a tight transcriptional control is required at the onset of oncogenic signal, coordinating both positive and negative regulation of gene expression. Not surprisingly, numerous chromatin modifiers contribute to the cellular response to oncogenic stress. While these chromatin modifiers were initially thought of as mere mediators of the cellular response to oncogenic stress, recent studies have uncovered a direct and specific regulation of chromatin modifiers by oncogenic signals. We review here the diverse functions of chromatin modifiers in the cellular response to oncogenic stress, and discuss the implications of these findings on the regulation of cell cycle progression and proliferation by activated oncogenes.

The p38 mitogen activated protein kinase pathway (MAPK) is known to promote cell survival, endocrine therapy resistance and hormone independent breast cancer cell proliferation. Therefore, we utilized the novel p38 inhibitor RWJ67657 to investigate the relevance of targeting this pathway in the ER (+) breast cancer cell line MCF-7. Our results show that RWJ67657 inhibits both basal and estrogen stimulated phosphorylation of p38α, resulting in decreased activation of the downstream p38α targets hsp27 and MAPAPK. Furthermore, inhibition of p38α by RWJ67657 blocks clonogenic survival of MCF-7 cells with little effect on non-cancerous breast epithelial cells. Even though p38α is known to phosphorylate ERα at residue within ER's hinge region at Thr311, resulting in increased ERα transcriptional activation, our results suggest RWJ67657 inhibits the p38α-induced activation of ER by targeting both the AF-1 and AF-2 activation domains within ERα. We further show that RWJ67657 decreases the transcriptional activity of the ER coactivators SRC-1, SRC-2 and SRC-3. Taken together, our results strongly suggest that in addition to phosphorylating Thr311 within ERα, p38α indirectly activates the ER by phosphorylation and stimulation of the known ERα coactivators, SRC-1, -2 and-3. Overall, our data underscore the therapeutic potential of targeting the p38 MAPK pathway in the treatment of ER (+) breast cancer.

<h4>Background</h4>Chronic alcohol use causes widespread changes in the cellular biology of the amygdala's central nucleus (CeA), a GABAergic center that integrates autonomic physiology with the emotional aspects of motivation and learning. While alcohol-induced neurochemical changes play a role in dependence and drinking behavior, little is known about the CeA's dynamic changes during withdrawal, a period of emotional and physiologic disturbance.<h4>Methods</h4>We used a qRT-PCR platform to measure 139 transcripts in 92 rat CeA samples from control (N = 33), chronically alcohol exposed (N = 26), and withdrawn rats (t = 4, 8, 18, 32, and 48 hours; N = 5, 10, 7, 6, 5). This focused transcript set allowed us to identify significant dynamic expression patterns during the first 48 hours of withdrawal and propose potential regulatory mechanisms.<h4>Results</h4>Chronic alcohol exposure causes a limited number of small magnitude expression changes. In contrast, withdrawal results in a greater number of large changes within 4 hours of removal of the alcohol diet. Sixty-five of the 139 measured transcripts (47%) showed differential regulation during withdrawal. Over the 48-hour period, dynamic changes in the expression of γ-aminobutyric acid type A (GABA(A) ), ionotropic glutamate and neuropeptide system-related G-protein-coupled receptor subunits, and the Ras/Raf signaling pathway were seen as well as downstream transcription factors (TFs) and epigenetic regulators. Four temporally correlated gene clusters were identified with shared functional roles including NMDA receptors, MAPKKK and chemokine signaling cascades, and mediators of long-term potentiation, among others. Cluster promoter regions shared overrepresented binding sites for multiple TFs including Cebp, Usf-1, Smad3, Ap-2, and c-Ets, suggesting a potential regulatory role.<h4>Conclusions</h4>During alcohol withdrawal, the CeA experiences rapid changes in mRNA expression of these functionally related transcripts that were not predicted by measurement during chronic exposure. This study provides new insight into dynamic expression changes during alcohol withdrawal and suggests novel regulatory relationships that potentially impact the aspects of emotional modulation.

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.

Amphiphilic macromolecules (AMs) based on carbohydrate domains functionalized with poly(ethylene glycol) can inhibit the uptake of oxidized low density lipoprotein (oxLDL) mediated by scavenger receptor A (SR-A) and counteract foam cell formation, the characteristic "atherosclerotic" phenotype. A series of AMs was prepared by altering the carbohydrate chemistry to evaluate the influence of backbone architecture on the physicochemical and biological properties. Upon evaluating the degree of polymer-based inhibition of oxLDL uptake in human embryonic kidney cells expressing SR-A, two AMs (2a and 2c) were found to have the most efficacy. Molecular modeling and docking studies show that these same AMs have the most favorable binding energies and most close interactions with the molecular model of the SR-A collagen-like domain. Thus, minor changes in the AMs' architecture can significantly affect the physicochemical properties and inhibition of oxLDL uptake. These insights can be critical for designing optimal AM-based therapeutics for the management of cardiovascular disease.

Abnormal insoluble ubiqitinated protein aggregates are found in the brains of Huntington's disease (HD) patients and in mice transgenic for the HTT mutation. Here, we describe the earliest stages of visible NII formation in brains of R6/2 mice killed between 2 and 6 weeks of age. We found that huntingtin-positive aggregates formed rapidly (within 24-48 hours) in a spatiotemporal manner similar to that we described previously for ubiquitinated inclusions. However, in most neurons, aggregates are not ubiquitinated when they first form. It has always been assumed that mutant huntingtin is recognised as 'foreign' and consequently ubiquitinated and targeted for degradation by the ubiquitin-proteasome system pathway. Our data, however, suggest that aggregation and ubiquitination are separate processes, and that mutant huntingtin fragment is not recognized as 'abnormal' by the ubiquitin-proteasome system before aggregation. Rather, mutant Htt appears to aggregate before it is ubiquitinated, and then either aggregated huntingtin is ubiquitinated or ubiquitinated proteins are recruited into aggregates. Our findings have significant implications for the role of the ubiquitin-proteasome system in the formation of aggregates, as they suggest that this system is not involved until after the first aggregates form.

<h4>Objective</h4>The objective of this study is to develop a method for selective detection of the calcific (hydroxyapatite) component in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues ex vivo. This method uses a novel optical molecular imaging contrast dye, Cy-HABP-19, to target calcified cells and tissues.<h4>Methods</h4>A peptide that mimics the binding affinity of osteocalcin was used to label hydroxyapatite in vitro and ex vivo. Morphological changes in vascular smooth muscle cells were evaluated at an early stage of the mineralization process induced by extrinsic stimuli, osteogenic factors and a magnetic suspension cell culture. Hydroxyapatite components were detected in monolayers of these cells in the presence of osteogenic factors and a magnetic suspension environment.<h4>Results</h4>Atherosclerotic plaque contains multiple components including lipidic, fibrotic, thrombotic, and calcific materials. Using optical imaging and the Cy-HABP-19 molecular imaging probe, we demonstrated that hydroxyapatite components could be selectively distinguished from various calcium salts in human aortic smooth muscle cells in vitro and in calcified cardiovascular tissues, carotid endarterectomy samples and aortic valves, ex vivo.<h4>Conclusion</h4>Hydroxyapatite deposits in cardiovascular tissues were selectively detected in the early stage of the calcification process using our Cy-HABP-19 probe. This new probe makes it possible to study the earliest events associated with vascular hydroxyapatite deposition at the cellular and molecular levels. This target-selective molecular imaging probe approach holds high potential for revealing early pathophysiological changes, leading to progression, regression, or stabilization of cardiovascular diseases.

An adequate supply of iron is essential for normal development of the fetus and newborn child. Iron deficiency and iron deficiency anemia (IDA) during pregnancy increase the risk of preterm birth and low birth weight. Iron is important for development of the fetal brain and cognitive abilities of the newborn. Children born to iron-deficient mothers will start their lives suffering from iron deficiency or even IDA. Oral iron prophylaxis to pregnant women improves iron status and prevents development of IDA. The Danish National Board of Health has since 1992 recommended prophylactic oral iron supplements to all pregnant women and the currently advocated dose is 40-50 mg ferrous iron taken between meals from 10 weeks gestation to delivery. However, 30-40 mg ferrous iron is probably an adequate dose in most affluent societies. In developed countries, individual iron prophylaxis guided by iron status (serum ferritin) has physiological advantages compared to general iron prophylaxis. In contrast, in most developing countries, general iron prophylaxis is indicated, and higher doses of oral iron, for example, 60 mg ferrous iron or even more should be recommended, according to the present iron status situation in the specific populations of women of fertile age and pregnant women.

Colorectal cancer (CRC) is one of the most common causes of death, despite decades of research. Initially considered as a disease due to genetic mutations, it is now viewed as a complex malignancy because of the involvement of epigenetic abnormalities. A functional equivalence between genetic and epigenetic mechanisms has been suggested in CRC initiation and progression. A hallmark of CRC is its pathogenetic heterogeneity attained through at least three distinct pathways: a traditional (adenoma-carcinoma sequence), an alternative, and more recently the so-called serrated pathway. While the alternative pathway is more heterogeneous and less characterized, the traditional and serrated pathways appear to be more homogeneous and clearly distinct. One unsolved question in colon cancer biology concerns the cells of origin and from which crypt compartment the different pathways originate. Based on molecular and pathological evidences, we propose that the traditional and serrated pathways originate from different crypt compartments explaining their genetic/epigenetic and clinicopathological differences. In this paper, we will discuss the current knowledge of CRC pathogenesis and, specifically, summarize the role of genetic/epigenetic changes in the origin and progression of the multiple CRC pathways. Elucidation of the link between the molecular and clinico-pathological aspects of CRC would improve our understanding of its etiology and impact both prevention and treatment.

Prognosis in colorectal cancer patients is quite variable, even after adjustment for clinical parameters such as disease stage and microsatellite instability status. It is possible that the psychological distress experienced by patients, including anxiety and depression, may be correlated with poor prognosis. In the present study, we hypothesize that genetic variations within three genes biologically linked to the stress response, namely serotonin transporter (SLC6A4), brain-derived neurotrophic factor (BDNF), and arginine vasopressin receptor (AVPR1B) genes are associated with prognosis in colorectal cancer patients. We used a population-based cohort of 280 patients who were followed for up to 12.5 years after diagnosis. Our multivariate analysis showed that a tagSNP in the SLC6A4 gene (rs12150214) was a predictor of shorter overall survival (HR: 1.572, 95%CI: 1.142-2.164, p = 0.005) independent of stage, age, grade and MSI status. Additionally, a multivariate analysis using the combined genotypes of three polymorphisms in this gene demonstrated that the presence of any of the minor alleles at these polymorphic loci was an independent predictor of both shorter overall survival (HR: 1.631, 95%CI: 1.190-2.236, p = 0.002) and shorter disease specific survival (HR: 1.691, 95%CI: 1.138-2.512, p = 0.009). The 5-HTT protein coded by the SLC6A4 gene has also been implicated in inflammation. While our results remain to be replicated in other patient cohorts, we suggest that the genetic variations in the SLC6A4 gene contribute to poor survival in colorectal cancer patients.

<h4>Background</h4>High-throughput methods that ascribe a cellular or physiological function for each gene product are useful to understand the roles of genes that have not been extensively characterized by molecular or genetic approaches. One method to infer gene function is "guilt-by-association", in which the expression pattern of a poorly characterized gene is shown to co-vary with the expression of better-characterized genes. The function of the poorly characterized gene is inferred from the known function(s) of the well-described genes. For example, genes co-expressed with transcripts that vary during the cell cycle, development, environmental stresses, and with oncogenesis have been implicated in those processes.<h4>Findings</h4>While examining the expression characteristics of several poorly characterized genes, we noted that we could associate each of the genes with a cellular phenotype by correlating individual gene expression changes with gene set enrichment scores from individual samples. We evaluated the effectiveness of this approach using a modest sized gene expression data set (expO) and a compendium of gene expression phenotypes (MSigDBv3.0). We found the transcripts that correlated best with enrichment in mitochondrial and lysosomal gene sets were mostly related to those processes (89/100 and 44/50, respectively). The reciprocal evaluation, ranking gene sets according to correlation of enrichment with an individual gene's expression, also reflected known associations for prominent genes in the biomedical literature (16/19). In evaluating the model, we also found that 4% of the genome encodes proteins that are associated with small molecule and small peptide signal transduction gene sets, implicating a large number of genes in both internal and external environmental sensing.<h4>Conclusions</h4>Our results show that this approach is useful to infer functions of disparate sets of genes. This method mirrors the biological experimental approaches used by others to associate individual genes with defined gene expression changes. Moreover, the approach can be used beyond discovering genes related to a cellular process to discover meaningful expression phenotypes from a compendium that are associated with a given gene. The effectiveness, versatility, and breadth of this approach make possible its application in a variety of contexts and with a variety of downstream analyses.

Attention deficit/hyperactivity disorder (ADHD) in children is a neurobehavioral disorder characterized by inattention, hyperactivity, and/or impulsivity. The biochemical abnormalities and genetic factors play significant roles in the etiology of ADHD. These symptoms affect the behavior performance and social relationships of children in school and at home. Recently, many studies about biochemical abnormalities in ADHD have been published. Several research groups have also suggested the genetic contribution to ADHD, and attempted to identify susceptibility and candidate genes for this disorder through the genetic linkage and association studies. To date, these studies have reported substantial evidence implicating several genes (dopaminergic: DRD4, DAT1, DRD5, COMT; noradrenergic: DBH, ADRA2A; serotonergic: 5-HTT, HTR1B, HTR2A; cholinergic: CHRNA4, and central nervous system development pathway: SNAP25, BDNF) in the etiology of ADHD. Understanding the biochemistry and genetics of ADHD will allow us to provide a useful addition with other treatment procedures for ADHD.

Whole exome/genome sequencing has been fundamental in the identification of somatic mutations in the spliceosome machinery in myelodysplastic syndromes (MDSs) and other hematologic disorders. SF3B1, splicing factor 3b subunit 1 is mutated in 60%-80% of refractory anemia with ring sideroblasts (RARS) and RARS associated with thrombocytosis (RARS-T), 2 distinct subtypes of MDS and MDS/myeloproliferative neoplasms (MDSs/MPNs). An idiosyncratic feature of RARS/RARS-T is the presence of abnormal sideroblasts characterized by iron overload in the mitochondria, called RS. Based on the high frequency of mutations of SF3B1 in RARS/RARS-T, we investigated the consequences of SF3B1 alterations. Ultrastructurally, SF3B1 mutants showed altered iron distribution characterized by coarse iron deposits compared with wild-type RARS patients by transmission electron microscopy. SF3B1 knockdown experiments in K562 cells resulted in down-regulation of U2-type intron-splicing by RT-PCR. RNA-sequencing analysis of SF3B1 mutants showed differentially used genes relevant in MDS pathogenesis, such as ASXL1, CBL, EZH, and RUNX families. A SF3B pharmacologic inhibitor, meayamycin, induced the formation of RS in healthy BM cells. Further, BM aspirates of Sf3b1 heterozygous knockout mice showed RS by Prussian blue. In conclusion, we report the first experimental evidence of the association between SF3B1 and RS phenotype. Our data suggest that SF3B1 haploinsufficiency leads to RS formation.

HIP1 crystal structures solved in our laboratory revealed abnormalities in the coiled-coil region, suggesting intrinsic plasticity. To test this, specific amino acids in the coiled-coil were mutated. The apparent thermal stability of HIP1 was altered when Thr528 and Glu531 were replaced by leucine, and was enhanced when Lys510 was also mutated. In cells, HIP1 mutant expression produced aggregation. MTS and flow cytometry indicate a correlation between aggregated HIP1 and enhanced cell death. These data support the idea that flexibility of the HIP1 coiled-coil domain is important for normal function and may lead to new insights into Huntington's disease.

The development of a system for the continuous culture of Plasmodium vivax in vitro would benefit from the use of reticulocytes derived from differentiated hematopoietic stem cells (HCS). At present, the need to use both fresh reticulocytes and fresh P. vivax isolates represents a major obstacle towards this goal, particularly for laboratories located in non-endemic countries. Here, we describe a new method for the cryopreservation of HSC-derived reticulocytes to be used for both P. falciparum and P. vivax invasion tests. Cryopreserved P. falciparum and P. vivax isolates could invade both fresh and cryopreserved HSC-derived reticulocytes with similar efficiency. This new technique allows the storage of HSC-derived reticulocytes which can be used for later invasion tests and represents an important step towards the establishment of a continuous P. vivax culture.

Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs), estrogens exert rapid actions via cell membrane-localized ERs (mERs). We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA) and an ERα selective antagonist (MPP dihydrochloride). We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through which E2 participates in anterior pituitary cell renewal during the estrous cycle.

To date, genome-wide association studies (GWAS) have identified at least 32 novel loci for obesity and body mass-related traits. However, the causal genetic variant and molecular mechanisms of specific susceptibility genes in relation to obesity are yet to be fully confirmed and characterised. Here, we examined whether the candidate gene NEGR1 encoding the neuronal growth regulator 1, also termed neurotractin or Kilon, accounts for the obesity association. To characterise the function of NEGR1 for body weight control in vivo, we generated two novel mutant mouse lines, including a constitutive NEGR1-deficient mouse line as well as an ENU-mutagenised line carrying a loss-of-function mutation (Negr1-I87N) and performed metabolic phenotypic analyses. Ablation of NEGR1 results in a small but steady reduction of body mass in both mutant lines, accompanied with a small reduction in body length in the Negr1-I87N mutants. Magnetic resonance scanning reveals that the reduction of body mass in Negr1-I87N mice is due to a reduced proportion of lean mass. Negr1-I87N mutants display reduced food intake and physical activity while normalised energy expenditure remains unchanged. Expression analyses confirmed the brain-specific distribution of NEGR1 including strong expression in the hypothalamus. In vitro assays show that NEGR1 promotes cell-cell adhesion and neurite growth of hypothalamic neurons. Our results indicate a role of NEGR1 in the control of body weight and food intake. This study provides evidence that supports the link of the GWAS candidate gene NEGR1 with body weight control.

Candida albicans, the most prevalent fungal pathogen, undergoes yeast-to-hyphal switch which has long been identified as a key fungal virulence factor. We showed here that the lichen-derived small molecule retigeric acid B (RAB) acted as an inhibitor that significantly inhibited the filamentation of C. albicans, leading to the prolonged survival of nematodes infected by C. albicans. Quantitative real-time PCR analysis and intracellular cAMP measurement revealed RAB regulated the Ras1-cAMP-Efg1 pathway by reducing cAMP level to inhibit the hyphae formation. Confocal microscopic observation showed RAB induced the expression of Dpp3, synthesizing more farnesol, which was confirmed by gas chromatography-mass spectroscopy detection. An adenylyl cyclase activity assay demonstrated RAB could repress the activity of Cdc35 through stimulating farnesol synthesis, thus causing a decrease in cAMP synthesis, leading to retarded yeast-to-hyphal transition. Moreover, reduced levels of intracellular cAMP resulted in the inhibition of downstream adhesins. Together, these findings indicate that RAB stimulates farnesol production that directly inhibits the Cdc35 activity, reducing the synthesis of cAMP and thereby causing the disruption of the morphologic transition and attenuating the virulence of C. albicans. Our work illustrates the underlying mechanism of RAB-dependent inhibition of the yeast-to-hyphal switch and provides a potential application in treating the infection of C. albicans.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.

Valid animal models of psychopathology need to include behavioural readouts informed by human findings. In the probabilistic reversal learning (PRL) task, human subjects are confronted with serial reversal of the contingency between two operant stimuli and reward/punishment and, superimposed on this, a low probability (0.2) of punished correct responses/rewarded incorrect responses. In depression, reward-stay and reversals completed are unaffected but response-shift following punished correct response trials, referred to as negative feedback sensitivity (NFS), is increased. The aims of this study were to: establish an operant spatial PRL test appropriate for mice; obtain evidence for the processes mediating reward-stay and punishment-shift responding; and assess effects thereon of genetically- and pharmacologically-altered serotonin (5-HT) function. The study was conducted with wildtype (WT) and heterozygous mutant (HET) mice from a 5-HT transporter (5-HTT) null mutant strain. Mice were mildly food deprived and reward was sugar pellet and punishment was 5-s time out. Mice exhibited high motivation and adaptive reversal performance. Increased probability of punished correct response (PCR) trials per session (p = 0.1, 0.2 or 0.3) led to monotonic decrease in reward-stay and reversals completed, suggesting accurate reward prediction. NFS differed from chance-level at p PCR = 0.1, suggesting accurate punishment prediction, whereas NFS was at chance-level at p = 0.2-0.3. At p PCR = 0.1, HET mice exhibited lower NFS than WT mice. The 5-HTT blocker escitalopram was studied acutely at p PCR = 0.2: a low dose (0.5-1.5 mg/kg) resulted in decreased NFS, increased reward-stay and increased reversals completed, and similarly in WT and HET mice. This study demonstrates that testing PRL in mice can provide evidence on the regulation of reward and punishment processing that is, albeit within certain limits, of relevance to human emotional-cognitive processing, its dysfunction and treatment.

Photoinitiated polymerization remains a robust method for fabrication of hydrogels, as these reactions allow facile spatial and temporal control of gelation and high compatibility for encapsulation of cells and biologics. The chain-growth reaction of macromolecular monomers, such as acrylated PEG and hyaluronan, is commonly used to form hydrogels, but there is growing interest in step-growth photopolymerizations, such as the thiol-ene "click" reaction, as an alternative. Thiol-ene reactions are not susceptible to oxygen inhibition and rapidly form hydrogels using low initiator concentrations. In this work, we characterize the differences in recovery of bioactive proteins when exposed to similar photoinitiation conditions during thiol-ene versus acrylate polymerizations. Following exposure to chain polymerization of acrylates, lysozyme bioactivity was approximately 50%; after step-growth thiol-ene reaction, lysozyme retained nearly 100% of its prereaction activity. Bioactive protein recovery was enhanced 1000-fold in the presence of a thiol-ene reaction, relative to recovery from solutions containing identical primary radical concentrations, but without the thiol-ene components. When the cytokine TGFβ was encapsulated in PEG hydrogels formed via the thiol-ene reaction, full protein bioactivity was preserved.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

<h4>Objective</h4>The aim of this study was to determine whether tumor necrosis factor superfamily 4 (TNFSF4) and TNF receptor-associated factor 1-complement 5 (TRAF1-C5) polymorphisms confer susceptibility to systemic lupus erythematosus (SLE).<h4>Methods</h4>The authors conducted meta-analyses on associations between polymorphisms of the TNFSF4 (rs2205960, rs1234315, rs10489265) and TRAF1-C5 (rs10818488, rs3761847) genes and SLE susceptibility, using fixed and random effects models.<h4>Results</h4>A total of 21 comparative studies were included in this meta-analysis; meta-analysis showed an association between the minor allele of rs2205960 of TNFSF4 and SLE in all study subjects (odds ratio [OR]=1.356, 95% confidence interval [CI]=1.275-1.442, p<1.0×10(-9)). Meta-analysis revealed an association between the minor alleles of rs1234315 and rs10489265 of TNFSF4 and SLE in Asians (OR=1.366, 95% CI=1.295-1.440, p<1.0×10(-9); OR=1.463, 95% CI=1.208-1.771, p=9.7×10(-5)). The minor allele of rs10818488 of TRAF1-C5 was found to be significantly associated with SLE in Europeans (OR=1.210, 95% CI=1.115-1.313, p=5.0×10(-6)). The association p-values remained significant after multiple corrections.<h4>Conclusions</h4>This meta-analysis confirms that TNFSF4 polymorphisms are associated with susceptibility to SLE in Asians and Europeans. An association was found between the rs10818488 polymorphism of TRAF1-C5 and susceptibility to SLE in Europeans.

The chondroitin sulfate proteoglycan core protein aggrecan is the major protein constituent of cartilage aside from collagen, and is largely responsible for its distinctive mechanical properties. Aggrecan is required both for proper cartilage formation in development and maintenance of mature cartilage. Prominent ACAN transcription is a conserved feature of vertebrate cartilage, although little is known about its specific transcriptional regulation. We examined the genomic interval containing human ACAN for transcriptional enhancers directing expression to cartilage, using a functional assay in transgenic zebrafish. We tested 24 conserved non-coding sequences, representing ~6% of the total sequence in the interval, and identified eleven independently capable of regulating reporter gene expression in cartilage. These enhancers were widely spaced, from >100kb upstream of the gene to within the first intron. While the majority displayed broad cartilage expression in zebrafish larvae, several were restricted to a subset of cartilage cells in the craniofacial skeleton. In older fish, the enhancers displayed differential activity; some maintained expression, either in all cartilage or preferentially in articular cartilage at the joints, while others were not active. This remarkable degree of overlapping regulatory control has been highly conserved; we identified clear orthologues of six enhancers at the chicken ACAN locus, arranged in the same order relative to the gene. These were also functional in directing expression to cartilage in transgenic zebrafish. Several enhancers contain potential binding sites for Sox9, consistent with its described role as an upstream regulator of ACAN expression. However, others lacked Sox9 consensus binding sites, implicating additional pathways and transcription factors as regulators of ACAN expression in cartilage, either in development or adult tissue. Our identification of these enhancer sequences is the necessary first step in detailed examination of the upstream regulators of ACAN expression.

Increases in intracellular free Ca(2+) play a major role in many cellular processes. The deregulation of Ca(2+) signaling is a feature of a variety of diseases, and modulators of Ca(2+) signaling are used to treat conditions as diverse as hypertension to pain. The Ca(2+) signal also plays a role in processes important in cancer, such as proliferation and migration. Many studies in cancer have identified alterations in the expression of proteins involved in the movement of Ca(2+) across the plasma membrane and subcellular organelles. In some cases, these Ca(2+) channels or pumps are potential therapeutic targets for specific cancer subtypes or correlate with prognosis.

Our aim was to employ novel analytical methods to investigate the therapeutic treatment of the energy regulation dysfunction occurring in a Huntington disease (HD) mouse model. HD is a neurodegenerative disorder that is characterized by progressive motor impairment and cognitive alterations. Changes in neuroendocrine function, body weight, energy metabolism, euglycemia, appetite function, and gut function can also occur. It is likely that the locus of these alterations is the hypothalamus. We determined the effects of three different euglycemic agents on HD progression using standard physiological and transcriptomic signature analyses. N171-82Q HD mice were treated with insulin, Exendin-4, and the newly developed GLP-1-Tf to determine whether these agents could improve energy regulation and delay disease progression. Blood glucose, insulin, metabolic hormone levels, and pancreatic morphology were assessed. Hypothalamic gene transcription, motor coordination, and life span were also determined. The N171-82Q mice exhibited significant alterations in hypothalamic gene transcription signatures and energy metabolism that were ameliorated, to varying degrees, by the different euglycemic agents. Exendin-4 or GLP-1-Tf (but not insulin) treatment also improved pancreatic morphology, motor coordination, and increased life span. Using hypothalamic transcription signature analyses, we found that the physiological efficacy variation of the drugs was evident in the degree of reversal of the hypothalamic HD pathological signature. Euglycemic agents targeting hypothalamic and energy regulation dysfunction in HD could potentially alter disease progression and improve quality of life in HD.

Coherent anti-Stokes Raman scattering (CARS) microscopy is applied for the first time for the evaluation of the protein secondary structure of polyglutamine (polyQ) aggregates in vivo. Our approach demonstrates the potential for translating information about protein structure that has been obtained in vitro by X-ray diffraction into a microscopy technique that allows the same protein structure to be detected in vivo. For these studies, fibres of polyQ containing peptides (D(2)Q(15)K(2)) were assembled in vitro and examined by electron microscopy and X-ray diffraction methods; the fibril structure was shown to be cross β-sheet. The same polyQ fibres were evaluated by Raman spectroscopy and this further confirmed the β-sheet structure, but indicated that the structure is highly rigid, as indicated by the strong Amide I signal at 1659 cm(-1). CARS spectra were simulated using the Raman spectrum taking into account potential non-resonant contributions, providing evidence that the Amide I signal remains strong, but slightly shifted to lower wavenumbers. Combined CARS (1657 cm(-1)) and multi-photon fluorescence microscopy of chimeric fusions of yellow fluorescent protein (YFP) with polyQ (Q40) expressed in the body wall muscle cells of Caenorhabditis elegans nematodes (1 day old adult hermaphrodites) revealed diffuse and foci patterns of Q40-YFP that were both fluorescent and exhibited stronger CARS (1657 cm(-1)) signals than in surrounding tissues at the resonance for the cross β-sheet polyQ in vitro.

Posttraumatic stress disorder (PTSD) is a pathological response to a traumatic event. A number of risk and vulnerability factors predicting PTSD development have been identified in the literature. Many of these variables are specific factors occurring during and after exposure to a traumatic event or are not measured prospectively to assess temporal sequence. Recent research, however, has begun to focus on pre-trauma individual differences that could contribute to risk for developing PTSD. The present review proposes that a number of biological and cognitive vulnerability factors place individuals at risk for PTSD development prior to the actual experience of trauma. Accordingly, this review provides a summary of evidence for a select number of these factors as pre-trauma vulnerabilities to PTSD. Included is a discussion of biological factors, including molecular genetic studies of systems regulating serotonin, catecholamines, and glucocorticoids as well as aspects of the neuroendocrine system. Specific cognitive factors are also considered, including intelligence, neuropsychological functioning and cognitive biases such as negative attributional style and appraisals. For each factor, the present review summarizes evidence to date regarding PTSD vulnerability and highlights directions for future research in this area.

The ability to generate human pluripotent stem cells (hPSCs) holds great promise for the understanding and the treatment of human neurological diseases in modern medicine. The hPSCs are considered for their in vitro use as research tools to provide relevant cellular model for human diseases, drug discovery, and toxicity assays and for their in vivo use in regenerative medicine applications. In this review, we highlight recent progress, promises, and challenges of hPSC applications in human neurological disease modeling and therapies.

Juvenile Huntington's disease (JHD) is usually defined as Huntington's disease with an onset ≤ 20 years. The proportion of JHD cases reported in studies of Huntington's disease (HD) varies. A review of the literature found 62 studies that reported the proportion of JHD cases amongst all HD cases. The proportion of JHD cases in these studies ranged from 1% to 15%, and in a meta-analysis the pooled proportion of JHD cases was 4.92% (95% confidence interval of 4.07% to 5.84%). Limiting the analysis to the 25 studies which used multiple methods of ascertainment resulted in a similar pooled proportion of 5.32%, (95% confidence interval 4.18% to 6.60%). A small difference was observed when the meta-analysis was restricted to studies from countries defined by the World Bank as high income, that used multiple methods of ascertainment, and that were conducted since 1980 (4.81%, 95% confidence interval 3.31% to 6.58%, n=11). This contrasts with the pooled result from three post 1980 studies using multiple methods of ascertainment from South Africa and Venezuela, defined by the World Bank as upper middle income, where the estimated mean proportion was 9.95%, (95% confidence interval 6.37% to 14.22%). These results, which are expected to be more robust than those from a single study alone, may be helpful in estimating the proportion of JHD cases in a given population. Key Words: Juvenile Huntington's disease, prevalence, epidemiology.

The ubiquitous filamentous fungus Aspergillus fumigatus secretes a number of allergens with protease activity and has been linked to a variety of allergic conditions such as Severe Asthma with Fungal Sensitization (SAFS) and Allergic Bronchopulmonary Aspergillosis (ABPA). However, it is unclear which allergen proteases are being secreted during fungal invasion and whether the local biological environment regulates their expression. Understanding the dynamic expression of allergen proteases during growth of A. fumigatus may lead to further characterisation of the pathogenesis of these disorders as well as improved standardisation in the commercial production of these allergens. Secretion of proteases during germination and early growth of A. fumigatus was investigated in response to various complex protein sources (pig lung homogenate, mucin or casein). Protease inhibitor studies demonstrated that A. fumigatus (AF293 strain) secretes predominately serine proteases during growth in pig lung based medium and mainly metalloproteases during growth in casein based medium but suppressed protease secretion in unmodified Vogel's minimal medium and secreted both types in mucin based medium. Analysis of gene transcription and protein identification by mass spectrometry showed that the matrix metalloprotease, Mep/Asp f 5 and the serine protease, Alp1/Asp f 13, were upregulated and secreted during growth in pig lung medium, whereas Alp1 was predominately expressed and secreted in mucin based medium. In casein medium, the matrix metalloprotease, Lap1, was also upregulated and secreted in addition to Mep and Alp1. These findings suggest that A. fumigatus is able to detect different complex proteins available as substrates in its environment and regulate protease secretion accordingly. There is a requirement for the standardisation of A. fumigatus allergen extracts used both in clinical diagnosis of A. fumigatus allergy and in research studies.

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.

<h4>Introduction</h4>The estrogen receptor (ER) co-regulator proline glutamic acid and leucine-rich protein 1 (PELP1) is a proto-oncogene that modulates epigenetic changes on ER target gene promoters via interactions with lysine-specific histone demethylase 1 (KDM1). In this study, we assessed the therapeutic potential of targeting the PELP1-KDM1 axis in vivo using liposomal (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine; DOPC) siRNA to downregulate PELP1 expression and KDM1 inhibitors, pargyline and N-((1S)-3-(3-(trans-2-aminocyclopropyl)phenoxy)-1-(benzylcarbamoyl)propyl)benzamide using preclinical models.<h4>Methods</h4>Preclinical xenograft models were used to test the efficacy of drugs in vivo. Ki-67 and terminal deoxynucleotidyl transferase dUTP nick end-labeling immunohistochemical analysis of epigenetic markers was performed on tumor tissues. The in vitro effect of PELP1-KDM axis blockers was tested using proliferation, reporter gene, chromatin immunoprecipitation and real-time RT-PCR assays. The efficacy of the KDM1 targeting drugs alone or in combination with letrozole and tamoxifen was tested using therapy-resistant model cells.<h4>Results</h4>Treatment of ER-positive xenograft-based breast tumors with PELP1-siRNA-DOPC or pargyline reduced tumor volume by 58.6% and 62%, respectively. In a postmenopausal model, in which tumor growth is stimulated solely by local estrogen synthesis, daily pargyline treatment reduced tumor volume by 78%. Immunohistochemical analysis of excised tumors revealed a combined decrease in cellular proliferation, induction of apoptosis and upregulation of inhibitory epigenetic modifications. Pharmacological inhibition of KDM1 in vitro increased inhibitory histone mark dimethylation of histone H3 at lysine 9 (H3K9me2) and decreased histone activation mark acetylation of H3K9 (H3K9Ac) on ER target gene promoters. Combining KDM1 targeting drugs with current endocrine therapies substantially impeded growth and restored sensitivity of therapy-resistant breast cancer cells to treatment.<h4>Conclusion</h4>Our results suggest inhibition of PELP1-KDM1-mediated histone modifications as a potential therapeutic strategy for blocking breast cancer progression and therapy resistance.

<h4>Background</h4>The genetic architecture responsible for chronic kidney disease (CKD) remains incompletely described. The Oligosyndactyly (Os) mouse models focal and segmental glomerulosclerosis (FSGS), which is associated with reduced nephron number caused by the Os mutation. The Os mutation leads to FSGS in multiple strains including the ROP-Os/+. However, on the C57Bl/6J background the mutation does not cause FSGS, although nephron number in these mice are equivalent to those in ROP-Os/+ mice. We exploited this phenotypic variation to identify genes that potentially contribute to glomerulosclerosis.<h4>Methods</h4>To identify such novel genes, which regulate susceptibility or resistance to renal disease progression, we generated and compared the renal transcriptomes using serial analysis of gene expression (SAGE) from the sclerosis-prone ROP-Os/+ and sclerosis resistant C57-Os/+ mouse kidneys. We confirmed the validity of the differential gene expression using multiple approaches. We also used an Ingenuity Pathway Analysis engine to assemble differentially regulated molecular networks. Cell culture techniques were employed to confirm functional relevance of selected genes.<h4>Results</h4>A comparative analysis of the kidney transcriptomes revealed multiple genes, with expression levels that were statistically different. These novel, candidate, renal disease susceptibility/resistance genes included neuropilin2 (Nrp2), glutathione-S-transferase theta (Gstt1) and itchy (Itch). Of 34 genes with the most robust statistical difference in expression levels between ROP-Os/+ and C57-Os/+ mice, 13 and 3 transcripts localized to glomerular and tubulointerstitial compartments, respectively, from micro-dissected human FSGS biopsies. Network analysis of all significantly differentially expressed genes identified 13 connectivity networks. The most highly scored network highlighted the roles for oxidative stress and mitochondrial dysfunction pathways. Functional analyses of these networks provided evidence for activation of transforming growth factor beta (TGFβ) signaling in ROP-Os/+ kidneys despite similar expression of the TGFβ ligand between the tested strains.<h4>Conclusions</h4>These data demonstrate the complex dysregulation of normal cellular functions in this animal model of FSGS and suggest that therapies directed at multiple levels will be needed to effectively treat human kidney diseases.

The majority of proteins in eukaryotes are composed of multiple domains, and the number and order of these domains is an important determinant of protein function. Although multidomain proteins with a particular domain architecture were initially considered to have a common evolutionary origin, recent comparative studies of protein families or whole genomes have reported that a minority of multidomain proteins could have appeared multiple times independently. Here, we test this scenario in detail for the signaling molecules netrin and secreted frizzled-related proteins (sFRPs), two groups of netrin domain-containing proteins with essential roles in animal development. Our primary phylogenetic analyses suggest that the particular domain architectures of each of these proteins were present in the eumetazoan ancestor and evolved a second time independently within the metazoan lineage from laminin and frizzled proteins, respectively. Using an array of phylogenetic methods, statistical tests, and character sorting analyses, we show that the polyphyly of netrin and sFRP is well supported and cannot be explained by classical phylogenetic reconstruction artifacts. Despite their independent origins, the two groups of netrins and of sFRPs have the same protein interaction partners (Deleted in Colorectal Cancer/neogenin and Unc5 for netrins and Wnts for sFRPs) and similar developmental functions. Thus, these cases of convergent evolution emphasize the importance of domain architecture for protein function by uncoupling shared domain architecture from shared evolutionary history. Therefore, we propose the terms merology to describe the repeated evolution of proteins with similar domain architecture and discuss the potential of merologous proteins to help understanding protein evolution.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in humans. Recent studies revealed that patterns of microRNA (miRNA) expression in GBM tissue samples are different from those in normal brain tissues, suggesting that a number of miRNAs play critical roles in the pathogenesis of GBM. However, little is yet known about which miRNAs play central roles in the pathology of GBM and their regulatory mechanisms of action. To address this issue, in this study, we systematically explored the main regulation format (feed-forward loops, FFLs) consisting of miRNAs, transcription factors (TFs) and their impacting GBM-related genes, and developed a computational approach to construct a miRNA-TF regulatory network. First, we compiled GBM-related miRNAs, GBM-related genes, and known human TFs. We then identified 1,128 3-node FFLs and 805 4-node FFLs with statistical significance. By merging these FFLs together, we constructed a comprehensive GBM-specific miRNA-TF mediated regulatory network. Then, from the network, we extracted a composite GBM-specific regulatory network. To illustrate the GBM-specific regulatory network is promising for identification of critical miRNA components, we specifically examined a Notch signaling pathway subnetwork. Our follow up topological and functional analyses of the subnetwork revealed that six miRNAs (miR-124, miR-137, miR-219-5p, miR-34a, miR-9, and miR-92b) might play important roles in GBM, including some results that are supported by previous studies. In this study, we have developed a computational framework to construct a miRNA-TF regulatory network and generated the first miRNA-TF regulatory network for GBM, providing a valuable resource for further understanding the complex regulatory mechanisms in GBM. The observation of critical miRNAs in the Notch signaling pathway, with partial verification from previous studies, demonstrates that our network-based approach is promising for the identification of new and important miRNAs in GBM and, potentially, other cancers.

Histone deacetylase (HDAC) inhibitors, especially vorinostat, are currently under investigation as potential adjuncts in the treatment of neuroblastoma. The effect of vorinostat co-treatment on the development of resistance to other chemotherapeutic agents is unknown. In the present study, we treated two human neuroblastoma cell lines [SK-N-SH and SK-N-Be(2)C] with progressively increasing doses of doxorubicin under two conditions: with and without vorinsotat co-therapy. The resultant doxorubicin-resistant (DoxR) and vorinostat-treated doxorubicin resistant (DoxR-v) cells were equally resistant to doxorubicin despite significantly lower P-glycoprotein expression in the DoxR-v cells. Whole genome analysis was performed using the Ilumina Human HT-12 v4 Expression Beadchip to identify genes with differential expression unique to the DoxR-v cells. We uncovered a number of genes whose differential expression in the DoxR-v cells might contribute to their resistant phenotype, including hypoxia inducible factor-2. Finally, we used Gene Ontology to categorize the biological functions of the differentially expressed genes unique to the DoxR-v cells and found that genes involved in cellular metabolism were especially affected.

There is increasing evidence that dysregulation of CD4(+) T cell populations leads to intestinal inflammation, but the regional distribution of these populations throughout the intestinal tract in healthy individuals remains unclear. Here, we show that T(H)17, T(H)22 and T(Reg) cells are enriched in the healthy human cecum compared to the terminal ileum and sigmoid colon, whereas T(H)1 and T(H)2 cells do not significantly vary by location. Transcriptional profiling analysis of paired pinch biopsies from different regions of the intestine identified significant differences in the metabolic state of the terminal ileum, cecum, and sigmoid colon. An increased proportion of T(H)17 cells was positively associated with expression of resistin (RETN) and negatively associated with expression of trefoil factor 1 (TFF1). These results suggest that CD4(+) T helper cells that are important in maintaining mucosal barrier function may be enriched in the cecum as a result of metabolic differences of the surrounding microenvironment.

Remarkable advances in cellular reprogramming have made it possible to generate pluripotent stem cells from somatic cells, such as fibroblasts obtained from human skin biopsies. As a result, human diseases can now be investigated in relevant cell populations derived from induced pluripotent stem cells (iPSCs) of patients. The rapid growth of iPSC technology has turned these cells into multipurpose basic and clinical research tools. In this paper, we highlight the roles of iPSC technology that are helping us to understand and potentially treat neurological diseases. Recent studies using iPSCs to model various neurogenetic disorders are summarized, and we discuss the therapeutic implications of iPSCs, including drug screening and cell therapy for neurogenetic disorders. Although iPSCs have been used in animal models with promising results to treat neurogenetic disorders, there are still many issues associated with reprogramming that must be addressed before iPSC technology can be fully exploited with translation to the clinic.

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

Impairment of energy metabolism is a key feature of Huntington disease (HD). Recently, we reported longitudinal neurochemical changes in R6/2 mice measured by in-vivo proton magnetic resonance spectroscopy ((1)H MRS; Zacharoff et al, 2012). Here, we present similar (1)H MRS measurements at an early stage in the milder Q111 mouse model. In addition, we measured the concentration of ATP and inorganic phosphate (P(i)), key energy metabolites not accessible with (1)H MRS, using (31)P MRS both in Q111 and in R6/2 mice. Significant changes in striatal creatine and phosphocreatine were observed in Q111 mice at 6 weeks relative to control, and these changes were largely reversed at 13 weeks. No significant change was detected in ATP concentration, in either HD mouse, compared with control. Calculated values of [ADP], phosphorylation potential, relative rate of ATP synthase (v/V(max)(ATP)), and relative rate of creatine kinase (v/V(max)(CK)) were calculated from the measured data. ADP concentration and v/V(max)(ATP) were increased in Q111 mice at 6 weeks, and returned close to normal at 13 weeks. In contrast, these parameters were normal in R6/2 mice. These results suggest that early changes in brain energy metabolism are followed by compensatory shifts to maintain energetic homeostasis from early ages through manifest disease.

In eukaryotic cells, the shape of mitochondria can be tuned to various physiological conditions by a balance of fusion and fission processes termed mitochondrial dynamics. Mitochondrial dynamics controls not only the morphology but also the function of mitochondria, and therefore is crucial in many aspects of a cell's life. Consequently, dysfunction of mitochondrial dynamics has been implicated in a variety of human diseases including cancer. Several proteins important for mitochondrial fusion and fission have been discovered over the past decade. However, there is emerging evidence that there are as yet unidentified proteins important for these processes and that the fusion/fission machinery is not completely conserved between yeast and vertebrates. The recent characterization of several mammalian proteins important for the process that were not conserved in yeast, may indicate that the molecular mechanisms regulating and controlling the morphology and function of mitochondria are more elaborate and complex in vertebrates. This difference could possibly be a consequence of different needs in the different cell types of multicellular organisms. Here, we review recent advances in the field of mitochondrial dynamics. We highlight and discuss the mechanisms regulating recruitment of cytosolic Drp1 to the mitochondrial outer membrane by Fis1, Mff, and MIEF1 in mammals and the divergences in regulation of mitochondrial dynamics between yeast and vertebrates.

Hydroxyapatite (Hap) is a calcium phosphate with a chemical formula that closely resembles that of the mineral constituents found in hard tissues, thereby explaining its natural biocompatibility and wide biomedical use. Nanostructured Hap materials appear to present a good performance in bone tissue applications because of their ability to mimic the dimensions of bone components. However, bone cell response to individual nanoparticles and/or nanoparticle aggregates lost from these materials is largely unknown and shows great variability. This work addresses the preparation and characterization of two different Hap nanoparticles and their interaction with osteoblastic cells. Hap particles were produced by a wet chemical synthesis (WCS) at 37°C and by hydrothermal synthesis (HS) at 180°C. As the ultimate in vivo applications require a sterilization step, the synthesized particles were characterized 'as prepared' and after sterilization (autoclaving, 120°C, 20 min). WCS and HS particles differ in their morphological (size and shape) and physicochemical properties. The sterilization modified markedly the shape, size and aggregation state of WCS nanoparticles. Both particles were readily internalized by osteoblastic cells by endocytosis, and showed a low intracellular dissolution rate. Concentrations of WCS and HS particles less than 500 μg ml(-1) did not affect cell proliferation, F-actin cytoskeleton organization and apoptosis rate and increased the gene expression of alkaline phosphatase and BMP-2. The two particles presented some differences in the elicited cell response. In conclusion, WCS and HS particles might exhibit an interesting profile for bone tissue applications. Results suggest the relevance of a proper particle characterization, and the interest of an individual nanoparticle targeted research.

To characterize somatic alterations in colorectal carcinoma, we conducted a genome-scale analysis of 276 samples, analysing exome sequence, DNA copy number, promoter methylation and messenger RNA and microRNA expression. A subset of these samples (97) underwent low-depth-of-coverage whole-genome sequencing. In total, 16% of colorectal carcinomas were found to be hypermutated: three-quarters of these had the expected high microsatellite instability, usually with hypermethylation and MLH1 silencing, and one-quarter had somatic mismatch-repair gene and polymerase ε (POLE) mutations. Excluding the hypermutated cancers, colon and rectum cancers were found to have considerably similar patterns of genomic alteration. Twenty-four genes were significantly mutated, and in addition to the expected APC, TP53, SMAD4, PIK3CA and KRAS mutations, we found frequent mutations in ARID1A, SOX9 and FAM123B. Recurrent copy-number alterations include potentially drug-targetable amplifications of ERBB2 and newly discovered amplification of IGF2. Recurrent chromosomal translocations include the fusion of NAV2 and WNT pathway member TCF7L1. Integrative analyses suggest new markers for aggressive colorectal carcinoma and an important role for MYC-directed transcriptional activation and repression.

<h4>Objective</h4>The effect of mHTT on human development was examined by evaluating measures of growth in children at risk for Huntington disease (HD).<h4>Methods</h4>Children at risk for HD with no manifest symptoms (no juvenile HD included) were enrolled and tested for gene expansion for research purposes only. Measurements of growth (height, weight, body mass index [BMI], and head circumference) in children tested as gene-expanded (n = 20, 7-18 years of age, CAG repeats ≥39) were compared to those of a large database of healthy children (n = 152, 7-18 years of age).<h4>Results</h4>Gene-expanded children had significantly lower measures of head circumference, weight, and BMI. Head circumference was abnormally low even after correcting for height, suggesting a specific deficit in brain growth, rather than a global growth abnormality.<h4>Conclusions</h4>These results indicate that, compared to a control population, children who were estimated to be decades from HD diagnosis have significant differences in growth. Further, they suggest that mHTT may play a role in atypical somatic, and in particular, brain development.

The mammalian JAM family is composed of three cell surface receptors. Interactions between the proteins have well-characterised roles in inflammation and tight junction formation, but little is known about their function in early development. Recently, we identified a role for jamb and jamc in zebrafish myocyte fusion. Genome duplication in the teleost lineage raised the possibility that additional JAM family paralogues may also function in muscle development. To address this, we searched the zebrafish genome to identify potential paralogues and confirmed their homology, bringing the total number of zebrafish jam family members to six. We then compared the physical binding properties of each paralogue by surface plasmon resonance and determined the gene expression patterns of all zebrafish jam genes at different stages of development. Our results suggest a significant sub-functionalisation of JAM-B and JAM-C orthologues with respect to binding strength (but not specificity) and gene expression. The paralogous genes, jamb2 and jamc2, were not detected in the somites or myotome of wild-type embryos. We conclude that it is unlikely that the paralogues have a function in primary myogenesis.

Huntington's disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients, animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression. An understanding of the effects of mHtt may lead to strategies to slow the progression of HD. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of the current models using two N-mHtt-affected promoters in two cell models of HD using overexpression of known N-mHtt-interacting transcription factors, promoter deletion and mutation analyses and in vitro promoter binding assays. Consequently, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at promoters. In this model, N-mHtt interacts with multiple partners whose presence and affinity for N-mHtt influence the severity of gene dysregulation. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as why some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

<h4>Objective</h4>Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, and early tumor detection is the most promising approach for improving the EOC survival rate. The goal of this study was to identify the biomarkers underlying ovarian carcinogenesis.<h4>Study design</h4>To mimic the onset and progression of human ovarian cancer, we established a rat model of ovarian neoplasm by implanting 7,12-dimethylbenz(a)anthracene (DMBA)-coated silk cloth strips onto the ovaries. Sera collected from rats bearing serous ovarian carcinoma (SOC) at baseline, 12 and 24 weeks after DMBA treatment and from controls were analyzed using iTRAQ combined with two-dimensional liquid chromatography and tandem mass spectrometry. The data were analyzed with ProteinPilot software for peptide matching, protein identification, and protein quantitation. Ingenuity pathway analysis software was used to identify the canonical pathways and biological interaction networks of differentially expressed proteins.<h4>Results</h4>The cumulative ovarian tumor incidence rate reached 75% at 32 weeks after DMBA treatment. Out of all tumors, 94% were EOC, and 51% of the EOC cases were SOC. A total of 225 unique, non-redundant proteins were identified with 95% confidence. Twenty-seven differentially expressed proteins were significantly up- or down-regulated during the early or advanced carcinogenesis of SOC. Fifteen proteins were previously reported to be involved in ovarian cancer, and 12 proteins, including MMRN1, SERPINC1, TLN1, AHSG, PLG, APOA2, HPX, APOC1, APOC2, FERMT3, FETUB and HBB, were identified for the first time in our study.<h4>Conclusion</h4>The discovery of these differentially expressed proteins provides valuable clues for understanding the molecular mechanism underlying the dynamic carcinogenic process of ovarian cancer. These proteins could be used as diagnostic biomarkers for early detection, disease monitoring and therapeutic targets.

Mesenchymal stem cells (MSCs) are used in many of the current stem cell-based clinical trials and their therapeutic efficacy has increasingly been attributed to secretion of paracrine factors. We have previously demonstrated that a therapeutic constituent of this secretion is exosome, a secreted bilipid membrane vesicle of ~50-100 nm with a complex cargo that is readily internalized by H9C2 cardiomyocytes. It reduces infarct size in a mouse model of myocardial ischemia/reperfusion (MI/R) injury. We postulate that this therapeutic efficacy is derived from the synergy of a select permutation of individual exosome components. To identify protein candidates in this permutation, the proteome was profiled and here we identified 20S proteasome as a protein candidate. Mass spectrometry analysis detected all seven α and seven β chains of the 20S proteasome, and also the three beta subunits of "immunoproteasome" with a very high confidence level. We demonstrated that a functional proteasome copurified with MSC exosomes with a density of 1.10-1.18 g/mL, and its presence correlated with a modest but significant reduction in oligomerized protein in a mouse model of myocardial infarction. Circulating proteasomes in human blood also copurified with exosomes. Therefore, 20S proteasome is a candidate exosome protein that could synergize with other constituents to ameliorate tissue damage.

Huntington's disease (HD) is a fatal inherited disorder leading to selective neurodegeneration and neuropsychiatric symptoms. Currently, there is no treatment to slow down or to stop the disease. There is also no therapy to effectively reduce the symptoms. In the investigation of novel therapies, different animal models of Huntington's disease, varying from insects to nonhuman primates, have been created and used. Few years ago, the first transgenic rat model of HD, carrying a truncated huntingtin cDNA fragment with 51 CAG repeats under control of the native rat huntingtin promoter, was introduced. We have been using this animal model in our research and review here our experience with the behavioural, neurophysiological, and histopathological phenotype of the transgenic Huntington's disease rats with relevant literature.

A chemically dynamic spermine-based polymer: polyspermine imidazole-4, 5-amide (PSIA, Mw > 7 kDa) was designed, synthesized, and evaluated in terms of its ability to deliver nucleic acids. This polymer was made from an endogenous monomer professionally condensing genes in sperms, spermine, and a known safety drug metabolite, imidazole-4, 5-dicarboxylic acid, through a bis-amide bond conjugated with the imidazole ring. This polymer can condense pDNA at a W/W ratio above 10 to form polyplexes (100-200 nm in diameter), which is consistent with the observation by transmission electron microscopy (TEM), and the zeta potential was in the range of 10-20 mV. The pDNA packaged polymer was stable in phosphate buffer solution (PBS) at pH 7.4 (simulated body fluid) while the polyplexes were releasing pDNA into the solution at pH 5.8 (simulated endo-lysosomes) due to the degradation of the bis-amide linkages in response to changes in pH values. PSIA-polyplexes were able to achieve efficient cellular uptake and luciferase gene silencing by co-transfection of pDNA and siRNA in COS-7 cells and HepG2 cells with negligible cytotoxicity. Biodistribution of Rhodamine B-labeled PSIA-polyplexes after being systemically injected in BALB/c nude-mice showed that the polyplexes circulated throughout the body, accumulated mainly in the kidney at 4 hours of sample administration, and moved to the liver and spleen after 24 hours. All the results suggested that PSIA offered a promising example to balance the transfection efficiency and toxicity of a synthetic carrier system for the delivery of therapeutic nucleic acids.

A folate conjugated ternary copolymer, FA-PEG-PEI-PCL, of poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), and poly(ɛ-caprolactone) (PCL) was synthesized. The copolymer self-assembled into cationic micelles capable of co-delivering siRNA and the anticancer drug doxorubicin (DOX). This dual functional nanocarrier demonstrated low cytotoxicity and high performance in drug/siRNA delivery. Upon the codelivery of siRNA, targeting the Bcl-2 gene, and DOX, using the folate-targeted nanocarrier, DOX-induced apoptosis in the skov-3 cells overexpressing folate receptor was significantly enhanced through a mechanism of downregulating the antiapoptotic protein Bcl-2, while simultaneously upregulating the proapoptotic protein Bax. This work suggested that the combination of Bcl-2 siRNA and DOX therapies is feasible, based on our dual functional nanocarrier, which set up a good basis for a future in vivo test.

New approaches consisting of 'multistage' vaccines against (TB) are emerging that combine early antigenic proteins with latency-associated antigens. In this study, HspX was tested for its potential to elicit both short- and long-term protective immune responses. HspX is a logical component in vaccine strategies targeting protective immune responses against primary infection, as well as against reactivation of latent infection, because as previously shown, it is produced during latency, and as our studies show, it elicits protection within 30 days of infection. Recent studies have shown that the current TB vaccine, bacilli Calmette-Guerin (BCG), does not induce strong interferon-γ T-cell responses to latency-associated antigens like HspX, which may be in part why BCG fails to protect against reactivation disease. We therefore tested HspX protein alone as a prophylactic vaccine and as a boost to BCG vaccination, and found that HspX purified from M. tuberculosis cell lysates protected mice against aerosol challenge and improved the protective efficacy of BCG when used as a booster vaccine. Native HspX was highly immunogenic and protective, in a dose-dependent manner, in both short- and long-term infection models. Based on these promising findings, HspX was produced as a recombinant protein in E. coli, as this would enable facile purification; however, recombinant HspX (rHspX) alone consistently failed to protect against aerosol challenge. Incubation of rHspX with mycobacterial cell lysate and re-purification following incubation restored the capacity of the protein to confer protection. These data suggest the possibility that the native form may chaperone an immunogenic and protective antigen that is mycobacteria-specific.

The Eph family of receptors is the largest family of receptor tyrosine kinases, but it remains poorly studied in lung cancer. We aimed to systematically explore the human Eph receptors and their ligands, the ephrins, in lung adenocarcinoma. The prognostic impact of Eph receptor and ephrin gene expression was analyzed using 2 independent cohorts of lung adenocarcinoma. Gene expression profiles in lung adenocarcinoma compared with normal adjacent lung were studied in 3 independent cohorts and in cell lines. Gene expression profiles were validated with quantitative polymerase chain reaction (qPCR) and Western blotting in cell lines. Functional studies to assess the role of Eph receptor A4 (EphA4) were carried out in vitro. The biological effects of EphA4 in lung cancer cell lines were assayed following overexpression and knockdown. Of the 11 Eph receptors and 8 ephrins analyzed, only EphA4 and ephrin A1 gene expression were consistently associated with an improved outcome in patients with lung adenocarcinoma. Expression levels of EphA4 by microarray correlated well with expression levels measured by qPCR and Western blotting. EphA4 overexpression reduced cell migration and invasion but did not affect cell cycle, apoptosis, or drug sensitivity. Surprisingly, EphA4 was expressed at higher levels in cancer compared with non-cancer tissues and cell lines. EphA4 gene expression is associated with an improved outcome in patients with resected lung adenocarcinoma, possibly by affecting cancer cell migration and invasion.

Changes in adipose tissue metabolism are central to adaptation of whole body energy homeostasis to pregnancy. To gain insight into the molecular mechanisms supporting tissue remodeling, we have characterized the longitudinal changes of the adipose transcriptome in human pregnancy. Healthy nonobese women recruited pregravid were followed in early (8-12 wk) and in late (36-38 wk) pregnancy. Adipose tissue biopsies were obtained in the fasting state from the gluteal depot. The adipose transcriptome was examined via whole genome DNA microarray. Expression of immune-related genes and extracellular matrix components was measured using real-time RT-PCR. Adipose mass, adipocyte size, and cell number increased in late pregnancy compared with pregravid measurements (P < 0.001) but remained unchanged in early pregnancy. The adipose transcriptome evolved during pregnancy with 10-15% of genes being differently expressed compared with pregravid. Functional gene cluster analysis revealed that the early molecular changes affected immune responses, angiogenesis, matrix remodeling, and lipid biosynthesis. Increased expression of macrophage markers (CD68, CD14, and the mannose-6 phosphate receptor) emphasized the recruitment of the immune network in both early and late pregnancy. The TLR4/NF-κB signaling pathway was enhanced specifically in relation to inflammatory adipokines and chemokines genes. We conclude that early recruitment of metabolic and immune molecular networks precedes the appearance of pregnancy-related physiological changes in adipose tissue. This biphasic pattern suggests that physiological inflammation is an early step preceding the development of insulin resistance, which peaks in late pregnancy.

Enterotoxigenic Escherichia coli (ETEC) strains that produce heat-stable (ST) and/or heat-labile (LT) enterotoxins are cause of post-weaning diarrhea in piglets. However, the relative importance of the different enterotoxins in host immune responses against ETEC infection has been poorly defined. In the present study, several isogenic mutant strains of an O149:F4ac(+), LT(+) STa(+) STb(+) ETEC strain were constructed that lack the expression of LT in combination with one or both types of ST enterotoxins (STa and/or STb). The small intestinal segment perfusion (SISP) technique and microarray analysis were used to study host early immune responses induced by these mutant strains 4 h after infection in comparison to the wild type strain and a PBS control. Simultaneously, net fluid absorption of pig small intestinal mucosa was measured 4 h after infection, allowing us to correlate enterotoxin secretion with gene regulation. Microarray analysis showed on the one hand a non-toxin related general antibacterial response comprising genes such as PAP, MMP1 and IL8. On the other hand, results suggest a dominant role for STb in small intestinal secretion early after post-weaning infection, as well as in the induced innate immune response through differential regulation of immune mediators like interleukin 1 and interleukin 17.

Intra-articular delivery of therapeutics to modulate osteoarthritis (OA) is challenging. Delivery of interleukin-1 receptor antagonist (IL-1Ra), the natural protein inhibitor of IL-1, to modulate IL-1-based inflammation through gene therapy or bolus protein injections has emerged as a promising therapy for OA. However, these approaches suffer from rapid clearance and reduced potency over time. Nano/microparticles represent a promising strategy for overcoming the shortcomings of intra-articular drug delivery. However, these delivery vehicles are limited for delivery of protein therapeutics due to their hydrophobic character, low drug loading efficiency, and harsh chemical conditions during particle processing. We designed a new block copolymer that assembles into submicron-scale particles and provides for covalently tethering proteins to the particle surface for controlled intra-articular protein delivery. This block copolymer self-assembles into 300 nm-diameter particles with a protein tethering moiety for surface covalent conjugation of IL-1Ra protein. This copolymer particle system efficiently bound IL-1Ra and maintained protein bioactivity in vitro. Furthermore, particle-tethered IL-1Ra bound specifically to target synoviocyte cells via surface IL-1 receptors. Importantly, IL-1Ra nanoparticles inhibited IL-1-mediated signaling to equivalent levels as soluble IL-1Ra. Finally, the ability of nanoparticles to retain IL-1Ra in the rat stifle joint was evaluated by in vivo imaging over 14 days. IL-1Ra-tethered nanoparticles significantly increased the retention time of IL-1Ra in the rat stifle joint over 14 days with enhanced IL-1Ra half-life (3.01 days) compared to that of soluble IL-1Ra (0.96 days) and without inducing degenerative changes in cartilage structure or composition.

No abstract available.

3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogues were synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6 position, an iodine at the 3'or 4' position of the phenyl ring, and an N-allyl or c-propylmethyl group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA) with a N-allyl group displayed lower δ opioid receptor affinity than its naltrexamine analogue, was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or physical dependence, and only limited inhibition of gastrointestinal transit. Thus, the aryl-naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with advantageous side-effect profile and greater selectivity.

Transition metals occupy an essential niche in biological systems. Their electrostatic properties stabilize substrates or reaction intermediates in the active sites of enzymes, and their heightened reactivity is harnessed for catalysis. However, this heightened activity also renders transition metals toxic at high concentrations. Bacteria, like all living organisms, must regulate their intracellular levels of these elements to satisfy their physiological needs while avoiding harm. It is therefore not surprising that the host capitalizes on both the essentiality and toxicity of transition metals to defend against bacterial invaders. This Review discusses established and emerging paradigms in nutrient metal homeostasis at the pathogen-host interface.

Misfolding and aggregation of huntingtin is one of the hallmarks of Huntington disease, but the overall structure of these aggregates and the mechanisms by which huntingtin misfolds remain poorly understood. Here we used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the structural features of huntingtin exon 1 (HDx1) containing 46 glutamine residues in its polyglutamine (polyQ) region. Despite some residual structuring in the N terminus, we find that soluble HDx1 is highly dynamic. Upon aggregation, the polyQ domain becomes strongly immobilized indicating significant tertiary or quaternary packing interactions. Analysis of spin-spin interactions does not show the close contact between same residues that is characteristic of the parallel, in-register structure commonly found in amyloids. Nevertheless, the same residues are still within 20 Å of each other, suggesting that polyQ domains from different molecules come into proximity in the fibrils. The N terminus has previously been found to take up a helical structure in fibrils. We find that this domain not only becomes structured, but that it also engages in tertiary or quaternary packing interactions. The existence of spin-spin interactions in this region suggests that such contacts could be made between N-terminal domains from different molecules. In contrast, the C-terminal domain is dynamic, contains polyproline II structure, and lacks pronounced packing interactions. This region must be facing away from the core of the fibrils. Collectively, these data provide new constraints for building structural models of HDx1 fibrils.

BCL11A was the focus of recent studies on its inhibiting effect when bound onto the β-globin cluster in the mechanism of hemoglobin switching and HbF downregulation. We examined a cohort of 10 patients displaying different HbF levels and short deletions within the γβ-δ intergenic region to find a possible correlation with the BCL11A binding site located 5' to the δ-globin gene. Precise characterization of deletions was achieved using a custom DNA-array chip and breakpoint sequencing. The α-globin cluster and major SNP associated with HbF expression were genotyped. Our results show that the loss of the BCL11A binding domain located 5' to the δ-globin gene is correlated with a strong HbF difference (mean+2.7 g/dL, ratio 2.81). This result provides evidence for the use of BCL11A level down-regulation or this domain blockage for new therapies in sickle cell disease and β-thalassemia major patients.

Phospholipids are of critical importance in mammalian cell biology, both through providing a permeability barrier and acting as substrates for synthesis of lipid mediators. Recently, several new families of bioactive lipids were identified that form through the enzymatic oxidation of membrane phospholipids in circulating innate immune cells and platelets. These comprise eicosanoids attached to phosphatidylethanolamine and phosphatidylcholine and form within 2-5 minutes of cell activation by pathophysiologic agonists, via the coordinated action of receptors and enzymes. In this review, we summarize what is currently known regarding their structures, mechanisms of formation, cell biology, and signaling actions. We show that phospholipid oxidation by acutely activated immune cells is a controlled event, and we propose a central role in regulating membrane biology and innate immune function during health and disease. We also review the mass spectrometry methods used for identification of the lipids and describe how these approaches can be used for discovery of new lipid mediators in complex biologic samples.

RNA helicases are ubiquitous in plants and animals and function in many cellular processes. Retroviruses, such as human immunodeficiency virus (HIV-1), encode no RNA helicases in their genomes and utilize host cellular RNA helicases at various stages of their life cycle. Here, we briefly summarize the roles RNA helicases play in HIV-1 replication that have been identified recently, in part, through genome-wide screenings, proteomics, and molecular studies. Some of these helicases augment virus propagation while others apparently participate in antiviral defenses against viral replication.

Alzheimer's disease (AD) is a progressive neurodegenerative disease involving the alteration of gene expression at the whole genome level. Genome-wide transcriptional profiling of AD has been conducted by many groups on several relevant brain regions. However, identifying the most critical dys-regulated genes has been challenging. In this work, we addressed this issue by deriving critical genes from perturbed subnetworks. Using a recent microarray dataset on six brain regions, we applied a heaviest induced subgraph algorithm with a modular scoring function to reveal the significantly perturbed subnetwork in each brain region. These perturbed subnetworks were found to be significantly overlapped with each other. Furthermore, the hub genes from these perturbed subnetworks formed a connected hub network consisting of 136 genes. Comparison between AD and several related diseases demonstrated that the hub network was robustly and specifically perturbed in AD. In addition, strong correlation between the expression level of these hub genes and indicators of AD severity suggested that this hub network can partially reflect AD progression. More importantly, this hub network reflected the adaptation of neurons to the AD-specific microenvironment through a variety of adjustments, including reduction of neuronal and synaptic activities and alteration of survival signaling. Therefore, it is potentially useful for the development of biomarkers and network medicine for AD.

<h4>Background</h4>The characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research and relatively little is known governing these processes in allergic inflammation. Here we present novel findings defining the miRNA and mRNA transcriptome in eosinophilic esophagitis (EoE), an increasing recognized allergic disorder.<h4>Methods</h4>Esophageal epithelial miRNA and mRNA from five paired biopsies pre- and post-treatment with glucocorticosteroids were profiled using Taqman and Affymetrix arrays. Validation was performed on additional paired biopsies, untreated EoE specimens and normal controls. Differentially regulated miRNAs and mRNAs were generated, within which miRNA-mRNA target pairs with high predicted confidence were identified.<h4>Results</h4>Compared to the post-glucocorticoid treated esophageal mucosa, of all the 377 miRNA sequences examined, 32 miRNAs were significantly upregulated and four downregulated in the pre-treated biopsies. MiR-214 was the most upregulated (150 fold) and miR-146b-5b, 146a, 145, 142-3p and 21 were upregulated by at least 10 fold. Out of 12 miRNAs chosen for validation by qRT-PCR, five (miR-214, 146b-5p, 146a, 142-3p and 21) were confirmed and 11 shared the same trend. When the expression of the 12 miRNAs in the EoE mucosa was compared to unrelated normal mucosa, six (miR-214, 146b-5p, 146a, 21, 203, and 489) showed similar significant changes as in the paired samples and 10 of them shared the same trend. In the same five pairs of samples used to profile miRNA, 311 mRNAs were down-regulated and 35 were up-regulated in pre-treated EoE mucosa. Among them, 164 mRNAs were identified as potential targets of differentially regulated miRNAs. Further analysis revealed that immune-related genes, targeted and non-targeted by miRNAs, were among the most important genes involved in the pathogenesis of EoE.<h4>Conclusions</h4>Our findings add to the accumulating body of data defining a regulatory role for miRNA in immune and allergic processes.

<h4>Background</h4>High body iron store has been associated with an increased risk of type 2 diabetes (T2D); it remains unknown whether the genetic variants related to body iron status affect T2D risk. We aimed at comprehensively investigating the associations between the genetic variants related to body iron status and the T2D risk.<h4>Methodology/principal findings</h4>Six common SNPs related to body iron status from recent genome-wide association (GWA) studies were determined in the Nurses' Health Study (NHS; 1,467 diabetic cases and 1,754 controls) and the Health Professionals Follow-up Study (HPFS; 1,124, diabetic cases and 1,298 controls). Plasma levels of ferritin, soluble transferrin receptor (sTfR), and transferrin were measured in NHS. Significant associations were observed for loci in TPMRSS6 with sTfR (P = 3.47×10(-6)), TF with transferrin (P = 0.0002 to 1.72×10(-10)); and HFE with ferritin (P = 0.017 to 1.6×10(-8)), sTfR (P = 0.007 to 7.9×10(-6)), and transferrin (P = 0.006 to 0.0007). The six SNPs together explained 5.7%, 2.7%, and 13.3% of the variation in plasma levels of ferritin, sTfR, and transferrin. After adjustment for the conventional risk factors, the T allele of SNP rs855791 in the TPMRSS6 gene was significantly associated with a 19% decreased risk of T2D (OR = 0.81; 95% CI = 0.66-0.98; P = 0.03) in men. Multiple tests attenuated this significant association to null. No associations were observed in women. SNPs at HFE and TF were not associated with diabetes risk in either sex. Dietary iron intake did not modify the associations of the newly identified loci with diabetes risk.<h4>Conclusions/significance</h4>The newly identified iron-related SNP rs855791 in TPMRSS6 was nominally associated with a decreased risk of T2D in men but not in women. The apparent differences by gender warrant further study.

No abstract available.

<h4>Background</h4>We have recently identified a number of active regulatory networks involved in diabetes progression in Goto-Kakizaki (GK) rats by network screening. The networks were quite consistent with the previous knowledge of the regulatory relationships between transcription factors (TFs) and their regulated genes. To study the underlying molecular mechanisms directly related to phenotype changes, such as diseases, we also previously developed a computational procedure for identifying transcriptional master regulators (MRs) in conjunction with network screening and network inference, by effectively perturbing the phenotype states.<h4>Results</h4>In this work, we further improved our previous method for identifying MR candidates, by listing them in a more reliable manner, and applied the method to reveal the MR candidates for diabetes progression in GK rats from the active networks. Specifically, the active TF-gene pairs for different time periods in GK rats were first extracted from the networks by network screening. Another set of active TF-gene pairs was selected by network inference, by considering the gene expression signatures for those periods between GK and Wistar-Kyoto (WKY) rats. The TF-gene pairs extracted by the two methods were then further selected, from the viewpoints of the emergence specificity of TF in GK rats and the regulated-gene coverage of TF in the expression signature. Finally, we narrowed all of the genes down to only 5 TFs (Etv4, Fus, Nr2f1, Sp2, and Tcfap2b) as the candidates of MRs, with 54 regulated genes, by merging the selected TF-gene pairs.<h4>Conclusions</h4>The present method has successfully identified biologically plausible MR candidates, including the TFs related to diabetes in previous reports. Although the experimental verifications of the candidates and the present procedure are beyond the scope of this study, we narrowed down the candidates to 5 TFs, which can be used to perform the verification experiments relatively easily. The numerical results showed that our computational method is an efficient way to detect the key molecules responsible for biological phenomena.

The Drosha-DGCR8 complex (Microprocessor) is required for microRNA (miRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as the endonuclease. Using high-throughput sequencing and cross-linking immunoprecipitation (HITS-CLIP) we identified RNA targets of DGCR8 in human cells. Unexpectedly, miRNAs were not the most abundant targets. DGCR8-bound RNAs also comprised several hundred mRNAs as well as small nucleolar RNAs (snoRNAs) and long noncoding RNAs. We found that the Microprocessor controlled the abundance of several mRNAs as well as of MALAT1. By contrast, DGCR8-mediated cleavage of snoRNAs was independent of Drosha, suggesting the involvement of DGCR8 in cellular complexes with other endonucleases. Binding of DGCR8 to cassette exons is a new mechanism for regulation of the relative abundance of alternatively spliced isoforms. These data provide insights in the complex role of DGCR8 in controlling the fate of several classes of RNAs.

The Raf kinase inhibitor protein (RKIP) is a tumor suppressor that protects against metastasis and genomic instability. RKIP is downregulated in many types of tumors, although the mechanism for this remains unknown. MicroRNAs silence target genes via translational inhibition or target mRNA degradation, and are thus important regulators of gene expression. In the current study, we found that miR-224 expression is significantly upregulated in breast cancer cell lines, and especially in highly invasive MDA-MB-231 cells, compared to human normal breast epithelial cells. In addition, miR-224 inhibits RKIP gene expression by directly targeting its 3'-untranslated region (3'-UTR). Moreover, metastasis, as assayed by Transwell migration, 3D growth in Matrigel, and wound healing, was enhanced by ectopic expression of miR-224 and inhibited by miR-224 downregulation. Promotion of metastasis in response to miR-224 downregulation was associated with derepression of the stroma-associated RKIP target genes, CXCR4, MMP1, and OPN, which are involved in breast tumor metastasis to the bone. Taken together, our data indicate that miR-224 play an important role in metastasis of human breast cancer cells to the bone by directly suppressing the RKIP tumor suppressor.

Lanthanide ion luminescence has a long lifetime enabling highly sensitive detection in time-gated mode. The sensitivity can be further increased by using multiple luminescent labels attached to a carrier molecule, which can be conjugated to an object of interest. We found that up to 30 lanthanide chelates can be attached to avidin creating highly bright constructs. These constructs with Eu(3+) chelates display synergistic effect that enhance the brightness of heavily modified samples, while the opposite effect was observed for Tb(3+) chelates thereby significantly reducing their light emission. This undesirable quenching of Tb(3+) luminophores was completely suppressed by the introduction of an aromatic spacer between the chelate and the protein attachment site. The estimated detection limit for the conjugates is in the 10(-14)-10(-15) M range. We demonstrated a high sensitivity for the new probes by using them to label living cells of bacterial and mammalian origin.

CCT chaperonin is a highly conserved molecular chaperone, which plays an important role in the folding of complex proteins in mammalian cells. CCT chaperonin interacts with huntingtin and results in decrease of aggregate formation followed by increase of cell survival. Using yeast-two-hybrid system, we screen for specific CCT chaperonin subunit, which can recognize and bind to androgen receptor. We show that subunit 6 of CCT chaperonin interacts with androgen receptor. Interestingly, CCT chaperonin shows higher binding affinity to polyglutamine expanded androgen receptor than that of the wild-type. We prove this interaction in mammalian cell models, which show co-localization of androgen receptor and subunit 6 of CCT in cellular cytosol. Therefore, not only huntingtin but also androgen receptor is a polyglutamine expanded protein, which is a substrate of CCT chaperonin. Our results suggest that CCT might play an essential role in modulation of folding of polyglutamine expanded proteins and could be another target for further therapeutic studies.

To investigate the effects of genetic and non-genetic factors on bone mineral densities (BMDs) and osteoporotic fractures. This was a cross-sectional study to investigate the relationships between 18 SNPs and non-genetic factors with BMDs and osteoporotic fractures in 1012 Chinese Han women. Five SNPs in genes GPR177, CTNNB1, MEF2C, SOX6, and TNFRSF11B were associated with L1-4 or total hip BMDs. rs11898505 in SPTBN1 gene was associated with osteoporotic fractures. Subjects carrying the largest number of risk alleles (highest 10 %) not only had lower BMD values as compared to those carrying the least number of risk alleles (lowest 10 %), they also had a higher risk of fracture [P = 0.002, OR = 2.252, 95 %CI (1.136, 4.463)]. Results from multivariate stepwise regression analysis revealed that age [P < 0.001, OR = 1.038, 95 % CI (1.018, 1.058)], number of falls in a year [P < 0.001, OR = 2.347, 95 % CI (1.459, 3.774)], the G risk allele in rs11898505 [P = 0.023, OR = 1.559, 95 % CI (1.062, 2.290)], and the L1-4 BMD [P = 0.017, OR = 0.286, 95 % CI (0.102, 0.798)] were associated with the occurrence of osteoporotic fractures. Genetic (rs11898505) and non-genetic factors (age, number of falls in a year and L1-4 BMD) could work in concert to contribute to the risk of osteoporotic fractures.

Chromosomal rearrangements affecting the MLL gene are associated with high-risk pediatric, adult and therapy-associated acute leukemia. In this study, conventional cytogenetic, fluorescence in situ hybridization, and molecular genetic studies were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloid leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant.

Tumor necrosis factor (TNF-α) is a cytokine involved in systemic inflammation during acute phase reactions. The current study was designed to investigate the levels of pro-inflammatory cytokine (TNF-α) along with the anti-inflammatory cytokine (IL-10) during progression of non-alcoholic fatty liver disease (NAFLD) from simple steatosis to non-alcoholic steatohepatitis (NASH) and fibrosis in diabetic patients, and correlate the levels of cytokines with the progression of NAFLD. Fifty-two diabetic patients compared to 18 healthy controls were participated in this study. Based on clinical diagnosis, patients were divided into three groups: simple steatosis, NASH and fibrosis. Serum liver function tests, fasting blood glucose, bilirubin, ALT, AST, TNF-α, IL-10 and lipid profile were measured. TNF-α levels were significantly higher in NAFLD patients compared to control subjects with a significant positive correlation with body mass index and fasting blood glucose (FBG) but with negative correlation with IL-10. Serum IL-10 levels were significantly lower in NAFLD patients compared with controls. A positive correlation between IL-10 and HDL-C with concomitant negative correlation between IL-10 and FBG and triacylglycerides was found. Cytokine analyses showed that there was a prominent imbalance between TNF-α and IL-10 in patients with NAFLD, and this imbalance increase by increasing the progression of NAFLD especially in obese diabetic patients. TNF-α and IL-10 could be used in diagnosis and follow-up of NAFLD stages in a way to avoid liver biopsies in greater proportion of patients.

<h4>Background</h4>Genome-wide association studies (GWAS) are useful to reveal an association between single nucleotide polymorphisms and different measures of obesity. A multitude of new loci has recently been reported, but the exact function of most of the according genes is not known. The aim of our study was to start elucidating the function of some of these genes.<h4>Methods</h4>We performed an expression analysis of fourteen genes, namely BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MCR4, MTCH2, NEGR1, NRXN3, TMEM18, SEC16B and TFAP2B, via real-time RT-PCR in adipose tissue of the kidney capsule, the mesenterium and subcutaneum as well as the hypothalamus of obese Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats at an age of 22 weeks.<h4>Results</h4>All of our target genes except for SEC16B showed the highest expression in the hypothalamus. This suggests a critical role of these obesity-related genes in the central regulation of energy balance. Interestingly, the expression pattern in the hypothalamus showed no differences between obese ZDF and lean ZL rats. However, LYPLAL1, TFAP2B, SEC16B and FAIM2 were significantly lower expressed in the kidney fat of ZDF than ZL rats. NEGR1 was even lower expressed in subcutaneous and mesenterial fat, while MTCH2 was higher expressed in the subcutaneous and mesenterial fat of ZDF rats.<h4>Conclusion</h4>The expression pattern of the investigated obesity genes implies for most of them a role in the central regulation of energy balance, but for some also a role in the adipose tissue itself. For the development of the ZDF phenotype peripheral rather than central mechanisms of the investigated genes seem to be relevant.

Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30-250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells (P < 0.001). Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.

Non-coding RNAs (ncRNAs) have emerged as critical regulators of transcription, epigenetic processes, and gene silencing, which make them ideal candidates for insight into molecular evolution and a better understanding of the molecular pathways of neuropsychiatric disease. Here, we provide an overview of the current state of knowledge regarding various classes of ncRNAs and their role in neural plasticity and cognitive function, and highlight the potential contribution they may make to the development of a variety of neuropsychiatric disorders, including schizophrenia, addiction, and fear-related anxiety disorders.

Liver transplantation is a therapeutic option of choice for acute and chronic end-stage liver disease. Indications, contraindications, and surgical procedures for the liver transplantation have become well established. In most part of the world, the main source of liver for transplantation remains the donation after brain death (DBD), but in view of increasing death on the waiting list due to shortage of brain dead organs other options such as split liver transplantation, living donor liver transplantation (LDLT), and donation after cardiac death (DCD) have been used. In the pretransplantation era, liver failure was nearly universally fatal, with mortality from fulminant hepatic failure of 80-90 %, and 1-year mortality in decompensated cirrhosis of more than 50 %. In contrast, liver transplantation patient survival is presently more than 85 % at 1 year and more than 70 % at 5 years, emphasizing the clinical benefit of liver transplantation for either acute or chronic liver failure.

<h4>Background</h4>Ipilimumab induces long-lasting clinical responses in a minority of patients with metastatic melanoma. To better understand the mechanism(s) of action and to identify novel biomarkers associated with the clinical benefit and toxicity of ipilimumab, baseline characteristics and changes in CD4(+) and CD8(+) T cells from melanoma patients receiving ipilimumab were characterized by gene profiling and flow cytometry.<h4>Methods</h4>Microarray analysis of flow-cytometry purified CD4(+) and CD8(+) T cells was employed to assess gene profiling changes induced by ipilimumab. Selected molecules were further investigated by flow cytometry on pre, 3-month and 6-month post-treatment specimens.<h4>Results</h4>Ipilimumab up-regulated Ki67 and ICOS on CD4(+) and CD8(+) cells at both 3- and 6-month post ipilimumab (p ≤ 0.001), decreased CCR7 and CD25 on CD8(+) at 3-month post ipilimumab (p ≤ 0.02), and increased Gata3 in CD4(+) and CD8(+) cells at 6-month post ipilimumab (p ≤ 0.001). Increased EOMES(+)CD8(+), GranzymeB(+)EOMES(+)CD8(+) and decreased Ki67(+)EOMES(+)CD4(+) T cells at 6 months were significantly associated with relapse (all p ≤ 0.03). Decreased Ki67(+)CD8(+) T cells were significantly associated with the development of irAE (p = 0.02). At baseline, low Ki67(+)EOMES(+)CD8(+) T cells were associated with relapse (p ≤ 0.001), and low Ki67(+)EOMES(+)CD4(+) T cells were associated with irAE (p ≤ 0.008).<h4>Conclusions</h4>Up-regulation of proliferation and activation signals in CD4(+) and CD8(+) T cells were pharmacodynamic markers for ipilimumab. Ki67(+)EOMES(+)CD8(+) and Ki67(+)EOMES(+)CD4(+)T cells at baseline merit further testing as biomarkers associated with outcome and irAEs, respectively.

The inborn errors of heme biosynthesis, the porphyrias, are 8 genetically distinct metabolic disorders that can be classified as "acute hepatic," "hepatic cutaneous," and "erythropoietic cutaneous" diseases. Recent advances in understanding their pathogenesis and molecular genetic heterogeneity have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias, new understanding of the pathogenesis of the acute hepatic porphyrias, identification of the iron overload-induced inhibitor of hepatic uroporphyrin decarboxylase activity that causes the most common porphyria, porphyria cutanea tarda, the identification of an X-linked form of erythropoietic protoporphyria due to gain-of-function mutations in erythroid-specific 5-aminolevulinate synthase (ALAS2), and new and experimental treatments for the erythropoietic porphyrias. Knowledge of these advances is relevant for hematologists because they administer the hematin infusions to treat the acute attacks in patients with the acute hepatic porphyrias, perform the chronic phlebotomies to reduce the iron overload and clear the dermatologic lesions in porphyria cutanea tarda, and diagnose and treat the erythropoietic porphyrias, including chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for congenital erythropoietic protoporphyria. These developments are reviewed to update hematologists on the latest advances in these diverse disorders.

A hyperferritinemia has to be interpreted in relation with age and sex. The clinical evaluation begins with the interpretation of transferrine saturation which has to be controlled with a second fasting blood test. In case of high transferrine saturation associated with hyperferritinemia, HFE testing has first to be realized since the first diagnosis suspected is a HFE hemochromatosis. In case of normal transferrine saturation associated with a hyperferritinemia, the more frequent diagnosis is a metabolic syndrome, an inflammatory syndrome, a syndrome of cellular lysis or an excessive alcohol consumption. In case of HFE hemochromatosis, phlebotomy prevents complications. The goal is to obtain and to maintain a normal-low ferritin level. In case of metabolic syndrome, phlebotomy could be useful in case of high hepatic iron concentration measured with MRI or in case of on-alcoholic steato-hepatitis.

Heterogametic sex chromosomes have evolved independently in various lineages of vertebrates. Such sex chromosome pairs often contain nonrecombining regions, with one of the chromosomes harboring a master sex-determining (SD) gene. It is hypothesized that these sex chromosomes evolved from a pair of autosomes that diverged after acquiring the SD gene. By linkage and association mapping of the SD locus in fugu (Takifugu rubripes), we show that a SNP (C/G) in the anti-Müllerian hormone receptor type II (Amhr2) gene is the only polymorphism associated with phenotypic sex. This SNP changes an amino acid (His/Asp384) in the kinase domain. While females are homozygous (His/His384), males are heterozygous. Sex in fugu is most likely determined by a combination of the two alleles of Amhr2. Consistent with this model, the medaka hotei mutant carrying a substitution in the kinase domain of Amhr2 causes a female phenotype. The association of the Amhr2 SNP with phenotypic sex is conserved in two other species of Takifugu but not in Tetraodon. The fugu SD locus shows no sign of recombination suppression between X and Y chromosomes. Thus, fugu sex chromosomes represent an unusual example of proto-sex chromosomes. Such undifferentiated X-Y chromosomes may be more common in vertebrates than previously thought.

<h4>Background</h4>Several studies have hypothesized that genes regulating the components of the serotonin system, including serotonin transporter (5-HTTLPR) and serotonin 1 B receptor (5-HT1B), may be associated with alcoholism, but their results are contradictory because of alcoholism's heterogeneity. Therefore, we examined whether the 5-HTTLPR gene and 5-HT1B gene G861C polymorphism are susceptibility factors for a specific subtype of alcoholism, antisocial alcoholism in Han Chinese in Taiwan.<h4>Methods</h4>We recruited 273 Han Chinese male inmates with antisocial personality disorder (ASPD) [antisocial alcoholism (AS-ALC) group (n=120) and antisocial non-alcoholism (AS-N-ALC) group (n=153)] and 191 healthy male controls from the community. Genotyping was done using PCR-RFLP.<h4>Results</h4>There were no significant differences in the genotypic frequency of the 5-HT1B G861C polymorphism between the 3 groups. Although AS-ALC group members more frequently carried the 5-HTTLPR S/S, S/LG, and LG/LG genotypes than controls, the difference became non-significant after controlling for the covarying effects of age. However, the 5-HTTLPR S/S, S/LG, and LG/LG genotypes may have interacted with the 5-HT1B G861C C/C polymorphism and increased the risk of becoming antisocial alcoholism.<h4>Conclusion</h4>Our study suggests that neither the 5-HTTLPR gene nor the 5-HT1B G861C polymorphism alone is a risk factor for antisocial alcoholism in Taiwan's Han Chinese population, but that the interaction between both genes may increase susceptibility to antisocial alcoholism.

Disordered gambling is a moderately heritable trait, but the underlying genetic basis is largely unknown. We performed a genome-wide association study (GWAS) for disordered gambling using a quantitative factor score in 1312 twins from 894 Australian families. Association was conducted for 2 381 914 single-nucleotide polymorphisms (SNPs) using the family-based association test in Merlin followed by gene and pathway enrichment analyses. Although no SNP reached genome-wide significance, six achieved P-values < 1 × 10(-5) with variants in three genes (MT1X, ATXN1 and VLDLR) implicated in disordered gambling. Secondary case-control analyses found two SNPs on chromosome 9 (rs1106076 and rs12305135 near VLDLR) and rs10812227 near FZD10 on chromosome 12 to be significantly associated with lifetime Diagnostic and Statistical Manual of Mental Disorders, fourth edition pathological gambling and South Oaks Gambling Screen classified probable pathological gambling status. Furthermore, several addiction-related pathways were enriched for SNPs associated with disordered gambling. Finally, gene-based analysis of 24 candidate genes for dopamine agonist-induced gambling in individuals with Parkinson's disease suggested an enrichment of SNPs associated with disordered gambling. We report the first GWAS of disordered gambling. While further replication is required, the identification of susceptibility loci and biological pathways will be important in characterizing the biological mechanisms that underpin disordered gambling.

Identification of genomic loci and segments that are identical by descent (IBD) allows inference on problems such as relatedness detection, IBD disease mapping, heritability estimation and detection of recent or ongoing positive selection. Here, employing a novel statistical method, we use IBD to find signals of selection in the Maasai from Kinyawa, Kenya (MKK). In doing so, we demonstrate the advantage of statistical tools that can probabilistically estimate IBD sharing without having to thin genotype data because of linkage disequilibrium (LD), and that allow for both inbreeding and more than one allele to be shared IBD. We use our novel method, GIBDLD, to estimate IBD sharing between all pairs of individuals at all genotyped SNPs in the MKK, and, by looking for genomic regions showing excess IBD sharing in unrelated pairs, find loci that are known to have undergone recent selection (eg, the LCT gene and the HLA region) as well as many novel loci. Intriguingly, those loci that show the highest amount of excess IBD, with the exception of HLA, also show a substantial number of unrelated pairs sharing all four of their alleles IBD. In contrast to other IBD detection methods, GIBDLD provides accurate probabilistic estimates at each locus for all nine possible IBD sharing states between a pair of individuals, thus allowing for consanguinity, while also modeling LD, thus removing the need to thin SNPs. These characteristics will prove valuable for those doing genetic studies, and estimating IBD, in the wide variety of human populations.

Monoamine oxidases (MAOs) are flavoproteins of the outer mitochondrial membrane that catalyze the oxidative deamination of biogenic and xenobiotic amines. In mammals there are two isoforms (MAO-A and MAO-B) that can be distinguished on the basis of their substrate specificity and their sensitivity towards specific inhibitors. Both isoforms are expressed in most tissues, but their expression in the central nervous system and their ability to metabolize monoaminergic neurotransmitters have focused MAO research on the functionality of the mature brain. MAO activities have been related to neurodegenerative diseases as well as to neurological and psychiatric disorders. More recently evidence has been accumulating indicating that MAO isoforms are expressed not only in adult mammals, but also before birth, and that defective MAO expression induces developmental abnormalities in particular of the brain. This review is aimed at summarizing and critically evaluating the new findings on the developmental functions of MAO isoforms during embryogenesis.

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.

Monte Carlo simulations of a mesoscale model of oligonucleosomes are analyzed to examine the role of dynamic-linker histone (LH) binding/unbinding in high monovalent salt with divalent ions, and to further interpret noted chromatin fiber softening by dynamic LH in monovalent salt conditions. We find that divalent ions produce a fiber stiffening effect that competes with, but does not overshadow, the dramatic softening triggered by dynamic-LH behavior. Indeed, we find that in typical in vivo conditions, dynamic-LH binding/unbinding reduces fiber stiffening dramatically (by a factor of almost 5, as measured by the elasticity modulus) compared with rigidly fixed LH, and also the force needed to initiate chromatin unfolding, making it consistent with those of molecular motors. Our data also show that, during unfolding, divalent ions together with LHs induce linker-DNA bending and DNA-DNA repulsion screening, which guarantee formation of heteromorphic superbeads-on-a-string structures that combine regions of loose and compact fiber independently of the characteristics of the LH-core bond. These structures might be important for gene regulation as they expose regions of the DNA selectively. Dynamic control of LH binding/unbinding, either globally or locally, in the presence of divalent ions, might constitute a mechanism for regulation of gene expression.

The medial prefrontal cortex (MPFC) is central to self-regulation and has been implicated in generating a cluster of event-related potential components, collectively referred to as medial frontal negativities (MFNs). These MFNs are elicited while individuals monitor behavioral and environmental consequences, and include the error-related negativity, Nogo N2, and the feedback-related negativity. A growing cognitive and affective neuroscience literature indicates that the activation of the anterior cingulate cortex (ACC) and surrounding medial prefrontal regions during performance monitoring is not only influenced by task context, but that these patterns of activity also vary as a function of individual differences (e.g., personality, temperament, clinical and non-clinical symptomatology, socio-political orientation, and genetic polymorphisms), as well as interactions between individual differences and task context. In this review we survey the neuroscience literature on the relations between performance monitoring, personality, task context, and brain functioning with a focus on the MPFC. We relate these issues to the role of affect in the paradigms used to elicit performance-monitoring neural responses and highlight some of the theoretical and clinical implications of this research. We conclude with a discussion of the complexity of these issues and how some of the basic assumptions required for their interpretation may be clarified with future research.

Microarray data analysis has been shown to provide an effective tool for studying cancer and genetic diseases. Although classical machine learning techniques have successfully been applied to find informative genes and to predict class labels for new samples, common restrictions of microarray analysis such as small sample sizes, a large attribute space and high noise levels still limit its scientific and clinical applications. Increasing the interpretability of prediction models while retaining a high accuracy would help to exploit the information content in microarray data more effectively. For this purpose, we evaluate our rule-based evolutionary machine learning systems, BioHEL and GAssist, on three public microarray cancer datasets, obtaining simple rule-based models for sample classification. A comparison with other benchmark microarray sample classifiers based on three diverse feature selection algorithms suggests that these evolutionary learning techniques can compete with state-of-the-art methods like support vector machines. The obtained models reach accuracies above 90% in two-level external cross-validation, with the added value of facilitating interpretation by using only combinations of simple if-then-else rules. As a further benefit, a literature mining analysis reveals that prioritizations of informative genes extracted from BioHEL's classification rule sets can outperform gene rankings obtained from a conventional ensemble feature selection in terms of the pointwise mutual information between relevant disease terms and the standardized names of top-ranked genes.

Diseases of the cornea are common and refer to conditions like infections, injuries and genetic defects. Morphologically, many corneal diseases affect only certain layers of the cornea and separate analysis of the individual layers is therefore of interest to explore the basic molecular mechanisms involved in corneal health and disease. In this study, the three main layers including, the epithelium, stroma and endothelium of healthy human corneas were isolated. Prior to analysis by LC-MS/MS the proteins from the different layers were either (i) separated by SDS-PAGE followed by in-gel trypsinization, (ii) in-solution digested without prior protein separation or, (iii) in-solution digested followed by cation exchange chromatography. A total of 3250 unique Swiss-Prot annotated proteins were identified in human corneas, 2737 in the epithelium, 1679 in the stroma, and 880 in the endothelial layer. Of these, 1787 proteins have not previously been identified in the human cornea by mass spectrometry. In total, 771 proteins were quantified, 157 based on in-solution digestion and 770 based on SDS-PAGE separation followed by in-gel digestion of excised gel pieces. Protein analysis showed that many of the identified proteins are plasma proteins involved in defense responses.

Psychiatric disorders are among the most intractable enigmas in medicine. In the past 5 years, there has been unprecedented progress on the genetics of many of these conditions. In this Review, we discuss the genetics of nine cardinal psychiatric disorders (namely, Alzheimer's disease, attention-deficit hyperactivity disorder, alcohol dependence, anorexia nervosa, autism spectrum disorder, bipolar disorder, major depressive disorder, nicotine dependence and schizophrenia). Empirical approaches have yielded new hypotheses about aetiology and now provide data on the often debated genetic architectures of these conditions, which have implications for future research strategies. Further study using a balanced portfolio of methods to assess multiple forms of genetic variation is likely to yield many additional new findings.

<h4>Unlabelled</h4><h4>Background</h4>Genome-wide association studies (GWAS) have recently identified several new genetic variants associated with obesity. The majority of the variants are within introns or between genes, suggesting they affect gene expression, although it is not clear which of the nearby genes they affect. Understanding the regulation of these genes will be key to determining the role of these variants in the development of obesity and will provide support for a role of these genes in the development of obesity.<h4>Methods</h4>We examined the expression of 19 GWAS obesity genes in the brain and specifically the hypothalamus, adipose tissue and liver of mice by real-time quantitative PCR. To determine whether these genes are nutritionally regulated, as may be expected for genes affecting obesity, we compared tissues from fasting and non-fasting animals and tissues from mice consuming a high fat high sucrose diet in comparison to standard rodent chow.<h4>Results</h4>We found complex, tissue-dependent patterns of nutritional regulation of most of these genes. For example, Bat2 expression was increased ~10-fold in the brain of fed mice but was lower or unchanged in the hypothalamus and adipose tissue. Kctd15 expression was upregulated in the hypothalamus, brain and adipose tissue of fed mice and downregulated by high fat feeding in liver, adipose tissue and the hypothalamus but not the remainder of the brain. Sh2b1 expression in the brain and Faim2 expression in adipose tissue were specifically increased >20-fold in fed mice. Tmem18 expression in adipose tissue but not the brain was reduced 80% by high fat feeding. Few changes in the expression of these genes were observed in liver.<h4>Conclusions</h4>These data show nutritional regulation of nearly all these GWAS obesity genes, particularly in the brain and adipose tissue, and provide support for their role in the development of obesity. The complex patterns of nutritional and tissue-dependent regulation also highlight the difficulty that may be encountered in determining how the GWAS genetic variants affect gene expression and consequent obesity risk in humans where access to tissues is constrained.

<h4>Purpose</h4>We tested the combination of a tumor lysate vaccine with a panel of costimulatory molecules to identify an immunotherapeutic approach capable of curing established murine gliomas.<h4>Experimental design</h4>Glioma-bearing mice were primed with a tumor lysate vaccine, followed by systemic administration of the following costimulatory ligands: OX40L, CD80, 4-1BBL, and GITRL, which were fused to the Fc portion of human immunoglobulin. Lymphocytes and mRNA were purified from the brain tumor site for immune monitoring studies. Numerous variations of the vaccine and Fc-OX40L regimen were tested alone or in combination with temozolomide.<h4>Results</h4>Lysate vaccinations combined with Fc-OX40L led to the best overall survival, yielding cure rates of 50% to 100% depending on the timing, regimen, and combination with temozolomide. Cured mice that were rechallenged with glioma cells rejected the challenge, showing immunologic memory. Lymphocytes isolated from the draining lymph nodes of vaccine/Fc-OX40L-treated mice had superior tumoricidal function relative to all other groups. Vaccine/Fc-OX40L-treated mice exhibited a significant increase in proliferation of brain-infiltrating CD4 and CD8 T cells, as indicated by Ki67 staining. Fc-OX40L had single-agent activity in transplanted and spontaneous glioma models, and the pattern of inflammatory gene expression in the tumor predicted the degree of therapeutic response.<h4>Conclusions</h4>These data show that Fc-OX40L has unique and potent activity against experimental gliomas and warrants further testing.

A bivalent dynamic covalent chemistry (DCC) system has been designed to selectively target members of the homodimeric glutathione-S-transferase (GST) enzyme family. The dynamic covalent libraries (DCLs) use aniline-catalysed acylhydrazone exchange between bivalent hydrazides and glutathione-conjugated aldehydes and the bis-hydrazides act as linkers to bridge between each glutathione binding site. The resultant DCLs were found to be compatible and highly responsive to templating with different GST isozymes, with the best results coming from the M and Schistosoma japonicum (Sj) class of GSTs, targets in cancer and tropical disease, respectively. The approach yielded compounds with selective, nanomolar affinity (K(i) =61 nM for mGSTM1-1) and demonstrates that DCC can be used to simultaneously interrogate binding sites on different subunits of a dimeric protein.

The multifunctional protein netrin-1 was initially discovered as the main attractive cue for commissural axon guidance by acting through its receptor DCC. Recently, we have shown that netrin-1 also interacts with the orphan transmembrane receptor amyloid precursor protein (APP). APP is cleaved by proteases, generating amyloid-β peptide, the main component of the amyloid plaques that are associated with Alzheimer disease. Our previous work demonstrated that via its interaction with APP, netrin-1 is a negative regulator of amyloid-β production in adult brain, but the biological relevance of APP/netrin-1 interaction under non-pathological conditions was unknown. We show here that during commissural axon navigation, APP, expressed at the growth cone, is part of the DCC receptor complex mediating netrin-1-dependent axon guidance. APP interacts with DCC in the presence of netrin-1 and enhances netrin-1-mediated DCC intracellular signaling, such as MAPK activation. Inactivation of APP in mice is associated with reduced commissural axon outgrowth. Thus, APP functionally acts as a co-receptor for DCC to mediate axon guidance.

Mitochondrial enoyl-CoA isomerase (ECI1) is an auxiliary enzyme involved in unsaturated fatty acid oxidation. In contrast to most of the other enzymes involved in fatty acid oxidation, a deficiency of ECI1 has yet to be identified in humans. We used wild-type (WT) and Eci1-deficient knockout (KO) mice to explore a potential presentation of human ECI1 deficiency. Upon food withdrawal, Eci1-deficient mice displayed normal blood β-hydroxybutyrate levels (WT 1.09 mM vs. KO 1.10 mM), a trend to lower blood glucose levels (WT 4.58 mM vs. KO 3.87 mM, P=0.09) and elevated blood levels of unsaturated acylcarnitines, in particular C12:1 acylcarnitine (WT 0.03 μM vs. KO 0.09 μM, P<0.01). Feeding an olive oil-rich diet induced an even greater increase in C12:1 acylcarnitine levels (WT 0.01 μM vs. KO 0.04 μM, P<0.01). Overall, the phenotypic presentation of Eci1-deficient mice is mild, possibly caused by the presence of a second enoyl-CoA isomerase (Eci2) in mitochondria. Knockdown of Eci2 in Eci1-deficient fibroblasts caused a more pronounced accumulation of C12:1 acylcarnitine on incubation with unsaturated fatty acids (12-fold, P<0.05). We conclude that Eci2 compensates for Eci1 deficiency explaining the mild phenotype of Eci1-deficient mice. Hypoglycemia and accumulation of C12:1 acylcarnitine might be diagnostic markers to identify ECI1 deficiency in humans.

Tyrosine phosphorylation is a hallmark for activation of STAT proteins, but their transcriptional activity also depends on other secondary modifications. Type I IFNs can activate both the ISGF3 (STAT1:STAT2:IRF9) complex and STAT3, but with cell-specific, selective triggering of only the ISGF3 transcriptional program. Following a genome-wide RNAi screen, we identified the SIN3 transcription regulator homolog A (Sin3a) as an important mediator of this STAT3-targeted transcriptional repression. Sin3a directly interacts with STAT3 and promotes its deacetylation. SIN3A silencing results in a prolonged nuclear retention of activated STAT3 and enhances its recruitment to the SOCS3 promoter, concomitant with histone hyperacetylation and enhanced STAT3-dependent transcription. Conversely, Sin3a is required for ISGF3-dependent gene transcription and for an efficient IFN-mediated antiviral protection against influenza A and hepatitis C viruses. The Sin3a complex therefore acts as a context-dependent ISGF3/STAT3 transcriptional switch.

Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA.

The highly diverse serotonergic system with at least 16 different receptor subtypes is implicated in the pathophysiology of most neuropsychiatric disorders including affective and anxiety disorders, obsessive compulsive disorder, post-traumatic stress disorder, eating disorders, sleep disturbance, attention deficit/hyperactivity disorder, drug addiction, suicidal behavior, schizophrenia, Alzheimer, etc. Alterations of the interplay between various pre- and postsynaptic receptor subtypes might be involved in the pathogenesis of these disorders. However, there is a lack of comprehensive in vivo values using standardized procedures. In the current PET study we quantified 3 receptor subtypes, including the major inhibitory (5-HT(1A) and 5-HT(1B)) and excitatory (5-HT(2A)) receptors, and the transporter (5-HTT) in the brain of healthy human subjects to provide a database of standard values. PET scans were performed on 95 healthy subjects (age=28.0 ± 6.9 years; 59% males) using the selective radioligands [carbonyl-(11)C]WAY-100635, [(11)C]P943, [(18)F]altanserin and [(11)C]DASB, respectively. A standard template in MNI stereotactic space served for region of interest delineation. This template follows two anatomical parcellation schemes: 1) Brodmann areas including 41 regions and 2) AAL (automated anatomical labeling) including 52 regions. Standard values (mean, SD, and range) for each receptor and region are presented. Mean cortical and subcortical binding potential (BP) values were in good agreement with previously published human in vivo and post-mortem data. By means of linear equations, PET binding potentials were translated to post-mortem binding (provided in pmol/g), yielding 5.89 pmol/g (5-HT(1A)), 23.5 pmol/g (5-HT(1B)), 31.44 pmol/g (5-HT(2A)), and 11.33 pmol/g (5-HTT) being equivalent to the BP of 1, respectively. Furthermore, we computed individual voxel-wise maps with BP values and generated average tracer-specific whole-brain binding maps. This knowledge might improve our interpretation of the alterations taking place in the serotonergic system during neuropsychiatric disorders.

Huntington disease (HD), a neurodegenerative disorder caused by an expanded CAG repeat in the HTT gene, remains without a treatment to modify the course of the illness. Lithium, a drug widely used for the treatment of bipolar disorder, has been shown to exert neuroprotective effects in a number of models of neurological disease but may have various toxic effects at conventional therapeutic doses. We examined whether NP03, a novel low-dose lithium microemulsion, would improve the disease phenotypes in the YAC128 mouse model of HD. We demonstrate that NP03 improves motor function, ameliorates the neuropathological deficits in striatal volume, neuronal counts, and DARPP-32 expression, and partially rescues testicular atrophy in YAC128 mice. These positive effects were accompanied by improvements in multiple biochemical endpoints associated with the pathogenesis of HD, including normalization of caspase-6 activation and amelioration of deficits in BDNF levels, and with no lithium-related toxicity. Our findings demonstrate that NP03 ameliorates the motor and neuropathological phenotypes in the YAC128 mouse model of HD, and represents a potential therapeutic approach for HD.

While many large-scale risk factors for calcific aortic valve disease (CAVD) have been identified, the molecular etiology and subsequent pathogenesis of CAVD have yet to be fully understood. Specifically, it is unclear what biological phenomena underlie the significantly higher occurrence of CAVD in the male population. We hypothesized the existence of intrinsic, cellular-scale differences between male and female valvular interstitial cells (VICs) that contribute to male sex being a risk factor for CAVD. Differences in gene expression profiles between healthy male and female porcine VICs were investigated via microarray analysis. Mean expression values of each probe set in the male samples were compared to the female samples, and biological processes were analyzed for overrepresentation using Gene Ontology term enrichment analysis. There were 183 genes identified as significantly (fold change>2; P<0.05) different in male versus female aortic valve leaflets. Within this significant gene list there were 298 overrepresented biological processes, several of which are relevant to pathways identified in CAVD pathogenesis. In particular, pathway analysis indicated that cellular proliferation, apoptosis, migration, ossification, angiogenesis, inflammation, and extracellular matrix reorganization were all significantly represented in the data set. These gene expression findings also translated into functional differences in VIC behavior in the in vitro environment, as sex-related differences in proliferation and apoptosis were confirmed in VIC populations cultured in vitro. These data suggest that a sex-related propensity for CAVD exists on the cellular level in healthy subjects, a phenomenon that could have significant clinical implications. These findings also strongly support discontinuing the use of mixed-sex VIC cultures, thereby changing the current standard in the field.

The molecular composition and drug responses of calcium-activated K(+) (BK) channels of skeletal muscle are unknown. Patch-clamp experiments combined with transcript scanning of the Kcnma1 gene encoding the alpha subunit of the BK channel were performed in rat slow-twitch soleus (Sol) and fast-twitch flexor digitorum brevis (FDB) skeletal muscles. Five splicing products of the Kcnma1 gene were isolated from Sol and FDB: the e17, e22, +29 aa, Slo27 and Slo0 variants. RT-PCR analysis demonstrated that the expression of e22 and Slo0 were 80-90% higher in FDB than Sol, whereas the expression of Slo27 was 60% higher in Sol than FDB, and the +29 aa variant was equally expressed in both muscle types. No beta 1-4 subunits were detected. In Sol, a large BK current with low Ca(2+) sensitivity was recorded. The BK channel of Sol also showed a reduced response to BK channel openers, such as NS1619, acetazolamide and related drugs. In FDB, a reduced BK current with high Ca(2+) sensitivity and an enhanced drug response was recorded. The total BK RNA content, which was 200% higher in Sol than in FDB, correlated with the BK currents in both muscles. Drug responses primarily correlated with e22 and Slo0 expression levels in FDB and to Slo27 expression in Sol muscle. In conclusion, phenotype-dependent BK channel biophysical and pharmacological properties correlated with the expression levels of the variants in muscles. These findings may be relevant to conditions affecting postural muscles, such as prolonged bed-rest, and to diseases affecting fast-twitch muscles, such as periodic paralysis. Down-regulation or up-regulation of the variants associated with pathological conditions may affect channel composition and drug responses.

Stem cell-based treatment for Huntington's disease (HD) is an expanding field of research. Although various stem cells have been shown to be beneficial in vivo, no long standing clinical effect has been demonstrated. To address this issue, we are developing a stem cell-based therapy designed to improve the microenvironment of the diseased tissue via delivery of neurotrophic factors (NTFs). Previously, we established that bone marrow derived human mesenchymal stem cells (MSCs) can be differentiated using medium based cues into NTF-secreting cells (NTF+ cells) that express astrocytic markers. NTF+ cells were shown to alleviate neurodegeneration symptoms in several disease models in vitro and in vivo, including the model for excitotoxicity. In the present study, we explored if the timing of intrastriatal transplantation of hNTF+ cells into the R6/2 transgenic mouse model for HD influences motor function and survival. One hundred thousand cells were transplanted bilaterally into the striatum of immune-suppressed mice at 4.5, 5.5 and 6.5 weeks of age. Contrary to our expectations, early transplantation of NTF+ cells did not improve motor function or overall survival. However, late (6.5 weeks) transplantation resulted in a temporary improvement in motor function and an extension of life span relative to that observed for PBS treated mice. We conclude that late transplantation of NTF+ cells induces a beneficial effect in this transgenic model for HD. Since no transplanted NTF+ cells could be detected in vivo, we suspect that the temporary nature of the beneficial effect is due to poor survival of transplanted cells. In general, we submit that NTF+ cells should be further evaluated for the therapy of HD.

There has been increasing interest lately in understanding how natural dietary antioxidants affect chemoprevention, and recently, there has been a merging of information about antioxidants, endogenous and exogenous reactive oxygen and nitrogen species (RONS), and inflammation. RONS normally serve the cells as second messengers to regulate many of the intracellular signaling cascades that govern multiple cellular activities. However, when the amount of RONS exceeds the cell's ability to metabolize/eliminate them, the cell becomes stressed and acquires genetic and epigenetic aberrations and dysregulated intracellular signaling cascades. In addition, there has been a better understanding of the role of tissue inflammation in the carcinogenesis process. Herein we integrate these fields to explain where RONS arise and how natural dietary antioxidants are principally working through refurbishing pathways that use RONS as second messengers.

<h4>Background</h4>In HIV and hepatitis C virus (HCV) coinfected patients, the role of antiretroviral therapy (ART) on hepatic steatosis (HS) remains controversial.<h4>Methods</h4>HIV/HCV coinfected patients receiving ART and previously untreated for HCV who underwent a liver biopsy were included. Cumulative duration of exposure to each antiretroviral was recorded up to liver biopsy date. Logistic regression analyses evaluated factors associated with steatosis and its severity.<h4>Results</h4>184 patients were included: median age 41 years, 84% male, 89% Caucasian, 61% with a past history of intravenous drug use. HCV genotypes were 1 (55%), 2 (6%), 3 (26%), and 4 (13%). Median HCV-RNA was 6.18 log10 IU/ml. HIV-RNA was undetectable (<400 copies/ml) in 67% of patients. Median CD4 count was 321/mm3. All patients had been exposed to nucleoside reverse transcriptase inhibitors (median cumulative exposure 56 months); 126 received protease inhibitors (23 months), and 79 non-nucleoside reverse transcriptase inhibitors (16 months). HS was observed in 102 patients (55%): 41% grade 1; 5% grade 2, and 9% grade 3. In multivariate analysis, HCV genotype 3 and HCV viral load were moderately associated with mild steatosis but strongly with grade 2-3 steatosis. After adjustment for the period of biopsy, no association was detected between HS and exposure to any antiretroviral class or drug, or duration of ART globally or comparing genotype 3 to others.<h4>Conclusions</h4>Among our ART-treated HIV-HCV cohort predominantly infected with genotype 1, 55% of patients had HS which was associated with HCV-related factors, but not ART class or duration of exposure.

Age-related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high- and low-functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low-functioning elderly (LFE) participants when compared to young and high-functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC-1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population.

Iron is vital for almost all organisms because of its ability to donate and accept electrons with relative ease. It serves as a cofactor for many proteins and enzymes necessary for oxygen and energy metabolism, as well as for several other essential processes. Mammalian cells utilize multiple mechanisms to acquire iron. Disruption of iron homeostasis is associated with various human diseases: iron deficiency resulting from defects in the acquisition or distribution of the metal causes anemia, whereas iron surfeit resulting from excessive iron absorption or defective utilization causes abnormal tissue iron deposition, leading to oxidative damage. Mammals utilize distinct mechanisms to regulate iron homeostasis at the systemic and cellular levels. These involve the hormone hepcidin and iron regulatory proteins, which collectively ensure iron balance. This review outlines recent advances in iron regulatory pathways as well as in mechanisms underlying intracellular iron trafficking, an important but less studied area of mammalian iron homeostasis.

Retinal glial (Müller) cells are involved in a wide range of developmental mechanisms, including axon guidance and angiogenesis. This study was undertaken to explore whether Netrin-4, an axonal guidance molecule, is expressed by Müller cells and promotes angiogenesis-related activities. Netrin-4 was found through all retinal layers, and its expression was demonstrated in Müller cells, retinal pigment epithelium cells and bovine retinal endothelial cells (BRECs). Co-localization of Netrin-4 with Müller cell-specific molecules [cellular retinaldehyde-binding protein (cRALBP), vimentin] was observed in the ganglion cell layer, nerve fiber layer, and at the outer limiting membrane. Under hypoxic conditions, the release of Netrin-4 from Müller cells was increased, with mRNA levels upregulated in a hypoxia-inducible factor-1-dependent manner and dependent on the concomitantly induced release of vascular endothelial growth factor. These findings were consistent with an intensified immunofluorescence of Netrin-4 labeling in the postischemic retinas after ischemia-reperfusion. Netrin-4 stimulated BRECs to increase phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK)-1/-2, and p38, in a dose-dependent manner. Synthetic inhibitors of the MAP kinases were able to suppress Netrin-4-induced migration and proliferation of BRECs suggesting that both MAP kinases are differentially involved in Netrin-4-induced angiogenesis. Two receptors for Netrins, i.e., deleted in colorectal cancer (DCC) and uncoordinated-5-homolog 1 (Unc5H1), were detected in BRECs. DCC is at least partially required for Netrin-4-induced activation of ERK-1/-2. These data suggest that Müller glial cells contribute to, and may modulate, retinal Netrin-4 levels. This may be a novel pathway of Müller cell-mediated control of retinal angiogenesis, particularly under hypoxic/ischemic conditions when the cells upregulate Netrin-4 expression.

Inhibitory neurons are critical for regulating effective transfer of sensory information and network stability. The precision of inhibitory function likely derives from the existence of a variety of interneuron subtypes. Their specification is largely dependent on the locale of origin of interneuron progenitors. Neocortical and hippocampal inhibitory neurons originate the subpallium, namely in the medial and caudal ganglionic eminences (MGE and CGE), and in the preoptic area (POA). In the hippocampus, neuronal nitric oxide synthase (nNOS)-expressing cells constitute a numerically large GABAergic interneuron population. On the contrary, nNOS-expressing inhibitory neurons constitute the smallest of the known neocortical GABAergic neuronal subtypes. The origins of most neocortical GABAergic neuron subtypes have been thoroughly investigated, however, very little is known about the origin of, or the genetic programs underlying the development of nNOS neurons. Here, we show that the vast majority of neocortical nNOS-expressing neurons arise from the MGE rather than the CGE. Regarding their molecular signature, virtually all neocortical nNOS neurons co-express the neuropeptides somatostatin (SST) and neuropeptide Y (NPY), and about half of them express the calcium-binding protein calretinin (CR). nNOS neurons thus constitute a small cohort of the MGE-derived SST-expressing population of cortical inhibitory neurons. Finally, we show that conditional removal of the transcription factor Sox6 in MGE-derived GABAergic cortical neurons results in an absence of SST and CR expression, as well as reduced expression of nNOS in neocortical nNOS neurons. Based on their respective abundance, origin and molecular signature, our results suggest that neocortical and hippocampal nNOS GABAergic neurons likely subserve different functions and have very different physiological relevance in these two cortical structures.

Effects of 3-month exposure to microgravity environment on the expression of genes and proteins in mouse brain were studied. Moreover, responses of neurobiological parameters, nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF), were also evaluated in the cerebellum, hippocampus, cortex, and adrenal glands. Spaceflight-related changes in gene and protein expression were observed. Biological processes of the up-regulated genes were related to the immune response, metabolic process, and/or inflammatory response. Changes of cellular components involving in microsome and vesicular fraction were also noted. Molecular function categories were related to various enzyme activities. The biological processes in the down-regulated genes were related to various metabolic and catabolic processes. Cellular components were related to cytoplasm and mitochondrion. The down-regulated molecular functions were related to catalytic and oxidoreductase activities. Up-regulation of 28 proteins was seen following spaceflight vs. those in ground control. These proteins were related to mitochondrial metabolism, synthesis and hydrolysis of ATP, calcium/calmodulin metabolism, nervous system, and transport of proteins and/or amino acids. Down-regulated proteins were related to mitochondrial metabolism. Expression of NGF in hippocampus, cortex, and adrenal gland of wild type animal tended to decrease following spaceflight. As for pleiotrophin transgenic mice, spaceflight-related reduction of NGF occurred only in adrenal gland. Consistent trends between various portions of brain and adrenal gland were not observed in the responses of BDNF to spaceflight. Although exposure to real microgravity influenced the expression of a number of genes and proteins in the brain that have been shown to be involved in a wide spectrum of biological function, it is still unclear how the functional properties of brain were influenced by 3-month exposure to microgravity.

<h4>Unlabelled</h4>Wilson's disease (WND) and hereditary hemochromatosis (HH) are two metal loading diseases of copper and iron, respectively, and are both recessively inherited. In central Sweden, where HH is common, 9 Wilson kindred (14 members) were identified. Aims of the study were to test whether nine WND families shared a common origin, a common mutation and if carrying HFE mutations affected their phenotype.<h4>Results</h4>The nine families were traced through 13 generations to a common founder origin in the mid-seventeenth century. Despite identity of descent, four different ATP7B mutations appeared with homozygosity in four, with two different mutations, W779X and T977M. There were three compound heterozygotes, W779X/T977M, R1319X/H1069Q and one T977M combined with a new, previously not described mutation, probably of Finnish origin. The founder family also included 26 descendant kindred (55 members) with HH as shown by HFE mutations. This admixture coincided with a migration out of the original parish into hemochromatosis-rich localities. One WND patient had iron overload (serum ferritin 672 µg/l and raised liver enzymes), but lacked HFE mutations. In another family with serious hemochromatosis (two sons dying from bronze diabetes), the coinheritance of congenital spherocytosis was probably the cause rather than an additional effect of WND.<h4>Conclusions</h4>WND though a rare disease may become aggregated like HH in certain areas due to local founder effects. Despite extensive pedigree studies leading back to the local founder family, the authors were unable to find a single defining mutation of the ATP7B gene.

Studies elucidating how and why neurodegeneration unfolds suggest that a complex interplay between genetic and environmental factors is responsible for disease pathogenesis. Recent breakthroughs in the field of epigenetics promise to advance our understanding of these mechanisms and to promote the development of useful and effective pre-clinical risk stratification strategies, molecular diagnostic and prognostic methods, and disease-modifying treatments.

A model of osteoporosis based on induced inflammation (IMO) was applied on rabbit bones. The structural heterogeneity and molecular complexity of bone significantly affect bone mechanical properties. A tool like Fourier transform infrared spectroscopy, able to analyze both the inorganic and organic phase simultaneously, could provide compositional information regarding cortical and trabecular sections under normal and osteoporotic conditions. In this study, we assessed the mineral/matrix ratio, carbonate and phosphate content and labile (i.e., non-apatitic) species contribution to bone mineral and collagen cross-linking patterns. Clear differences were observed between cortical and trabecular bone regarding mineral and carbonate content. Induced inflammation lowers the mineral/matrix ratio and increases the overall carbonate accumulation. Elevated concentrations of labile species were detected in osteoporotic samples, especially in the trabecular sections. Collagen cross-linking patterns were indirectly observed through the 1660/1690 cm⁻¹ ratio in the amide I band and a positive correlation was found with the mineralization index. Principal component analysis (PCA) applied to female samples successfully clustered trabecular and osteoporotic cases. The important role played by the phosphate ions was confirmed by corresponding loadings plots. The results suggest that the application of the IMO model to rabbit bones effectively alters bone remodeling and forms an osteoporotic bone matrix with a dissimilar composition compared to the normal one.

The regulation of protein synthesis in discrete cellular subdomains, or local protein synthesis, has important roles in development as well as brain function. This review will discuss some recent findings that shed new light on mRNA translation regulation and how these layers of regulation may work together to elicit tissue specific and spatially restricted gene expression.

Robust methods for highly parallel, quantitative analysis of cellular protein tyrosine kinase activities may provide tools critically needed to decipher oncogenic signaling, discover new targeted drugs, diagnose cancer and monitor patients. Here, we describe proof-of-principle for a novel protein kinase assay with the potential to help overcome these challenges. MALDI-TOF mass spectrometry provides an ideal tool for label-free multiplexed analysis of peptide phosphorylation, but is poorly matched to homogeneous assays and complex samples. Thus, we conjugated a common oligonucleotide tag to multiple peptide substrates, offering efficient capture from solution-phase kinase reactions by annealing to the complementary sequence tethered to PEG-passivated superparamagnetic microparticles. To enable reversible conjugation, we developed a novel bifunctional cross-linker allowing simple and efficient preparation of photocleavable peptide-oligonucleotide conjugates. After washing away contaminants and following photorelease, MALDI-TOF analysis yielded relative phosphorylation of each peptide with high sensitivity and specificity. Validating the hybridization-mediated multiplexed kinase assay, when three peptide substrate-oligonucleotide conjugates were mixed with the tyrosine kinase c-Abl and ATP, we readily observed their differential phosphorylation yet measured a common IC(50) for the Abl kinase inhibitor imatinib. This new assay enables analysis of protein kinase activities in a multiplexed format amenable to screening inhibitors against multiple kinases in parallel, an important capability for drug discovery and predictive diagnostics.

<h4>Objective</h4>To determine in extremely low-birth-weight infants if elevated blood interferon-γ (IFN-γ), interleukin (IL)-1β, IL-18, tumor necrosis factor-α (TNF-α), and transforming growth factor-β are associated with need for shunt following severe intraventricular hemorrhage (IVH) or with ventricular dilation following milder grades/no IVH.<h4>Study design</h4>Whole blood cytokines were measured on postnatal days 1, 3, 7, 14, and 21. Maximum IVH grade in the first 28 days, and shunt surgery or ventricular dilation on subsequent ultrasound (28 days' to 36 weeks' postmenstrual age) were determined.<h4>Results</h4>Of 902 infants in the National Institute of Child Health and Human Development Neonatal Research Network Cytokine study who survived to 36 weeks or discharge, 3.1% had shunts. Of the 12% of infants with severe (grade III to IV) IVH, 26% had a shunt associated with elevated TNF-α. None of the infants without IVH (69%) or with grade I (12%) or II (7%) IVH received shunts, but 8.4% developed ventricular dilation, associated with lower IFN-γ and higher IL-18.<h4>Conclusion</h4>Statistically significant but clinically nondiscriminatory alterations in blood cytokines were noted in infants with severe IVH who received shunts and in those without severe IVH who developed ventricular dilation. Blood cytokines are likely associated with brain injury but may not be clinically useful as biomarkers for white matter damage.

<h4>Background</h4>Most patients with hereditary hemochromatosis in the Caucasian population are homozygous for the p.C282Y mutation in the HFE gene. The penetrance and expression of hereditary hemochromatosis differ largely among cases of homozygous p.C282Y. Genetic factors might be involved in addition to environmental factors.<h4>Design and methods</h4>In the present study, we analyzed 50 candidate genes involved in iron metabolism and evaluated the association between 214 single nucleotide polymorphisms in these genes and three phenotypic outcomes of iron overload (serum ferritin, iron removed and transferrin saturation) in a large group of 296 p.C282Y homozygous Italians. Polymorphisms were tested for genetic association with each single outcome using linear regression models adjusted for age, sex and alcohol consumption.<h4>Results</h4>We found a series of 17 genetic variants located in different genes with possible additive effects on the studied outcomes. In order to evaluate whether the selected polymorphisms could provide a predictive signature for adverse phenotype, we re-evaluated data by dividing patients in two extreme phenotype classes based on the three phenotypic outcomes. We found that only a small improvement in prediction could be achieved by adding genetic information to clinical data. Among the selected polymorphisms, a significant association was observed between rs3806562, located in the 5'UTR of CYBRD1, and transferrin saturation. This variant belongs to the same haplotype block that contains the CYBRD1 polymorphism rs884409, found to be associated with serum ferritin in another population of p.C282Y homozygotes, and able to modulate promoter activity. A luciferase assay indicated that rs3806562 does not have a significant functional role, suggesting that it is a genetic marker linked to the putative genetic modifier rs884409.<h4>Conclusions</h4>While our results support the hypothesis that polymorphisms in genes regulating iron metabolism may modulate penetrance of HFE-hereditary hemochromatosis, with emphasis on CYBRD1, they strengthen the notion that none of these polymorphisms alone is a major modifier of the phenotype of hereditary hemochromatosis.

Huntington's disease (HD) is a genetic neurodegenerative disease characterized by an exceedingly high number of contiguous glutamine residues in the translated protein, huntingtin (Htt). The primary site of cell toxicity is the nucleus, but mitochondria have been identified as key components of cell damage. The present work has been carried out in immortalized lymphocytes from patients with HD. These cells, in comparison with lymphoid cells from healthy subjects, displayed: i) a redistribution of mitochondria, forming large aggregates; ii) a constitutive hyperpolarization of mitochondrial membrane; and iii) a constitutive alteration of mitochondrial fission machinery, with high apoptotic susceptibility. Moreover, mitochondrial fission molecules, e.g., protein dynamin-related protein 1, as well as Htt, associated with mitochondrial raft-like microdomains, glycosphingolipid-enriched structures detectable in mitochondria. These findings, together with the observation that a ceramide synthase inhibitor and a raft disruptor are capable of impairing the peculiar mitochondrial remodeling in HD cells, suggest that mitochondrial alterations occurring in these cells could be due to raft-mediated defects of mitochondrial fission/fusion machinery.

CD38, a membrane protein expressed in airway smooth muscle (ASM) cells, plays a role in cellular Ca(2+) dynamics and ASM contractility. In human ASM (HASM) cells, TNF-α induces CD38 expression through activation of MAPKs, NF-κB, and AP-1, and its expression is differentially elevated in cells from asthmatic patients compared with cells from nonasthmatic subjects. The CD38 3'-untranslated region (UTR) has targets for miR-140-3p. We hypothesized that miR-140-3p regulates CD38 expression in HASM cells by altering CD38 mRNA stability. Basal and TNF-α-induced expression of miR-140-3p was determined in nonasthmatic ASM (NAASM) and asthmatic ASM (AASM) cells. NAASM and AASM cells were transfected with control, miR-140-3p mimic, or miR-140-3p antagomirs, and CD38 expression and CD38 mRNA stability were determined. Luciferase reporter assays were used to determine miR-140-3p binding to the CD38 3'-UTR. Activation of p38, ERK, and JNK MAPKs, NF-κB, and AP-1 was determined in miR-140-3p mimic-transfected NAASM. TNF-α attenuated miR-140-3p expression in NAASM and AASM cells, but at a greater magnitude in AASM cells. CD38 mRNA expression was attenuated by miR-140-3p mimic at comparable magnitude in NAASM and AASM cells. Mutated miR-140-3p target on the CD38 3'-UTR reversed the inhibition of luciferase activity by miR-140-3p mimic. CD38 mRNA stability was unaltered by miR-140-3p mimic in NAASM or AASM cells following arrest of transcription. TNF-α-induced activation of p38 MAPK and NF-κB was attenuated by miR-140-3p mimic. The findings indicate that miR-140-3p modulates CD38 expression in HASM cells through direct binding to the CD38 3'-UTR and indirect mechanisms involving activation of p38 MAPK and NF-κB. Furthermore, indirect mechanisms appear to play a major role in the regulation of CD38 expression.

Recent systematic studies using newly developed genomic approaches have revealed common mechanisms and principles that underpin the spatial organization of eukaryotic genomes and allow them to respond and adapt to diverse functional demands. Genomes harbor, interpret, and propagate genetic and epigenetic information, and the three-dimensional (3D) organization of genomes in the nucleus should be intrinsically linked to their biological functions. However, our understanding of the mechanisms underlying both the topological organization of genomes and the various nuclear processes is still largely incomplete. In this essay, we focus on the functional relevance as well as the biophysical properties of common organizational themes in genomes (e.g. looping, clustering, compartmentalization, and dynamics), and examine the interconnection between genome structure and function from this angle. Present evidence supports the idea that, in general, genome architecture reflects and influences genome function, and is relatively stable. However, the answer as to whether genome architecture is a hallmark of cell identity remains elusive.

<h4>Background</h4>Oxidative stress related genes modify the effects of ambient air pollution or tobacco smoking on lung function decline. The impact of interactions might be substantial, but previous studies mostly focused on main effects of single genes.<h4>Objectives</h4>We studied the interaction of both exposures with a broad set of oxidative-stress related candidate genes and pathways on lung function decline and contrasted interactions between exposures.<h4>Methods</h4>For 12679 single nucleotide polymorphisms (SNPs), change in forced expiratory volume in one second (FEV(1)), FEV(1) over forced vital capacity (FEV(1)/FVC), and mean forced expiratory flow between 25 and 75% of the FVC (FEF(25-75)) was regressed on interval exposure to particulate matter <10 µm in diameter (PM10) or packyears smoked (a), additive SNP effects (b), and interaction terms between (a) and (b) in 669 adults with GWAS data. Interaction p-values for 152 genes and 14 pathways were calculated by the adaptive rank truncation product (ARTP) method, and compared between exposures. Interaction effect sizes were contrasted for the strongest SNPs of nominally significant genes (p(interaction)<0.05). Replication was attempted for SNPs with MAF>10% in 3320 SAPALDIA participants without GWAS.<h4>Results</h4>On the SNP-level, rs2035268 in gene SNCA accelerated FEV(1)/FVC decline by 3.8% (p(interaction) = 2.5×10(-6)), and rs12190800 in PARK2 attenuated FEV1 decline by 95.1 ml p(interaction) = 9.7×10(-8)) over 11 years, while interacting with PM10. Genes and pathways nominally interacting with PM10 and packyears exposure differed substantially. Gene CRISP2 presented a significant interaction with PM10 (p(interaction) = 3.0×10(-4)) on FEV(1)/FVC decline. Pathway interactions were weak. Replications for the strongest SNPs in PARK2 and CRISP2 were not successful.<h4>Conclusions</h4>Consistent with a stratified response to increasing oxidative stress, different genes and pathways potentially mediate PM10 and tobacco smoke effects on lung function decline. Ignoring environmental exposures would miss these patterns, but achieving sufficient sample size and comparability across study samples is challenging.

This review summarizes evidence of dysregulated reward circuitry function in a range of neurodevelopmental and psychiatric disorders and genetic syndromes. First, the contribution of identifying a core mechanistic process across disparate disorders to disease classification is discussed, followed by a review of the neurobiology of reward circuitry. We next consider preclinical animal models and clinical evidence of reward-pathway dysfunction in a range of disorders, including psychiatric disorders (i.e., substance-use disorders, affective disorders, eating disorders, and obsessive compulsive disorders), neurodevelopmental disorders (i.e., schizophrenia, attention-deficit/hyperactivity disorder, autism spectrum disorders, Tourette's syndrome, conduct disorder/oppositional defiant disorder), and genetic syndromes (i.e., Fragile X syndrome, Prader-Willi syndrome, Williams syndrome, Angelman syndrome, and Rett syndrome). We also provide brief overviews of effective psychopharmacologic agents that have an effect on the dopamine system in these disorders. This review concludes with methodological considerations for future research designed to more clearly probe reward-circuitry dysfunction, with the ultimate goal of improved intervention strategies.

To obtain a global picture of how alveolar macrophages respond to influenza A virus (IAV) infection, we used a quantitative proteomics method to systematically examine protein expression in the IAV-infected primary human alveolar macrophages. Of the 1214 proteins identified, 43 were significantly up-regulated and 63 significantly down-regulated at >95% confidence. The expression of an array of interferon (IFN)-induced proteins was significantly increased in the IAV-infected macrophages. The protein with the greatest expression increase was ISG15, an IFN-induced protein that has been shown to play an important role in antiviral defense. Concomitantly, quantitative real-time PCR analysis revealed that the gene expression of type I IFNs increased substantially following virus infection. Our results are consistent with the notion that type I IFNs play a vital role in the response of human alveolar macrophages to IAV infection. In addition to the IFN-mediated responses, inflammatory response, apoptosis, and redox state rebalancing appeared also to be major pathways that were affected by IAV infection. Furthermore, our data suggest that alveolar macrophages may play a crucial role in regenerating alveolar epithelium during IAV infection.

In genetic association studies, it is typically thought that genetic variants and environmental variables jointly will explain more of the inheritance of a phenotype than either of these two components separately. Traditional methods to identify gene-environment interactions typically consider only one measured environmental variable at a time. However, in practice, multiple environmental factors may each be imprecise surrogates for the underlying physiological process that actually interacts with the genetic factors. In this paper, we develop a variant of L(2) boosting that is specifically designed to identify combinations of environmental variables that jointly modify the effect of a gene on a phenotype. Because the effect modifiers might have a small signal compared with the main effects, working in a space that is orthogonal to the main predictors allows us to focus on the interaction space. In a simulation study that investigates some plausible underlying model assumptions, our method outperforms the least absolute shrinkage and selection and Akaike Information Criterion and Bayesian Information Criterion model selection procedures as having the lowest test error. In an example for the Women's Health Initiative-Population Architecture using Genomics and Epidemiology study, the dedicated boosting method was able to pick out two single-nucleotide polymorphisms for which effect modification appears present. The performance was evaluated on an independent test set, and the results are promising.

Human peripheral Vγ9Vδ2 T cells are activated by phosphorylated metabolites (phosphoagonists [PAg]) of the mammalian mevalonate or the microbial desoxyxylulose-phosphate pathways accumulated by infected or metabolically distressed cells. The underlying mechanisms are unknown. We show that treatment of nonsusceptible target cells with antibody 20.1 against CD277, a member of the extended B7 superfamily related to butyrophilin, mimics PAg-induced Vγ9Vδ2 T-cell activation and that the Vγ9Vδ2 T-cell receptor is implicated in this effect. Vγ9Vδ2 T-cell activation can be abrogated by exposing susceptible cells (tumor and mycobacteria-infected cells, or aminobisphosphonate-treated cells with up-regulated PAg levels) to antibody 103.2 against CD277. CD277 knockdown and domain-shuffling approaches confirm the key implication of the CD277 isoform BTN3A1 in PAg sensing by Vγ9Vδ2 T cells. Fluorescence recovery after photobleaching (FRAP) experiments support a causal link between intracellular PAg accumulation, decreased BTN3A1 membrane mobility, and ensuing Vγ9Vδ2 T-cell activation. This study demonstrates a novel role played by B7-like molecules in human γδ T-cell antigenic activation and paves the way for new strategies to improve the efficiency of immunotherapies using Vγ9Vδ2 T cells.

<h4>Background</h4>Many aging changes seem similar to those elicited by sleep-deprivation and psychosocial stress. Further, sleep architecture changes with age suggest an age-related loss of sleep. Here, we hypothesized that sleep deprivation in young subjects would elicit both stress and aging-like transcriptional responses.<h4>Methodology/principal findings</h4>F344 rats were divided into control and sleep deprivation groups. Body weight, adrenal weight, corticosterone level and hippocampal CA1 transcriptional profiles were measured. A second group of animals was exposed to novel environment stress (NES), and their hippocampal transcriptional profiles measured. A third cohort exposed to control or SD was used to validate transcriptional results with Western blots. Microarray results were statistically contrasted with prior transcriptional studies. Microarray results pointed to sleep pressure signaling and macromolecular synthesis disruptions in the hippocampal CA1 region. Animals exposed to NES recapitulated nearly one third of the SD transcriptional profile. However, the SD-aging relationship was more complex. Compared to aging, SD profiles influenced a significant subset of genes. mRNA associated with neurogenesis and energy pathways showed agreement between aging and SD, while immune, glial, and macromolecular synthesis pathways showed SD profiles that opposed those seen in aging.<h4>Conclusions/significance</h4>We conclude that although NES and SD exert similar transcriptional changes, selective presynaptic release machinery and Homer1 expression changes are seen in SD. Among other changes, the marked decrease in Homer1 expression with age may represent an important divergence between young and aged brain response to SD. Based on this, it seems reasonable to conclude that therapeutic strategies designed to promote sleep in young subjects may have off-target effects in the aged. Finally, this work identifies presynaptic vesicular release and intercellular adhesion molecular signatures as novel therapeutic targets to counter effects of SD in young subjects.

Trichohepatoenteric syndrome (THES) is characterized by chronic diarrhea, dysmorphic facies and hair abnormalities. Hepatic involvement varies from no abnormality to cirrhosis and hemochromatosis. Recently, mutations in the tetratricopeptide repeat domain 37 (TTC37) gene were identified to cause THES. The c.2808G>A variation was suggested as a possible founder mutation among the South Asians. We further report 2 unrelated cases of Asian-Indian ethnicity (Gujrati) with THES, wherein targeted mutation analysis revealed the same mutation in homozygous form in both cases. These findings, as well as haplotype analysis, corroborate the founder mutation hypothesis amongst Asian Indo-Pakistani ethnic groups. A restriction enzyme-based method is also described to identify this founder mutation. One of our probands had multiple hepatic hemangiomas, a feature not previously observed in this syndrome.

The spatial and temporal regulation of intracellular signaling is determined by the spatial and temporal organization of complexes assembled on scaffold proteins, which can be modulated by their interactions with additional proteins as well as subcellular localization. The scaffold KSR1 protein interacts with MAPK forming a complex that conveys a differential signaling in response to growth factors. The aim of this work is to determine the unknown mechanism by which VRK2A downregulates MAPK signaling. We have characterized the multiprotein complex formed by KSR1 and the Ser-Thr kinase VRK2A. VRK2A is a protein bound to the endoplasmic reticulum (ER) and retains a fraction of KSR1 complexes on the surface of this organelle. Both proteins, VRK2A and KSR1, directly interact by their respective C-terminal regions. In addition, MEK1 is also incorporated in the basal complex. MEK1 independently interacts with the CA5 region of KSR1 and with the N-terminus of VRK2A. Thus, VRK2A can form a high molecular size (600-1,000 kDa) stable complex with both MEK1 and KSR1. Knockdown of VRK2A resulted in disassembly of these high molecular size complexes. Overexpression of VRK2A increased the amount of KSR1 in the particulate fraction and prevented the incorporation of ERK1/2 into the complex after stimulation with EGF. Neither VRK2A nor KSR1 interact with the VHR, MKP1, MKP2, or MKP3 phosphatases. The KSR1 complex assembled and retained by VRK2A in the ER can have a modulatory effect on the signal mediated by MAPK, thus locally affecting the magnitude of its responses, and can explain differential responses depending on cell type.

<h4>Background</h4>Recent advances in the treatment of cancer have focused on targeting genomic aberrations with selective therapeutic agents. In rare tumors, where large-scale clinical trials are daunting, this targeted genomic approach offers a new perspective and hope for improved treatments. Cancers of the ampulla of Vater are rare tumors that comprise only about 0.2% of gastrointestinal cancers. Consequently, they are often treated as either distal common bile duct or pancreatic cancers.<h4>Methods</h4>We analyzed DNA from a resected cancer of the ampulla of Vater and whole blood DNA from a 63 year-old man who underwent a pancreaticoduodenectomy by whole genome sequencing, achieving 37× and 40× coverage, respectively. We determined somatic mutations and structural alterations.<h4>Results</h4>We identified relevant aberrations, including deleterious mutations of KRAS and SMAD4 as well as a homozygous focal deletion of the PTEN tumor suppressor gene. These findings suggest that these tumors have a distinct oncogenesis from either common bile duct cancer or pancreatic cancer. Furthermore, this combination of genomic aberrations suggests a therapeutic context for dual mTOR/PI3K inhibition.<h4>Conclusions</h4>Whole genome sequencing can elucidate an oncogenic context and expose potential therapeutic vulnerabilities in rare cancers.

Sox9 acts together with Sox5 or Sox6 as a master regulator for chondrogenesis; however, the inter-relationship among these transcription factors remains unclear. Here, we show that the protein kinase MLTK plays an essential role in the onset of chondrogenesis through triggering the induction of Sox6 expression by Sox9. We find that knockdown of MLTK in Xenopus embryos results in drastic loss of craniofacial cartilages without defects in neural crest development. We also find that Sox6 is specifically induced during the onset of chondrogenesis, and that the Sox6 induction is inhibited by MLTK knockdown. Remarkably, Sox6 knockdown phenocopies MLTK knockdown. Moreover, we find that ectopic expression of MLTK induces Sox6 expression in a Sox9-dependent manner. Our data suggest that p38 and JNK pathways function downstream of MLTK during chondrogenesis. These results identify MLTK as a novel key regulator of chondrogenesis, and reveal its action mechanism in chondrocyte differentiation during embryonic development.

<h4>Introduction</h4>Click labeling using 2-[¹⁸F]fluoroethyl azide has been proven to be promising methods of radiolabeling small molecules and peptides, some of which are undergoing clinical evaluations. However, the previously reported method afforded low yield, poor purities and under desirable reproducibility.<h4>Methods</h4>A vacuum distillation method was used to isolate 2-[¹⁸F]fluoroethyl azide, and the solvent effect of acetonitrile and dimethylformamide (DMF) on the click labeling using Cu(I) from copper sulfate/sodium ascorbate was studied. The labeling conditions were optimized to radiosynthesize a hydroxysuccinimide ester (N-hydroxysuccinimide, or NHS).<h4>Results</h4>2-[¹⁸F]fluoroethyl azide was isolated by the vacuum distillation method with >80% yield within 10min in a "pure" and click-ready form. It was found that the amount of DMF was critical for maintaining high levels of Cu(I) from copper sulfate/sodium ascorbate in order to rapidly complete the click labeling reaction. The addition of bathophenanthrolinedisulfonic acid disodium salt to the mixture of copper sulfate/sodium ascorbate also greatly improved the click labeling efficiency. Through exploiting these optimizations, a base-labile NHS ester was rapidly radiosynthesized in 90% isolated yield with good chemical and radiochemical purities.<h4>Conclusions</h4>We have developed a general method to click-label small molecules efficiently using [¹⁸F]2 for research and clinical use. This NHS ester can be used for conjugation chemistry to label antibodies, peptides and small molecules as positron emission tomography tracers.

The sudden infant death syndrome (SIDS) is the main cause of postneonatal infant death, being defined as the sudden death of an infant under one year of age that remains unexplained after a complete clinical review, autopsy and death scene investigation. The neurotransmitter serotonin (5-HT) is involved in the regulation of a broad array of behavioral and biological functions. By controlling the reuptake of 5-HT from the extracellular space, the serotonin transporter (5-HTT) regulates the duration and strength of the interactions between 5-HT and its receptors. It has been shown that the activity of the human 5-HTT gene promoter is regulated by polymorphic repetitive elements, resulting in differences in the efficacy of 5-HTT reuptake among the allelic variants: the short (S) allele is associated with lower transcriptional efficiency of the promoter compared with the long (L) allele. Using qRT-PCR we studied the gene expression of 5-HTT in ten SIDS cases, previously analyzed at a molecular level and which showed the genetic S/S profile. In nine cases we observed 5-HTT expression levels comparable to those seen in the control case, while in one case there was a remarkable reduction in the expression of the gene. It is presumable that, despite the presence of the same S/S genotype, the different genetic background could influence the transcript stability and that the polimorphic variant of the 5-HTT gene could respond differently to the external environmental stimuli.

Huntington's disease (HD) is a progressive and fatal neurodegenerative disease, and the most common inherited CAG repeat disorder. A polyglutamine expansion in the N-terminus of the huntingtin protein (HTT) leads to protein misfolding and downstream pathogenic processes culminating in widespread functional impairment and neurodegeneration in the striatum, cortex and other brain areas. To date, only symptomatic treatments are available that address motor, psychiatric and cognitive deficits. Here we review recent strategies for developing disease-modifying therapies designed to limit or abolish the pathogenic activities of the primary molecular target in HD, the mutant HTT protein itself.

Besides its action as a neurotransmitter, serotonin has multiple physiological functions in several peripheral organs. Recently, Yadav et al. suggested that peripheral serotonin produced in the gut was a major negative regulator of osteoblast proliferation. These data were challenged by Cui et al. that showed no change in bone density in mature mice with a global invalidation of tryptophan hydroxylase 1, the enzyme responsible of serotonin synthesis in the periphery. In this context, we showed that osteoclasts are able to synthetize serotonin that acts locally to induce osteoclast precursors differentiation. Our data and previous results from others suggest that rather than acting as a hormone, serotonin produced in the bone could act locally on osteoclast and osteoblast realizing in the bone a complete micro-serotoninergic system.

BioA, a pyridoxal 5'-phosphate (PLP) dependent aminotransferase, catalyzes the second step of biotin biosynthesis, converting 7-keto-8-aminopelargonic acid (KAPA) into 7,8-diaminopelargonic acid (DAPA). Amiclenomycin (ACM) isolated from cultures of different Streptomyces strains is a potent mechanism-based inhibitor of BioA that operates via an aromatization mechanism, irreversibly labeling the PLP cofactor. However, ACM is plagued by inherent chemical stability. Herein we describe the synthesis of four inhibitors, inspired by ACM but containing an allylic amine as the chemical warhead, designed to both improve stability and operate via a complementary Michael addition-pathway upon enzymatic oxidation of the allylic amine substrate to an enimine. Acyclic analogue M-1 contains a terminal olefin as the pro-Michael acceptor. The synthesis of M-1 features an alkyne-zipper reaction and the Overman rearrangement as key synthetic operations. The cyclic analogues M-2/3/4 contain either an endocyclic or exocyclic olefin as the pro-Michael acceptor. These were all prepared using a common strategy employing DIBAL reduction of a precursor bicyclic lactam, followed by in situ Horner-Wadsworth-Emmons (HWE) olefination as the key synthetic steps.

Ouabain is a cardiotonic steroid and specific inhibitor of the Na(+)/K(+)-ATPase. The relationship between ouabain treatment and the unfolded protein response (UPR) in cells is not precisely understood. Therefore, we studied the possible effects of ouabain on proliferation, apoptosis, and the UPR. HepG2 cells were cultured overnight and then treated with various concentrations of ouabain (0.75 to 750 nM) in the absence or presence of 10 mM 2-deoxyglucose (2-DG) for 48 hours. We also used real-time polymerase chain reaction to obtain quantitative measurements of expression levels of Grp78, Grp94, CHOP, MTJ-1, HKII, MDR-1, MRP-1, HO-1, and Par-4. Cell number, viability, and proliferation of HepG2 cells were monitored with a real-time cell analyzer system (xCELLigence). We show that ouabain modulates the UPR transcription program and induces cell death in glucose-deprived tumor cells. Ouabain at all concentrations showed no cytotoxicity whereas all concentrations were very effective under 2-DG stress conditions. Our findings show that disruption of the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical basis for developing UPR-targeting drugs against solid tumors. Ouabain use as an adjunct to conventional cancer therapy also warrants vigorous investigation.

Preterm birth is a global health issue impacting millions of mothers and babies. However, the etiology of preterm birth is not clearly understood. Our recent finding that premature decidual senescence with terminal differentiation is a cause of preterm birth in mice with uterine Trp53 deletion, encoding p53 protein, led us to explore other potential factors that are related to preterm birth. Using proteomics approaches, here, we show that 183 candidate proteins show significant changes in deciduae with Trp53 deletion as compared with normal deciduae. Functional categorization of these proteins unveiled new pathways that are influenced by p53. In particular, down-regulation of a cluster of antioxidant enzymes in p53-deficient deciduae suggests that increased oxidative stress could be one cause of preterm birth in mice harboring uterine deletion of Trp53.

The aims of this study were to 1) identify the earliest transcriptional response of the bovine endometrium to the presence of the conceptus (using RNAseq), 2) investigate if these genes are regulated by interferon tau (IFNT) in vivo, and 3) determine if they are predictive of the pregnancy status of postpartum dairy cows. RNAseq identified 459 differentially expressed genes (DEGs) between pregnant and cyclic endometria on day 16. Quantitative real-time PCR analysis of selected genes revealed PARP12, ZNFX1, HERC6, IFI16, RNF213, and DDX58 expression increased in pregnant compared with cyclic endometria on day 16 and were directly upregulated by intrauterine infusion of IFNT in vivo for 2 h (P < 0.05). On day 13 following estrous endometrial expression of nine genes increased [ARHGAP1, MGC127874, LIMS2, TBC1D1, FBXL7, C25H16orf71, LOC507810, ZSWIM4, and one novel gene (ENSBTAT00000050193)] and seven genes decreased (SERBP1, SRGAP2, AL7A1, TBK1, F2RL2, MGC128929, and WBSCR17; P < 0.05) in pregnant compared with cyclic heifers. Of these DEGs, significant differences in expression between pregnant and cyclic endometria were maintained on day 16 for F2RL2, LIMS2, LOC507810, MGC127874, TBC1D1, WBSCR17, and ZSWIM4 (P < 0.05) both their expression was not directly regulated by IFNT in vivo. Analysis of the expression of selected interferon-stimulated genes in blood samples from postpartum dairy cows revealed a significant increase (P < 0.05) in expression of ZXFX1, PARP12, SAMD9, and HERC6 on day 18 following artificial insemination in cows subsequently confirmed pregnant compared with cyclic controls. In conclusion, RNAseq identified a number of novel pregnancy-associated genes in the endometrium of cattle during early pregnancy that are not regulated by IFNT in vivo. In addition, a number of genes that are directly regulated by short term exposure to IFNT in vivo are differentially expressed on day 18 following estrus detection in the blood of postpartum dairy cows depending on their pregnancy status.

A man in his late 40s, referred by his general practitioner (GP) to a psychology-led pain management programme, made a subjective and spontaneous report of cognitive impairment. He further mentioned a ten year history of erectile dysfunction, joint pain, occasional nausea and excessive fatigue. He underwent cognitive assessment. Advised to return to his GP to seek further investigation, he was ultimately subsequently diagnosed with haemochromatosis and began radical therapeutic venesection. Repeat cognitive assessment, a year later and following stabilisation of the condition, found improvement on some cognitive measures. Subjective report was of vastly improved cognitive function.

Human induced pluripotent stem cells (hiPSCs) are emerging as a tool for understanding human brain development at cellular, molecular, and genomic levels. Here we show that hiPSCs grown in suspension in the presence of rostral neuralizing factors can generate 3D structures containing polarized radial glia, intermediate progenitors, and a spectrum of layer-specific cortical neurons reminiscent of their organization in vivo. The hiPSC-derived multilayered structures express a gene expression profile typical of the embryonic telencephalon but not that of other CNS regions. Their transcriptome is highly enriched in transcription factors controlling the specification, growth, and patterning of the dorsal telencephalon and displays highest correlation with that of the early human cerebral cortical wall at 8-10 wk after conception. Thus, hiPSC are capable of enacting a transcriptional program specifying human telencephalic (pallial) development. This model will allow the study of human brain development as well as disorders of the human cerebral cortex.

Polyubiquitination of misfolded proteins, especially K63-linked polyubiquitination, is thought to be associated with the formation of inclusion bodies. However, it is not well explored whether appropriate editing of the different types of ubiquitin linkages by deubiquitinating enzymes (DUBs) affects the dynamics of inclusion bodies. In this study, we report that a specific DUB, ataxin-3, is required for the efficient recruitment of the neurodegenerative disease-associated protein copper-zinc superoxide dismutase (SOD1) to aggresomes. The overexpression of ataxin-3 promotes mutant SOD1 aggresome formation by trimming K63-linked polyubiquitin chains. Moreover, knockdown of ataxin-3 decreases mutant SOD1 aggresome formation and increases cell death induced by mutant SOD1. Thus, our data suggest that the sequestration of misfolded SOD1 into aggresomes, which is driven by ataxin-3, plays an important role in attenuating protein misfolding-induced cell toxicity.

MARCH E3 ligases play a key role in controlling MHC class II surface expression by regulated ubiquitination of a lysine residue in the β-chain. Little is known concerning how these enzymes target their specific substrates. Here we show that recognition of HLA-DR by MARCH proteins is complex. Several features associated with the transmembrane domain and bordering regions influence the overall efficiency of receptor internalization. A cluster of residues at the interface of the lipid bilayer and the cytosol plays the most important role in MARCH8 recognition of HLA-DRβ. Variation in this sequence also determines specificity of MARCH9 for HLA-DQ. Residues located in helical face four of HLA-DRβ together with a charged residue at the boundary with the stalk region also contribute significantly to recognition. Truncation analysis suggested that a dileucine-like motif in the DRβ cytoplasmic tail influences the efficiency of co-localization of HLA-DR with MARCH8. The DRβ-encoded acceptor lysine functioned optimally when placed in its natural location relative to the bilayer. In the DRα/DRβ dimer most other amino acids in the cytoplasmic tail could be substituted for alanine with minimal influence on function. Our data support a model whereby multiple features of HLA-DR are involved in substrate recognition by MARCH8. The single most important region is located at the interface between the transmembrane domain and the cytosol. Variation in sequence in this location between different class II isotypes controls efficiency of recognition by different MARCH E3 ligases.

Here we examine the roles of two isoforms of glycogen synthase kinase-3 (GSK-3), GSK-3α and GSK-3β, in skeletal development. Both isoforms were unphosphorylated and active in chondrocyte differentiation stages during SOX9 and type II collagen (COL2A1) expression. Although knock-out of both alleles of Gsk3a (Gsk3a(-/-)) or a single allele of Gsk3b (Gsk3b(+/-)) in mice did not significantly affect skeletal development, compound knock-out (Gsk3a(-/-);Gsk3b(+/-)) caused dwarfism with impairment of chondrocyte differentiation. GSK-3α and GSK-3β induced differentiation of cultured chondrocytes with functional redundancy in a cell-autonomous fashion, independently of the Wnt/β-catenin signal. Computational predictions followed by SOX9 and COL2A1 transcriptional assays identified RelA (NF-κB p65) as a key phosphorylation target of GSK-3. Among several phosphorylation residues in RelA, Thr-254 was identified as the critical phosphorylation site for GSK-3 that modulated chondrocyte differentiation. In conclusion, redundant functions of GSK-3α and GSK-3β through phosphorylation of RelA at Thr-254 play a crucial role in early stages of chondrocyte differentiation.

<h4>Unlabelled</h4>Nonalcoholic fatty liver disease (NAFLD) is related to risk factors of coronary artery disease, such as dyslipidemia, diabetes, and metabolic syndrome, which are closely linked with visceral adiposity. The aim of this study was to investigate whether NAFLD was associated with coronary artery calcification (CAC), which is used as a surrogate marker for coronary atherosclerosis independent of computed tomography (CT)-measured visceral adiposity. Out of 5,648 subjects who visited one of our health screening centers between 2003 and 2008, we enrolled 4,023 subjects (mean age, 56.9 ± 9.4 years; 60.7% males) without known liver disease or a history of ischemic heart disease. CAC score was evaluated using the Agatston method. On univariate analysis, the presence of CAC (score >0) was significantly associated with age, sex, body mass index, aspartate aminotransferase, alanine aminotransferase, high-density lipoprotein cholesterol, triglycerides, and increased risk of diabetes, hypertension, smoking, and NAFLD. Increasing CAC scores (0, <10, 10-100, ≥ 100) were associated with higher prevalence of NAFLD (odds ratio [OR], 1.84; 95% confidence interval [CI], 1.61-2.10; P<0.001). Multivariable ordinal regression analysis was adjusted for traditional risk factors, and CT-measured visceral adipose tissue area in a subgroup of subjects showed that the increased CAC scores were significantly associated with the presence of NAFLD (OR, 1.28, 95% CI, 1.04-1.59; P = 0.023) independent of visceral adiposity.<h4>Conclusion</h4>Patients with NAFLD are at increased risk for coronary atherosclerosis independent of classical coronary risk factors, including visceral adiposity. These data suggest that NAFLD might be an independent risk factor for coronary artery disease.

<h4>Unlabelled</h4>Bile acid sequestrants (BAS) lower plasma low density lipoprotein levels and improve glycemic control. Colestimide, a BAS, has been claimed by computed tomography to reduce liver fat. Therefore, we examined the efficacy of colesevelam, a potent BAS, to decrease liver fat in patients with biopsy-proven nonalcoholic steatohepatitis (NASH). Liver fat was measured by a novel magnetic resonance imaging (MRI) technique, the proton-density-fat-fraction (PDFF), as well as by conventional MR spectroscopy (MRS). Fifty patients with biopsy-proven NASH were randomly assigned to either colesevelam 3.75 g/day orally or placebo for 24 weeks. The primary outcome was change in liver fat as measured by MRI-PDFF in colocalized regions of interest within each of the nine liver segments. Compared with placebo, colesevelam increased liver fat by MRI-PDFF in all nine segments of the liver with a mean difference of 5.6% (P = 0.002). We cross-validated the MRI-PDFF-determined fat content with that assessed by colocalized MRS; the latter showed a mean difference of 4.9% (P = 0.014) in liver fat between the colesevelam and the placebo arms. MRI-PDFF correlated strongly with MRS-determined hepatic fat content (r(2) = 0.96, P < 0.0001). Liver biopsy assessment of steatosis, cellular injury, and lobular inflammation did not detect any effect of treatment.<h4>Conclusion</h4>Colesevelam increases liver fat in patients with NASH as assessed by MRI as well as MRS without significant changes seen on histology. Thus, MRI and MRS may be better than histology to detect longitudinal changes in hepatic fat in NASH. Underlying mechanisms and whether the small MR-detected increase in liver fat has clinical consequences is not known.

Influenza A virus exerts a large health burden during both yearly epidemics and global pandemics. However, designing effective vaccine and treatment options has proven difficult since the virus evolves rapidly. Therefore, it may be beneficial to identify host proteins associated with viral infection and replication to establish potential new antiviral targets. We have previously measured host protein responses in continuously cultured A549 cells infected with mouse-adapted virus strain A/PR/8/34(H1N1; PR8). We here identify and measure host proteins differentially regulated in more relevant primary human bronchial airway epithelial (HBAE) cells. A total of 3740 cytosolic HBAE proteins were identified by 2D LC-MS/MS, of which 52 were up-regulated ≥2-fold and 41 were down-regulated ≥2-fold after PR8 infection. Up-regulated HBAE proteins clustered primarily into interferon signaling, other host defense processes, and molecular transport, whereas down-regulated proteins were associated with cell death signaling pathways, cell adhesion and motility, and lipid metabolism. Comparison to influenza-infected A549 cells indicated some common influenza-induced host cell alterations, including defense response, molecular transport proteins, and cell adhesion. However, HBAE-specific alterations consisted of interferon and cell death signaling. These data point to important differences between influenza replication in continuous and primary cell lines and/or alveolar and bronchial epithelial cells.

<h4>Background</h4>Anthrax lethal toxin (LT), produced by the Gram-positive bacterium Bacillus anthracis, is a highly effective zinc dependent metalloprotease that cleaves the N-terminus of mitogen-activated protein kinase kinases (MAPKK or MEKs) and is known to play a role in impairing the host immune system during an inhalation anthrax infection. Here, we present the transcriptional responses of LT treated human monocytes in order to further elucidate the mechanisms of LT inhibition on the host immune system.<h4>Results</h4>Western Blot analysis demonstrated cleavage of endogenous MEK1 and MEK3 when human monocytes were treated with 500 ng/mL LT for four hours, proving their susceptibility to anthrax lethal toxin. Furthermore, staining with annexin V and propidium iodide revealed that LT treatment did not induce human peripheral monocyte apoptosis or necrosis. Using Affymetrix Human Genome U133 Plus 2.0 Arrays, we identified over 820 probe sets differentially regulated after LT treatment at the p <0.001 significance level, interrupting the normal transduction of over 60 known pathways. As expected, the MAPKK signaling pathway was most drastically affected by LT, but numerous genes outside the well-recognized pathways were also influenced by LT including the IL-18 signaling pathway, Toll-like receptor pathway and the IFN alpha signaling pathway. Multiple genes involved in actin regulation, signal transduction, transcriptional regulation and cytokine signaling were identified after treatment with anthrax LT.<h4>Conclusion</h4>We conclude LT directly targets human peripheral monocytes and causes multiple aberrant gene responses that would be expected to be associated with defects in human monocyte's normal signaling transduction pathways and function. This study provides further insights into the mechanisms associated with the host immune system collapse during an anthrax infection, and suggests that anthrax LT may have additional downstream targets outside the well-known MAPK pathway.

<h4>Background</h4>The pre B-cell leukemia transcription factor 1 (Pbx1) genes belong to the three amino acid loop extension family of homeodomain proteins that form hetero-oligomeric complexes with other homeodomain transcription factors, thereby modulating target specificity, DNA binding affinity and transcriptional activity of their molecular associates.<h4>Results</h4>Here, we provide evidence that Pbx1 is expressed in mesencephalic dopaminergic neurons from embryonic day 11 into adulthood and determines some of the cellular properties of this neuronal population. In Pbx1-deficient mice, the mesencephalic dopaminergic axons stall during mid-gestation at the border between di- and telencephalon before entering the ganglionic eminence, leading to a loose organization of the axonal bundle and partial misrouting. In Pbx1-deficient dopaminergic neurons, the high affinity netrin-1 receptor, deleted in colon cancer (DCC), is down-regulated. Interestingly, we found several conserved Pbx1 binding sites in the first intron of DCC, suggesting a direct regulation of DCC transcription by Pbx1.<h4>Conclusions</h4>The expression of Pbx1 in dopaminergic neurons and its regulation of DCC expression make it an important player in defining the axonal guidance of the midbrain dopaminergic neurons, with possible implications for the normal physiology of the nigro-striatal system as well as processes related to the degeneration of neurons during the course of Parkinson's disease.

Ejaculated spermatozoa undergo capacitation and acrosome reaction by responding to extrinsic clues and activate signalling cascades to induce protein tyrosine phosphorylation. In the present study, we investigated the existence, the Janus kinase (JAK) and activator of transcription (STAT) pathway and determined its physiological relevance. JAK1 and STAT1 are localised on the equatorial region and the midpiece of their human spermatozoa, JAK2 is detected on the sperm tail. Capacitation leads to phosphorylation of JAK2 but not JAK1 and STAT1. In the uncapacitated sperm, phosphorylated JAK2 (pJAK2) is localised mainly in the tail region; in response to capacitation, the JAK2 is phosphorylated in the midpiece and the head region along with the tail. Progesterone (5 μm) leads to phosphorylation of JAK1, JAK2 and STAT1 in a time-dependent manner. In progesterone-treated spermatozoa, the JAK2 in the tail is hyperphosphorylated, the JAK2 in the head and the midpiece is dephosphorylated. We conclude that in human spermatozoa, the JAK1/2 pathway is activated upon capacitation and is further modulated by progesterone; the biological processes controlled by this pathway in sperm need to be elucidated.

<h4>Background</h4>Improved methods are needed for predicting prognosis and the benefit of delivering adjuvant chemotherapy (ACT) in patients with non-small-cell lung cancer (NSCLC).<h4>Methods</h4>A novel prognostic algorithm was identified using genomic profiles from 332 stage I-III adenocarcinomas and independently validated on a separate series of 264 patients with stage I-II tumors, compiled from five previous studies. The prognostic algorithm was used to interrogate genomic data from a series of patients treated with adjuvant chemotherapy. Those genes associated with outcome in the adjuvant treatment setting, independent to prognosis were used to train an algorithm able to classify a patient as either a responder or non-responder to ACT. The performance of this signature was independently validated on a separate series of genomic profiles from patients enrolled in a randomized controlled trial of cisplatin/vinorelbine vs. observation alone (JBR.10).<h4>Results</h4>NSCLC patients exhibiting the high-risk, poor-prognosis form of the 160-gene prognosis signature experienced a 2.80-times higher rate of 5-year disease specific death (log rank P < 0.0001) compared to those with the low-risk, good prognosis profile, adjusted for covariates. The prognosis signature was found to especially accurate at identifying early stage patients at risk of disease specific death within 24 months of diagnosis when compared to traditional methods of outcome prediction.Separately, NSCLC patients with the 37-gene ACT-response signature (n = 70, 64 %), benefited significantly from cisplatin/vinorelbine (adjusted HR: 0.23, P = 0.0032). For those patients predicted to be responders, receiving this form of ACT conferred a 25 % improvement in the probability of 5-year-survival, compared to observation alone and adjusted for covariates. Conversely, in those patients predicted to be non-responders, ACT was observed to offer no significant survival benefit (adjusted HR: 0.55, P = 0.32).The two gene signatures overlap by one gene only SPSB3, which interacts with the oncogene MET. In this study, higher levels of SPSB3 which were associated with favorable prognosis and benefit from ACT.<h4>Conclusions</h4>These complimentary prognostic and predictive gene signatures may assist physicians in their management and treatment of patients with early stage lung cancer.