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ell understood. The enzyme catechol O-methyltransferase (COMT) catabolizes dopamine and norepinephrine in the prefrontal cortex and striatum, brain regions involved in behavioral control. The goal of this work was to investigate the association between genetic variation in COMT functioning and fronto-striatal brain functioning during successful inhibitory control, a critical aspect of behavioral control.
METHODS: Participants were 65 (22 female) 7-12 year olds who were genotyped for the functional COMT Val158Met (rs4680) single-nucleotide polymorphism and underwent functional magnetic resonance imaging while performing a go/no-go task. The majority of the sample (80%) had at least one parent with a history of alcohol use disorder and were thus at heightened risk for substance use disorders.
RESULTS: There was a significant main effect of genotype on brain activation in left and right putamen during successful versus failed inhibition and in right inferior frontal gyrus/insula during successful inhibition versus baseline. Follow-up tests revealed that Met homozygotes had greater activation in each region relative to Val homozygotes.
CONCLUSIONS: These results are relevant for understanding how specific genes influence brain functioning related to underlying risk factors for substance use disorders and other disinhibitory psychopathologies.

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ut RD. There are limited association studies performed between RD genes, specifically genes on 6p22, and regional brain activation during reading tasks. Using fourteen variants in DCDC2, KIAA0319, and TTRAP and exhaustive reading measures, we first tested for association with reading performance in 82 parent-offspring families (326 individuals). Next, we determined the association of these variants with activation of sixteen brain regions of interest during four functional magnetic resonance imaging-reading tasks. We nominally replicated associations between reading performance and variants of DCDC2 and KIAA0319. Furthermore, we observed a number of associations with brain activation patterns during imaging-reading tasks with all three genes. The strongest association occurred between activation of the left anterior inferior parietal lobe and complex tandem repeat BV677278 in DCDC2 (uncorrected p=0.00003, q=0.0442). Our results show that activation patterns across regions of interest in the brain are influenced by variants in the DYX2 locus. The combination of genetic and functional imaging data show a link between genes and brain functioning during reading tasks in subjects with RD. This study highlights the many advantages of imaging data as an endophenotype for discerning genetic risk factors for RD and other communication disorders and underscores the importance of integrating neurocognitive, imaging, and genetic data in future investigations.

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describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

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at biallelic variants in ribonuclease inhibitor (RNH1) confer susceptibility to a distinctive ANE subtype.
METHODS: This study aimed to evaluate clinical data, neuroradiological studies, genomic sequencing, and protein immunoblotting results in 8 children from 4 families who experienced acute febrile encephalopathy.
RESULTS: All 8 healthy children became acutely encephalopathic during a viral/febrile illness and received a variety of immune modulation treatments. Long-term outcomes varied from death to severe neurologic deficits to normal outcomes. The neuroradiological findings overlapped with ANE but had distinguishing features. All affected children had biallelic predicted damaging variants in RNH1: a subset that was studied had undetectable RNH1 protein. Incomplete penetrance of the RNH1 variants was evident in 1 family.
CONCLUSION: Biallelic variants in RNH1 confer susceptibility to a subtype of ANE (ANE2) in previously healthy children. Intensive immunological treatments may alter outcomes. Genomic sequencing in children with unexplained acute febrile encephalopathy can detect underlying genetic etiologies, such as RNH1, and improve outcomes in the probands and at-risk siblings.

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can effectively inhibit TNF bioactivity. METHODS: Fifty-three consecutive ambulatory or hospitalized SLE patients and 140 consecutive healthy subjects were enrolled in a prospective cohort study. Serum levels of sTNFR were measured by a unique 2-sided capture enzyme-linked immunosorbent assay using mouse monoclonal antibodies and rabbit antisera against the sTNFR. RESULTS: The mean +/- SD concentrations of both the p55 (type I) and p75 (type II) soluble receptors were significantly higher in a group of 46 SLE patients than in controls: 1.89 +/- 0.89 ng/ml versus 0.77 +/- 0.19 ng/ml and 7.25 +/- 3.89 ng/ml versus 3.02 +/- 0.57 ng/ml, respectively (P < 0.0001 for both). The incidence and the extent of the increase among the healthy subjects and these patients (as well as in 7 additional patients on whom sequential studies were performed) correlated with disease activity more than did the occurrence of serum anti-DNA antibodies (correlation coefficients with disease activity 0.81 and 0.85 for p55 and p75 sTNFR, respectively, and 0.51 for anti-DNA antibodies). The increase in sTNFR levels seems to reflect, largely, enhanced formation, and only to a minor extent, reduced clearance due to impairment of renal function. Sera of the SLE patients had a marked inhibitory effect on the in vitro cytocidal activity of TNF, and this was shown to result entirely from their higher sTNFR receptor concentration. CONCLUSION: An increase in serum levels of sTNFR may become a useful marker for SLE activity since it shows a stronger correlation than do any other laboratory or clinical parameters employed presently in the daily clinical setting. At the concentrations attained in the serum of SLE patients, sTNFR effectively inhibit the bioactivity of TNF and may thus be a significant determinant of the intensity of the manifestations of SLE.

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4 in a mouse model of P. aeruginosa keratitis. METHODS: Wild type (WT) and each of the Pglyrp-null genotypes were challenged with P. aeruginosa keratitis. The eyes were scored in a blinded manner 24 and 48h post infection. Viable bacterial counts and inflammatory factors (IL-12, TNF-alpha, IFN-gamma, CCL2, IL-6 and IL-10) were measured in whole eye homogenates using cytometric bead arrays. Expressions of Pglyrp-1-4, mouse beta defensins (mBD)-2,-3, cathelicidin-related antimicrobial peptide (CRAMP) were determined by qRTPCR in total RNA extracts of uninfected and infected eyes of WT and each of the Pglyrp-null mouse types. RESULTS: The Pglyrp-2-/- mice showed reduced disease and lower induction of pro-inflammatory TNF-alpha (p = 0.02) than WT or the other Pglyrp null mice. Viable bacterial yield was significantly lower in the Pglyrp-2-/- (p = 0.0007) and the Pglyrp-4-/- (p = 0.098) mice. With regards to expression of these antimicrobial genes, Pglyrp-2 expression was induced after infection in WT mice. Pglyrp-3 expression was low before and after infection in WT mice, while Pglyrp-4 expression was slightly elevated after infection in WT, Pglyrp-2 and -3 null mice. Pglyrp-1 expression was slightly elevated after infection in all genotypes without statistical significance. Transcripts for antimicrobial peptides mBD2, mBD3 and CRAMP were elevated in infected Pglyrp-2-/- males without statistical significance. CONCLUSIONS: Efficient resolution of keratitis in the Pglyrp-2-/- mice may be due to a reduced pro-inflammatory microenvironment and synergistic antibacterial activities of defensins, CRAMP and Pglyrp-1. Therefore, in ocular infections the pro-inflammatory functions of Pglyrp-2 must be regulated to benefit the host.

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l dominant neurodevelopmental disorders, while CHD5-related disorders are still unknown. Thanks to GeneMatcher and international collaborations, we assembled a cohort of 16 unrelated individuals harboring heterozygous CHD5 variants, all identified by exome sequencing. Twelve patients had de novo CHD5 variants, including ten missense and two splice site variants. Three familial cases had nonsense or missense variants segregating with speech delay, learning disabilities, and/or craniosynostosis. One patient carried a frameshift variant of unknown inheritance due to unavailability of the father. The most common clinical features included language deficits (81%), behavioral symptoms (69%), intellectual disability (64%), epilepsy (62%), and motor delay (56%). Epilepsy types were variable, with West syndrome observed in three patients, generalized tonic-clonic seizures in two, and other subtypes observed in one individual each. Our findings suggest that, in line with other CHD-related disorders, heterozygous CHD5 variants are associated with a variable neurodevelopmental syndrome that includes intellectual disability with speech delay, epilepsy, and behavioral problems as main features.

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, we found that augmented maternal care reduced glutamatergic synapses onto stress-sensitive hypothalamic neurons and repressed expression of the stress-responsive gene, Crh. In hypothalamus in vitro, reduced glutamatergic neurotransmission recapitulated the repressive effects of augmented maternal care on Crh, and this required recruitment of the transcriptional repressor repressor element-1 silencing transcription factor/neuron restrictive silencing factor (NRSF). Increased NRSF binding to chromatin was accompanied by sequential repressive epigenetic changes which outlasted NRSF binding. chromatin immunoprecipitation-seq analyses of NRSF targets identified gene networks that, in addition to Crh, likely contributed to the augmented care-induced phenotype, including diminished depression-like and anxiety-like behaviors. Together, we believe these findings provide the first causal link between enriched neonatal experience, synaptic refinement and induction of epigenetic processes within specific neurons. They uncover a novel mechanistic pathway from neonatal environment to emotional resilience.

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ements reveal that in aqueous solution (i.e., in the absence of structure-inducing organic solvents) CGRP preferentially populates conformations with short end-to-end distances. However, the end-to-end distance of CGRP is larger than that of IAPP. We find that electrostatic interactions can account for such a difference. At variance with previous reports on the secondary structure of CGRP, we find that the end-to-end distance of the peptide increases with decreasing pH and salt concentration, due to Coulomb repulsion by charged residues. Interestingly, our data show that the reconfiguration dynamics of CGRP is significantly slower than that of human IAPP in water but not in denaturant, providing experimental evidence for roughness in the energy landscape, or internal friction, in these peptides. The data reported here provide both structural and dynamical information that can be used to validate results from molecular simulations of calcitonin family peptides in aqueous solution.

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etion, thereby promoting interferon-gamma production and T helper 1 (T(H)1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in T cells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to "innate immune cells" but is an integral component of normal adaptive T(H)1 responses.

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role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS.

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tive RNA binding domain found in ribosomal proteins and a yeast suppressor of translation termination. By UV cross-linking and immunoprecipitation, we show that SBP2 specifically binds selenoprotein mRNAs both in vitro and in vivo. Using (75)Se-labeled Sec-tRNA(Sec), we developed an in vitro system for analyzing Sec incorporation in which the translation of a selenoprotein mRNA was both SBP2 and SECIS element dependent. Immunodepletion of SBP2 from the lysates abolished Sec insertion, which was restored when recombinant SBP2 was added to the reaction. These results establish that SBP2 is essential for the co-translational insertion of Sec into selenoproteins. We hypothesize that the binding activity of SBP2 may be involved in preventing termination at the UGA/Sec codon.

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assays for initiation revealed that although the smaller subunit contains catalytic function, initiation requires the presence of the larger subunit. A two-plasmid system was developed for the coexpression of both subunits in E. coli. This system was exploited to express and study truncations of the larger, human p58 subunit in order to investigate its role in primer formation. Analysis of the complexes formed between the truncated human p58 subunits and the native human p49 subunit revealed that protein-protein contacts between these two subunits occur over several regions of the human p58 subunit. Of four primase complexes containing different truncated p58 subunits only one complex supported initiation as measured by the formation of dinucleotides. All complexes supported the extension of oligoA-primed polydT, suggesting that the intrinsic RNA polymerase activity residing in the smaller subunit was not affected. These results suggest that several regions of the human p58 subunit are in contact with the human p49 subunit during the initiation of primer synthesis.

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d the activity of a 120-kDa RNA-binding protein that interacted specifically with the SECIS element in the phospholipid hydroperoxide glutathione peroxidase mRNA. This study reports the RNA binding and biochemical properties of this protein, SECIS-binding protein 2 (SBP2). We detected SBP2 binding activity in liver, hepatoma cell, and testis extracts from which SBP2 has been purified by anion exchange and RNA affinity chromatography. This scheme has allowed us to identify a 120-kDa polypeptide that co-elutes with SBP2 binding activity from wild-type but not mutant RNA affinity columns. A characterization of SBP2 biochemical properties reveals that SBP2 binding is sensitive to oxidation and the presence of heparin, rRNA, and poly(G). SBP2 activity elutes with a molecular mass of approximately 500 kDa during gel filtration chromatography, suggesting the existence of a large functional complex. Direct cross-linking and competition experiments demonstrate that the minimal phospholipid hydroperoxide glutathione peroxidase 3' UTR binding site is between 82 and 102 nucleotides, which correlates with the minimal sequence necessary for translational read-through. SBP2 also interacts specifically with the minimally functional 3' UTR of another selenoprotein mRNA, deiodinase 1.

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istone-post-translational modifications (PTMs) in various conditions and during cellular differentiation. However, our understanding of the abundance of histone PTMs and their regulatory mechanisms still remain unknown. RESULTS: Here, we present a quantitative and comprehensive study of the abundance levels of histone PTMs during the differentiation of mouse embryonic stem cells (ESCs) using mass spectrometry (MS). We observed dynamic changes of histone PTMs including increased H3K9 methylation levels in agreement with previously reported results. More importantly, we found a global decrease of multiply acetylated histone H4 peptides. Brd4 targets acetylated H4 with a strong affinity to multiply modified H4 acetylation sites. We observed that the protein levels of Brd4 decreased upon differentiation together with global histone H4 acetylation. Inhibition of Brd4:histone H4 interaction by the BET domain inhibitor (+)-JQ1 in ESCs results in enhanced differentiation to the endodermal lineage, by disrupting the protein abundance dynamics. Genome-wide ChIP-seq mapping showed that Brd4 and H4 acetylation are co-occupied in the genome, upstream of core pluripotency genes such as Oct4 and Nanog in ESCs and lineage-specific genes in embryoid bodies (EBs). CONCLUSIONS: Together, our data demonstrate the fundamental role of Brd4 in monitoring cell differentiation through its interaction with acetylated histone marks and disruption of Brd4 may cause aberrant differentiation.

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ent model of intrauterine growth restriction induced by chronic hypoxia and NO inhibition with N(G)-nitro-L-arginine methyl ester (L-NAME). STUDY DESIGN: Timed pregnant adult Sprague-Dawley rats were assigned to the following groups: (1) 20% (oxygen) O2 + saline (n = 7); (2) 20% O2 + L-NAME (n = 8); (3) 14% O2 + saline (n = 5); (4) 14% O2 + L-NAME (n = 5); (5) 10% O2 + saline (n = 6); and (6) 10% O2 + L-NAME (n = 6). Seven nulliparous females served as nonpregnant controls. L-NAME (50 mg/rat/day) or saline was administered via subcutaneous osmotic pumps, inserted on day 17 of gestation. A hypoxic chamber was used to assure mild (14% O2) or severe (10% O2) hypoxic environment after surgical placement of the minipumps and until the animals were killed on day 21 of gestation before the onset of labor. Maternal blood was collected preceding death. Free serum levels of VEGF, PlGF, and sFlt-1 were measured by highly specific immunoassays. Two composite indices were calculated (sFV: log [(sFlt-1)/VEGF] and sFP: log [(sFlt-1)/PlGF] and compared among groups. RESULTS: Fetal growth restriction was induced by both severe hypoxia (10% O2) and L-NAME infusion (2-way analysis of variance, P = .02 O2 levels, P < .001 L-NAME), whereas their combination proved to be the most damaging (P < .001). Pregnancy was characterized by higher maternal serum concentrations of VEGF (P < .001) and PlGF (P < .001), but lower levels of sFlt-1 (P = .037) compared with nonpregnant controls. Serum VEGF levels were not altered by either hypoxia or L-NAME infusion (P = .348 O2 levels, P = .205 L-NAME). In contrast, L-NAME significantly increased sFlt-1 serum levels independent of O2 levels (P = .032, L-NAME treatment, P = .991 O2 levels). Chronic hypoxia significantly decreases the circulating levels of PlGF (P < .001) independent of L-NAME treatment. The sFV ratio was neither altered by hypoxia nor by L-NAME infusion. In contrast, the sFP ratio was significantly increased by both L-NAME (P < .001) and severe hypoxia (P < .001), but the effect was not synergistic (P = .655). CONCLUSION: Chronic NO inhibition as well as hypoxia induce fetal growth restriction and significantly change maternal circulating levels of sFlt-1 and PlGF, but not of VEGF. The primary effect of chronic hypoxia is in decreasing circulating levels of PlGF that contrasts with that of NO inhibition, which selectively increases sFlt-1 levels. Their effect is thus not synergistic, suggesting independent pathways.

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on of neurodegenerative disorders. Few pharmacological studies use the transgenic model of PD to characterize the neuroprotection actions of a lead compound known to target HDAC in the brain.
METHODS: In our study, we investigated neuroprotective effects of liposomal-formulated curcumin: Lipocurc™ targeting HDAC inhibitor in the DJ-1(Park 7)-gene knockout rat model of PD. Group I (DJ-1-KO-Lipocurc™) received Lipocurc™ 20 mg/kg iv 3× weekly for 8 weeks; Group II: DJ-1 KO controls (DJ-1 KO-PBS) received i.v. phosphate-buffered saline (PBS). Group III: DJ-1-Wild Type (DJ-1 WT-PBS) received PBS. We monitored various components of motor behavior, rotarod, dyskinesia, and open-field behaviors, both at baseline and at regular intervals. Toward the end of the 8 weeks, we measured neuronal apoptosis and dopamine (DA) neuron-specific tyrosine hydroxylase levels by immunohistochemistry methods at post-mortem.
RESULTS: We found that DJ-KO Group I and Group II, as compared with DJ-1 WT group, exhibited moderate degree of motor impairment on the rotarod test. Lipocurc™ treatment improved the motor behavior motor impairment to a greater extent than the PBS treatment. There was marked apoptosis in the DJ-1 WT group. Lipocurc™ significantly blocked neuronal apoptosis: the apoptotic index of DJ-1-KO-Lipocurc™ group was markedly reduced compared with the DJ-KO-PBS group (3.3 vs 25.0, p<0.001). We found preliminary evidence Lipocurc™ stimulated DA neurons in the substantia nigra. The ratio of immature to mature DA neurons in substantia nigra was statistically higher in the DJ-1-KO-Lipocurc™ group (p<0.025).
CONCLUSIONS: We demonstrated for the first time Lipocurc™'s anti-apoptotic and neurotrophic effects in theDJ-1-KO rat model of PD. Our promising findings warrant randomized controlled trial of Lipocurc™ in translating the novel nanotechnology-based epigenetics-driven drug discovery platform toward efficacious therapeutics in PD.

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SC progeny, hinting at a constitutive role of DMT1 in ensuring the provision of iron in the PNS. In light of these previous results, the present work evaluates the participation of DMT1 in the remyelination process following a demyelinating lesion promoted by sciatic nerve crush--a reversible model of Wallerian degeneration. DMT1 was observed to colocalize with a SC marker S100beta at all survival times analyzed. In turn, the assessment of DMT1 mRNA expression exhibited an increase 7 days post-injury, while DMT1 protein levels showed an increase 14 days after crush at the lesion site and distal stump; finally, an increase in iron levels became evident as from 14 days post-injury, in parallel with DMT1 values. To sum up, the present work unveils the role of DMT1 in mediating the neuroregenerative action of iron.

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is finding and to evaluate whether combining the IL28B genotype and HLA class II alleles allow viral clearance to be accurately predicted. METHODS: Of 168 patients, 130 with complete clinical history were included in the analysis. According with their HCV antibodies status 13 were defined HCV resistant, and 117 infected. Infected patients were subdivided, giving 49 with self-limiting and 68 with ongoing infection. RESULTS: IL28B CC-genotype was observed in 32 patients with self-limiting and in 23 with ongoing infection (64% versus 34%; P=0.004). HLA DQB1*0301 allele was associated with viral clearance in 36 cases (73%; P<0.0001). Both DQB1*0301 and IL28B CC-genotype were found to be independent predictors of HCV clearance (OR=5.64, 95% CI 1.52-20.9 and OR=5.76, 95% CI 2.16-15.33, respectively). With the addition of DQB1*0301, the accuracy of the prediction increased from 63% to 69%. CONCLUSIONS: In addition to IL28B CC-genotype, HLA DQB1*0301 helps in predicting natural clearance of HCV after acute infection.

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c phenotypes. The aim of our study was to investigate the association between low-frequency TRIB3 coding variants and CAD in patients with type 2 diabetes (T2D). METHODS: Three case-control studies for CAD from Italy and US were analyzed, for a total of 1565 individuals, all with type 2 diabetes. Infrequent variants were identified by re-sequencing TRIB3 exons in 140 "extreme cases" and 140 "super-controls" and then genotyped in all study subjects. RESULTS: TRIB3 infrequent variants (n = 8), considered according to a collapsing rare variants framework, were significantly associated with CAD in diabetic patients from Italy (n = 700, OR = 0.43, 95% CI 0.20-0.91; p = 0.027), but not from the US (n = 865, OR = 1.22, 95% CI 0.69-2.18; p = 0.49). In the Italian sets, the association was especially strong among individuals who also carried the common R84 variant. CONCLUSION: Although preliminary, our finding suggests a role of TRIB3 low-frequency variants on CAD among Italian patients with T2D. Further studies are needed to address the role of TRIB3 infrequent variants in other populations of both European and non-European ancestries.

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inal/thoracic surgery. INTERVENTIONS: Standard multimodal POP treatment including opioids (tramadol) and nonsteroidal anti-inflammatory drugs (ketoprofen) at different dosages and infusion rates according to the predicted mild, moderate, or severe POP. OUTCOME MEASURES: Pain (Numeric Rating Scale-NRS) and sedation (Ramsay Sedation Scale-RSS) up to 24 hours after surgery. By genotyping 16 CYP2D6 alleles, the four CYP2D6 phenotypes poor metabolizer (PM), intermediate metabolizers (IM), extensive metabolizers (EM) and ultrarapid metabolizers (UM) were predicted. RESULTS: As compared with the CYP2D6-EM phenotype, in the early PO time (30 min) a higher RSS mean score in IM was observed (P = 0.035). A suggestion towards higher mean score in PM (P = 0.091) and a minor mean score in UM (P = 0.091) was also detected. No difference in the outcome of pain across the CYP2D6 phenotypes was observed. CONCLUSIONS: In respect to the normal CYP2D6 phenotype, our results suggested that slowly metabolizers (IMs and PMs) might have a major sedation, whereas more rapid metabolizers (UM) a minor sedation, in the early time after surgery. A minor role of CYP2D6 phenotype in PO analgesia may be suggested.

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, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS: Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80-2.70) in GHS, 2.31 (95% CI 1.22-4.34) in TVAS, and 1.36 (95% CI 0.88-2.10) in CREED, and 1.56 (95% CI 1.15-2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS: The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

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ies.
PATIENTS: Four children, 2 related and 2 unrelated, with the novel p.P1073L mutation (all patients) and either the p.A467T (2 patients), p.G848S (1 patient), or p.W748S (1 patient) mutation presented with psychomotor delay, encephalopathy, and liver failure.
INTERVENTIONS: Detailed clinical and laboratory examinations including brain magnetic resonance imaging, muscle biopsy, measurement of mitochondrial DNA, and sequencing of the POLG gene.
MAIN OUTCOME MEASURES: Definition of clinical variability.
RESULTS: All 4 patients had psychomotor delay, seizures, and liver disease. Three patients had severe gastrointestinal dysmotility, which may be associated with the new p.P1073L mutation.
CONCLUSIONS: The heterozygous presence of the novel p.P1073L mutation in trans with another recessive POLG mutation causes a hepatocerebral disorder identical or very similar to Alpers syndrome. This adds to the already striking clinical heterogeneity of POLG mutations. In the Belgian patients, the familial occurrence without consanguinity is related to the high frequency of the recessive p.A467T and p.W748S mutations in northwestern Europe and reveals a pitfall for diagnosis and genetic counseling.

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part of the malformative spectrum of tubulinopathies and X-linked lissencephaly with abnormal genitalia, but neurodevelopmental syndromes characterized by basal ganglia agenesis are not known to date. We ascertained two unrelated children (both female) presenting with spastic tetraparesis, severe generalized dystonia and intellectual impairment, sharing a unique brain malformation characterized by agenesis of putamina and globi pallidi, dysgenesis of the caudate nuclei, olfactory bulbs hypoplasia, and anomaly of the diencephalic-mesencephalic junction with abnormal corticospinal tract course. Whole-exome sequencing identified two novel homozygous variants, c.26C>A; p.(S9*) and c.752A>G; p.(Q251R) in the GSX2 gene, a member of the family of homeobox transcription factors, which are key regulators of embryonic development. GSX2 is highly expressed in neural progenitors of the lateral and median ganglionic eminences, two protrusions of the ventral telencephalon from which the basal ganglia and olfactory tubercles originate, where it promotes neurogenesis while negatively regulating oligodendrogenesis. The truncating variant resulted in complete loss of protein expression, while the missense variant affected a highly conserved residue of the homeobox domain, was consistently predicted as pathogenic by bioinformatic tools, resulted in reduced protein expression and caused impaired structural stability of the homeobox domain and weaker interaction with DNA according to molecular dynamic simulations. Moreover, the nuclear localization of the mutant protein in transfected cells was significantly reduced compared to the wild-type protein. Expression studies on both patients' fibroblasts demonstrated reduced expression of GSX2 itself, likely due to altered transcriptional self-regulation, as well as significant expression changes of related genes such as ASCL1 and PAX6. Whole transcriptome analysis revealed a global deregulation in genes implicated in apoptosis and immunity, two broad pathways known to be involved in brain development. This is the first report of the clinical phenotype and molecular basis associated to basal ganglia agenesis in humans.

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pulmonary inflammation and airway hyper-responsiveness in asthma. This study investigated whether an anti-TNF-alpha antibody would inhibit pulmonary inflammation and methacholine (Mch) hyper-responsiveness in a mouse model of asthma induced by a house dust extract containing both endotoxin and cockroach allergens. METHODS: A house dust sample was extracted with phosphate-buffered saline and then used for immunization and two additional pulmonary challenges of BALB/c mice. Mice were treated with an intravenous injection of anti-TNF-alpha antibody or control antibody 1 h before each pulmonary challenge. RESULTS: In a kinetic study, TNF-alpha levels within the bronchoalveolar lavage (BAL) fluid increased quickly peaking at 2 h while BAL levels of IL-4, IL-5, and IL-13 peaked at later time-points. Mch hyper-responsiveness was measured 24 h after the last challenge, and mice were killed 24 h later. TNF inhibition resulted in an augmentation of these Th2 cytokines. However, the allergic pulmonary inflammation was significantly reduced by anti-TNF-alpha antibody treatment as demonstrated by a substantial reduction in the number of BAL eosinophils, lymphocytes, macrophages, and neutrophils compared with rat IgG-treated mice. Mch hyper-responsiveness was also significantly reduced in anti-TNF-alpha antibody-treated mice and the pulmonary histology was also significantly improved. Inhibition of TNF significantly reduced eotaxin levels within the lung, suggesting a potential mechanism for the beneficial effects. These data indicate that anti-TNF-alpha antibody can reduce the inflammation and pathophysiology of asthma in a murine model of asthma induced by a house dust extract.

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per layer neurons. Bcl11a-deficient late-born neurons fail to correctly switch from multipolar to bipolar morphology, resulting in impaired radial migration. We show that the expression of Sema3c is increased in migrating Bcl11a-deficient neurons and that Bcl11a is a direct negative regulator of Sema3c transcription. In vivo gain-of-function and rescue experiments demonstrate that Sema3c is a major downstream effector of Bcl11a required for the cell polarity switch and for the migration of upper layer neurons. Our data uncover a novel Bcl11a/Sema3c-dependent regulatory pathway used by migrating cortical neurons.

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ortex, and its loss in neurons led to defects in neurite stability and migration. The αN-catenin paralog, αE-catenin, acts as a switch regulating the balance between β-catenin and Arp2/3 actin filament activities1. Loss of αN-catenin did not affect β-catenin signaling, but recombinant αN-catenin interacted with purified actin and repressed ARP2/3 actin-branching activity. The actin-binding domain of αN-catenin or ARP2/3 inhibitors rescued the neuronal phenotype associated with CTNNA2 loss, suggesting ARP2/3 de-repression as a potential disease mechanism. Our findings identify CTNNA2 as the first catenin family member with biallelic mutations in humans, causing a new pachygyria syndrome linked to actin regulation, and uncover a key factor involved in ARP2/3 repression in neurons.

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uencing in two consanguineous families affected by nonsyndromic autosomal-recessive intellectual disability, we identified truncating mutations in formin 2 (FMN2), encoding a protein that belongs to the formin family of actin cytoskeleton nucleation factors and is highly expressed in the maturing brain. We found that FMN2 localizes to punctae along dendrites and that germline inactivation of mouse Fmn2 resulted in animals with decreased spine density; such mice were previously demonstrated to have a conditioned fear-learning defect. Furthermore, patient neural cells derived from induced pluripotent stem cells showed correlated decreased synaptic density. Thus, FMN2 mutations link intellectual disability either directly or indirectly to the regulation of actin-mediated synaptic spine density.

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ase (a process known as GJ remodeling), producing an arrhythmogenic substrate. The main constituent of GJs in the ventricular myocardium is Connexin 43 (Cx43), an integral membrane protein that is rapidly turned over and shows decreased expression or function with age. We hypothesized that Wwp1, an ubiquitin ligase whose expression in known to increase in aging-related pathologies, may regulate Cx43 in vivo by targeting it for ubiquitylation and degradation and yield tissue-specific Cx43 loss of function phenotypes. When Wwp1 was globally overexpressed in mice under the control of a beta-actin promoter, the highest induction of Wwp1 expression was observed in the heart which was associated with a 90% reduction in cardiac Cx43 protein levels, left ventricular hypertrophy (LVH), and the development of lethal ventricular arrhythmias around 8weeks of age. This phenotype was completely penetrant in two independent founder lines. Cardiomyocyte-specific overexpression of Wwp1 confirmed that this phenotype was cell autonomous and delineated Cx43-dependent and -independent roles for Wwp1 in arrhythmogenesis and LVH, respectively. Using a cell-based system, it was determined that Wwp1 co-immunoprecipitates with and ubiquitylates Cx43, causing a decrease in the steady state levels of Cx43 protein. These findings offer new mechanistic insights into the regulation of Cx43 which may be exploitable in various gap junctionopathies.

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P. Here, we examine the contribution of ATF7IP to the in vitro activity and substrate specificity of SETDB1. SETDB1 and ATF7IP were co-expressed and 1:1 stoichiometric complexes were purified for comparison against SETDB1 enzyme alone. We employed both radiometric flashplate-based and SAMDI mass spectrometry assays to follow methylation on histone H3 15-mer peptides, where lysine 9 was either unmodified, monomethylated, or dimethylated. Results show that SETDB1 and the SETDB1:ATF7IP complex efficiently catalyze both monomethylation and dimethylation of H3K9 peptide substrates. The activity of the binary complex was 4-fold lower than SETDB1 alone. This difference was due to a decrease in the value of kcat as the substrate KM values were comparable between SETDB1 and the SETDB1:ATF7IP complex. H3K9 methylation by SETDB1 occurred in a distributive manner, and this too was unaffected by the presence of ATF7IP. This finding is important as H3K9 can be methylated by HMTs other than SETDB1 and a distributive mechanism would allow for interplay between multiple HMTs on H3K9. Our results indicate that ATF7IP does not directly modulate SETDB1 catalytic activity, suggesting alternate roles, such as affecting cellular localization or mediating interaction with additional binding partners.

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t imprinted. An examination of embryonic RNA expression patterns shows that Bmp7 is expressed in a variety of skeletal and nonskeletal tissues. Both embryonic expression patterns and the human chromosomal sublocalization inferred from its mouse location make Bmp7 a candidate for the gene affected in some patients with Holt-Oram syndrome.

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for Otf-1 and Otf-2 recognized single loci on mouse chromosomes 1 and 7, respectively, whereas probes for Otf-3 recognized a minimum of eight independently segregating loci (designated Otf-3a through Otf-3h). Members of the Otf-3 family mapped to mouse chromosomes 1, 2, 3, 6, 14, 17, and the X chromosome, indicating that the Otf family has become widely dispersed during evolution. Several Otf loci mapped near developmental mutations, raising the possibility that these mutations result from defects in Otf family members.

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defects. Y955C POLG was targeted transgenically (TG) to the murine heart. Survival was determined in four TG (+/-) lines and wild-type (WT) littermates (-/-). Left ventricle (LV) performance (echocardiography and MRI), heart rate (electrocardiography), mtDNA abundance (real time PCR), oxidation of mtDNA (8-OHdG), histopathology and electron microscopy defined the phenotype. Cardiac targeted Y955C POLG yielded a molecular signature of CPEO in the heart with cardiomyopathy (CM), mitochondrial oxidative stress, and premature death. Increased LV cavity size and LV mass, bradycardia, decreased mtDNA, increased 8-OHdG, and cardiac histopathological and mitochondrial EM defects supported and defined the phenotype. This study underscores the pathogenetic role of human mutant POLG and its gene product in mtDNA depletion, mitochondrial oxidative stress, and CM as it relates to the genetic defect in CPEO. The transgenic model pathophysiologically links human mutant Pol gamma, mtDNA depletion, and mitochondrial oxidative stress to the mtDNA replication apparatus and to CM.

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ECTIVE: We sought to determine whether there was evidence of haploinsufficiency in hypertrophic cardiomyopathy caused by MYBPC3 mutations by comparing left ventricular muscle from patients undergoing surgical myectomy with samples from donor hearts. METHODS AND RESULTS: MyBP-C protein and mRNA levels were quantitated using immunoblotting and RT-PCR. Nine of 37 myectomy samples had mutations in MYBPC3: 2 missense alleles (Glu258Lys, Arg502Trp) and 7 premature terminations. No specific truncated MyBP-C peptides were detected in whole muscle homogenates of hypertrophic cardiomyopathy tissue. However, the overall level of MyBP-C in myofibrils was significantly reduced (P<0.0005) in tissue containing either a truncation or missense MYBPC3 mutation: 0.76+/-0.03 compared with 1.00+/-0.05 in donor and 1.01+/-0.06 in non-MYBPC3 mutant myectomies. CONCLUSIONS: The absence of any detectable truncated MyBP-C argues against its incorporation in the myofiber and any dominant negative effect. In contrast, the lowered relative level of full length protein in both truncation and missense MYBPC3 mutations argues strongly that haploinsufficiency is sufficient to cause the disease.

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ssion is associated with poor patient survival. Although EVI1Delta324 is co-transcribed with EVI1, its activity in cancer cells is not fully understood. Previous reports described that unlike EVI1, EVI1Delta324 protein cannot transform fibroblasts because of its disrupted N-terminal zinc finger (ZNF) domain. To better understand EVI1Delta324 biology and function, we obtained genome-wide binding occupancies and expression data in ovarian cancer cells. We characterized its DNA-binding sites, binding motif and target genes. Comparative analyses with previous study show that EVI1 and EVI1Delta324 share similar transcriptional activities linked to their common C-terminus ZNF domain. They bind to an E-twenty-six family (ETS)-like motif, target to a large extent the same genes and cooperate with AP1 transcription factor. EVI1Delta324-occupied genes were 70.7% similar to EVI1-bound genes. More strikingly, EVI1 and EVI1Delta324 differentially expressed genes were 99.87% identical, indicating comparable transcriptional regulatory functions. Consistently with gene ontologies linked to these target genes, EVI1Delta324 expression in HeLa cells could enhance anchorage-independent growth, such as EVI1, showing that EVI1Delta324 expression also lead to pro-oncogenic effects. The main specific feature of EVI1 variant is its N-terminus ZNF domain that binds DNA through GATA-like motif. We found that most GATA-like EVI1 chromatin immunoprecipitation sequencing peaks are far from genes and are not involved in transcriptional regulation. These genomic regions were enriched in simple sequence repeats and displayed high meiotic recombination rates. Overall, our genomics analyses uncovered common and specific features of two major MECOM isoforms. Their influence on transcription and downstream cell proliferation was comparable. However, EVI1-specific GATA-like binding sites, from its N-terminus ZNF domain, associated with high recombination rates, suggesting possible additional oncogenic potential for EVI1 in modulating genomic stability.

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ated to each other by having three to six repeats of a cysteine-rich sequence in their extracellular domains. Members of this family of receptors have also been shown to bind to ligands which are structurally related to TNF. The mouse homologue of the rat OX40 protein was cloned at the cDNA and genomic levels. The gene structure shows that there are several intron/exon borders shared between OX40 and CD27, CD40, TNF receptor type I, CD95 and 4-1BB genes. This group of genes is less closely related structurally to the gene structure of the NGF receptor. The gene encoding murine OX40 has been placed on mouse chromosome 4, in an area which contains the genes for TNF receptor type II and 4-1BB, and is syntenic with a region of human chromosome 1 which contains human TNF receptor type II, OX40, and CD30 genes.

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olonization and infection lead to clinical decline. Although fungi are frequently recovered from cystic fibrosis patient sputum samples and have been associated with deterioration of lung function, understanding of species and population dynamics remains in its infancy. Here, we coupled high-throughput sequencing of the ribosomal RNA internal transcribed spacer 1 (ITS1) with phenotypic and genotypic analyses of fungi from 89 sputum samples from 28 cystic fibrosis patients. Fungal communities defined by sequencing were concordant with those defined by culture-based analyses of 1,603 isolates from the same samples. Different patients harbored distinct fungal communities. There were detectable trends, however, including colonization with Candida and Aspergillus species, which was not perturbed by clinical exacerbation or treatment. We identified considerable inter- and intra-species phenotypic variation in traits important for host adaptation, including antifungal drug resistance and morphogenesis. While variation in drug resistance was largely between species, striking variation in morphogenesis emerged within Candida species. Filamentation was uncoupled from inducing cues in 28 Candida isolates recovered from six patients. The filamentous isolates were resistant to the filamentation-repressive effects of Pseudomonas aeruginosa, implicating inter-kingdom interactions as the selective force. Genome sequencing revealed that all but one of the filamentous isolates harbored mutations in the transcriptional repressor NRG1; such mutations were necessary and sufficient for the filamentous phenotype. Six independent nrg1 mutations arose in Candida isolates from different patients, providing a poignant example of parallel evolution. Together, this combined clinical-genomic approach provides a high-resolution portrait of the fungal microbiome of cystic fibrosis patient lungs and identifies a genetic basis of pathogen adaptation.

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ns (GEOSTAT-1) Study was a genetic substudy of Secondary Prevention of Acute Coronary Events-Reduction of Cholesterol to Key European Targets (SPACE ROCKET) (a randomized, controlled trial comparing 40 mg of simvastatin and 10 mg of rosuvastatin) that recruited 601 patients after myocardial infarction. We genotyped the following functional single nucleotide polymorphisms in the genes coding for the cytochrome P450 (CYP) metabolic enzymes, CYP2C9*2 (430C>T), CYP2C9*3 (1075A>C), CYP2C19*2 (681G>A), CYP3A5*1 (6986A>G), and hepatic influx and efflux transporters SLCO1B1 (521T>C) and breast cancer resistance protein (BCRP; 421C>A). We assessed 3-month LDL cholesterol levels and the proportion of patients reaching the current LDL cholesterol target of <70 mg/dL (<1.81 mmol/L). An enhanced response to rosuvastatin was seen for patients with variant genotypes of either CYP3A5 (P=0.006) or BCRP (P=0.010). Furthermore, multivariate logistic-regression analysis revealed that patients with at least 1 variant CYP3A5 and/or BCRP allele (n=186) were more likely to achieve the LDL cholesterol target (odds ratio: 2.289; 95% CI: 1.157, 4.527; P=0.017; rosuvastatin 54.0% to target vs simvastatin 33.7%). There were no differences for patients with variants of CYP2C9, CYP2C19, or SLCO1B1 in comparison with their respective wild types, nor were differential effects on statin response seen for patients with the most common genotypes for CYP3A5 and BCRP (n=415; odds ratio: 1.207; 95% CI: 0.768, 1.899; P=0.415). CONCLUSION: The LDL cholesterol target was achieved more frequently for the 1 in 3 patients with CYP3A5 and/or BCRP variant genotypes when prescribed rosuvastatin 10 mg, compared with simvastatin 40 mg. Clinical Trial Registration- URL: http://isrctn.org. Unique identifier: ISRCTN 89508434.

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ne out. The most striking characteristic of HCM mutations in MYBPC3 is that many are within introns and are predicted to cause aberrant splicing leading to a frameshift and a premature chain termination, yet the truncated peptides have never been identified in human heart tissue carrying these mutations. Instead of expression of a poison peptide we consistently observe haploinsufficiency of MyBP-C in MYBPC3 mutant human heart muscle. In this review we investigate the mechanism for MyBP-C haploinsufficiency and consider how this haploinsufficiency could cause hypertrophic cardiomyopathy.

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l localization to 2q34-q36 was established by hybridizing the CRYBA2 probe to microcell and radiation hybrids containing defined fragments of chromosome 2 as the only human contribution. The CRYBA2 probe was also used to localize, by interspecific backcross mapping, the mouse gene (Cryba2) to the central portion of chromosome 1 in a region of known human chromosome 2 homology. Finally, we demonstrate that in both species the beta A2 crystallin gene is linked but separable from the gamma A crystallin gene. The beta A2 crystallin gene is a candidate gene for human and mouse hereditary cataract.

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tudy using Veterans Affairs administrative data including subjects >or=65 years of age hospitalized with a COPD exacerbation. Our primary analysis was a multilevel model with the dependent variable of 90-day mortality and hospital as a random effect, controlling for preexisting comorbid conditions, demographics, and other medications prescribed. RESULTS: We identified 11,212 subjects with a mean age of 74.0 years, 98% were male, and 12.4% of subjects died within 90-days of hospital presentation. In this cohort, 20.3% of subjects were using statins, 32.0% were using ACE inhibitors or angiotensin II receptor blockers (ARB). After adjusting for potential confounders, current statin use (odds ratio 0.51, 95% confidence interval 0.40-0.64) and ACE inhibitor/ARB use (0.55, 0.46-0.66) were significantly associated with decreased 90-day mortality. CONCLUSION: Use of statins and ACE inhibitors prior to admission is associated with decreased mortality in subjects hospitalized with a COPD exacerbation. Randomized controlled trials are needed to examine whether the use of these medications are protective for those patients with COPD exacerbations.

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cids) of huntingtin with 82, 44 or 18 glutamines. Mice expressing relatively low steady-state levels of N171 huntingtin with 82 glutamine repeats (N171-82Q) develop behavioral abnormalities, including loss of coordination, tremors, hypokinesis and abnormal gait, before dying prematurely. In mice exhibiting these abnormalities, diffuse nuclear labeling, intranuclear inclusions and neuritic aggregates, all immunoreactive with an antibody to the N-terminus (amino acids 1-17) of huntingtin (AP194), were found in multiple populations of neurons. None of these behavioral or pathological phenotypes were seen in mice expressing N171-18Q. These findings are consistent with the idea that N-terminal fragments of huntingtin with a repeat expansion are toxic to neurons, and that N-terminal fragments are prone to form both intranuclear inclusions and neuritic aggregates.

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hway, previously shown to control F-actin turnover. Activation of SRF by overexpression of wild-type LIMK or ROCK-insensitive LIMK mutants also requires functional RhoA, indicating that a second RhoA-dependent signal is involved. This is provided by the RhoA effector mDia: dominant interfering mDia1 derivatives inhibit both serum- and LIMK-induced SRF activation and reduce the ability of LIMK to induce F-actin accumulation. These results demonstrate a role for LIMK in SRF activation, and functional cooperation between RhoA-controlled LIMK and mDia effector pathways.

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n in the mouse neural crest leads to an altered sympatho-vagal regulation of cardiac rhythmicity in adults characterized by a chronotropic incompetence and cardiac conduction defects, thus increasing the susceptibility to SCD. We demonstrated that Meis1 is a major regulator of sympathetic target-field innervation and that Meis1 deficient sympathetic neurons die by apoptosis from early embryonic stages to perinatal stages. In addition, we showed that Meis1 regulates the transcription of key molecules necessary for the endosomal machinery. Accordingly, the traffic of Rab5(+) endosomes is severely altered in Meis1-inactivated sympathetic neurons. These results suggest that Meis1 interacts with various trophic factors signaling pathways during postmitotic neurons differentiation.

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sorders (MCHS), 2) Alpers syndrome, 3) Ataxia Neuropathy Spectrum (ANS) disorders, 4) Myoclonus Epilepsy Myopathy Sensory Ataxia (MEMSA), 5) autosomal recessive Progressive External Ophthalmoplegia (arPEO), and 6) autosomal dominant Progressive External Ophthalmoplegia (adPEO). Due to the clinical heterogeneity, time-dependent evolution of symptoms, overlapping phenotypes, and inconsistencies in muscle pathology findings, definitive diagnosis relies on the molecular finding of deleterious mutations. We sequenced the exons and flanking intron region from approximately 350 patients displaying a phenotype consistent with POLG related mitochondrial disease and found informative mutations in 61 (17%). Two mutant alleles were identified in 31 unrelated index patients with autosomal recessive POLG-related disorders. Among them, 20 (67%) had Alpers syndrome, 4 (13%) had arPEO, and 3 (10%) had ANS. In addition, 30 patients carrying one altered POLG allele were found. A total of 25 novel alterations were identified, including 6 null mutations. We describe the predicted structural/functional and clinical importance of the previously unreported missense variants and discuss their likelihood of being pathogenic. In conclusion, sequence analysis allows the identification of mutations responsible for POLG-related disorders and, in most of the autosomal recessive cases where two mutant alleles are found in trans, finding deleterious mutations can provide an unequivocal diagnosis of the disease.

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asured by in vitro motility assay (2.3-fold for mice and 1.3-fold for humans) and in skinned papillary muscle. The mutation also abolished the change in Ca(2+) sensitivity normally linked to troponin I phosphorylation. MyBP-C and troponin I phosphorylation levels were the same as controls in transgenic mice and human carrier heart samples. ACTC E99K mice exhibited a high death rate between 28 and 45 days (48% females and 22% males). At 21 weeks, the hearts of the male survivors had enlarged atria, increased interstitial fibrosis, and sarcomere disarray. MRI showed hypertrophy, predominantly at the apex of the heart. End-diastolic volume and end-diastolic pressure were increased, and relaxation rates were reduced compared with nontransgenic littermates. End-systolic pressures and volumes were unaltered. ECG abnormalities were present, and the contractile response to beta-adrenergic stimulation was much reduced. Older mice (29-week-old females and 38-week-old males) developed dilated cardiomyopathy with increased end-systolic volume and continuing increased end-diastolic pressure and slower contraction and relaxation rates. ECG showed atrial flutter and frequent atrial ectopic beats at rest in some ACTC E99K mice. We propose that the ACTC E99K mutation causes higher myofibrillar Ca(2+) sensitivity that is responsible for the sudden cardiac death, apical hypertrophy, and subsequent development of heart failure in humans and mice.

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ogous regions between human and mouse chromosomes and provide additional information regarding human/mouse comparative mapping.

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had not been identified. Here we describe molecular cloning of murine OSM as an immediate early gene induced by a subset of cytokines including IL2, IL3 and erythropoietin (EPO) in myeloid and lymphoid cell lines. The induction kinetics of OSM are rapid and transient, reaching a maximal level within 30-60 min and decreasing thereafter. Induction of OSM depends on the signals generated by the membrane-proximal region of the EPO receptor as well as that of the beta chain of the IL3/GM-CSF receptor, which activate JAK2 and STAT5. About 100 bases upstream of the transcription initiation site of the OSM gene contains a possible STAT5 binding site which is essential for IL2, IL3 and EPO-dependent promoter activity of the OSM gene. Expression of STAT5 and the EPO receptor in COS cells conferred EPO-dependent activation of the OSM promoter. Moreover, the mutant IL2 receptor lacking the ability to activate STAT5 induced c-myc but failed to induce OSM. Thus OSM is one of the common targets of a subset of cytokines that activate STAT5. The murine OSM gene is located near to the LIF gene, expressed at high levels in bone marrow and possesses similar biological activity to human OSM. Identification of murine OSM as a cytokine-inducible immediate early gene provides a new insight into the physiological function of this unique cytokine.

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nesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.

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(XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.

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docytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

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antly underestimated. However, single gene defects occur only in a minority of cases, and multigenetic causes of congenital heart diseases have not been fully demonstrated. Here, we show that interactions between alleles of 3 Pbx genes, which encode homeodomain transcription factors, are sufficient to determine the phenotypic presentation of congenital heart diseases in mice. A major role is served by Pbx1, whose inactivation results in persistent truncus arteriosus. Reduction or absence of Pbx2 or Pbx3 leads to Pbx1 haploinsufficiency and specific malformations that resemble tetralogy of Fallot, overriding aorta with ventricular septal defect, and bicuspid aortic valves. Disruption of Meis1, which encodes a Pbx DNA-binding partner, results in cardiac anomalies that resemble those caused by Pbx mutations. Each of the observed cardiac defects represents developmental abnormalities affecting distinct stages of cardiac outflow tract development and corresponds to specific types of human congenital heart disease. Thus, varied deficiencies in the Pbx gene family produce a full spectrum of cardiac defects involving the outflow tract, providing a framework for determining multigenetic causes of congenital heart anomalies.

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pectively), two 'mimic' mouse models of HPS. We also show that these genes are orthologs of the genes mutated in individuals with HPS types 5 and 6, respectively, and that their protein products directly interact. Both genes are previously unknown and are found only in higher eukaryotes, and together represent a new class of genes that have evolved in higher organisms to govern the synthesis of highly specialized lysosome-related organelles.

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3 (thioredoxin reductases), GPX1, GPX2, GPX3, GPX4 and GPX6 (glutathione peroxidases), DIO1, DIO2, and DIO3 (iodothyronine deiodinases), MSRB1 (methionine-R-sulfoxide reductase 1) and SEPHS2 (selenophosphate synthetase 2). Selenoproteins without known functions have traditionally been denoted by SEL or SEP symbols. However, these symbols are sometimes ambiguous and conflict with the approved nomenclature for several other genes. Therefore, there is a need to implement a rational and coherent nomenclature system for selenoprotein-encoding genes. Our solution is to use the root symbol SELENO followed by a letter. This nomenclature applies to SELENOF (selenoprotein F, the 15 kDa selenoprotein, SEP15), SELENOH (selenoprotein H, SELH, C11orf31), SELENOI (selenoprotein I, SELI, EPT1), SELENOK (selenoprotein K, SELK), SELENOM (selenoprotein M, SELM), SELENON (selenoprotein N, SEPN1, SELN), SELENOO (selenoprotein O, SELO), SELENOP (selenoprotein P, SeP, SEPP1, SELP), SELENOS (selenoprotein S, SELS, SEPS1, VIMP), SELENOT (selenoprotein T, SELT), SELENOV (selenoprotein V, SELV) and SELENOW (selenoprotein W, SELW, SEPW1). This system, approved by the HUGO Gene Nomenclature Committee, also resolves conflicting, missing and ambiguous designations for selenoprotein genes and is applicable to selenoproteins across vertebrates.

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the immune system. Here we report the structure and chromosomal location of the mouse Mif gene and the partial characterization of five Mif pseudogenes. The mouse Mif gene spans less than 0.7 kb of chromosomal DNA and is composed of three exons. A comparison between the mouse and the human genes shows a similar gene structure and common regulatory elements in both promoter regions. The mouse Mif gene maps to the middle region of chromosome 10, between Bcr and S100b, which have been mapped to human chromosomes 22q11 and 21q22.3, respectively. The entire sequence of two pseudogenes demonstrates the absence of introns, the presence of the 5' untranslated region of the cDNA, a 3' poly(A) tail, and the lack of sequence similarity with untranscribed regions of the gene. The five pseudogenes are highly homologous to the cDNA, but contain a variable number of mutations that would produce mutated or truncated MIF-like proteins. Phylogenetic analyses of MIF genes and pseudogenes indicate several independent genetic events that can account for multiple genomic integrations. Three of the Mif pseudogenes were also mapped by interspecific backcross to chromosomes 1, 9, and 17. These results suggest that Mif pseudogenes originated by retrotransposition.

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hile in humans, there are two highly related genes (A and B) that were generated by homologous recombination. Chromosomal mapping indicated that the mouse gene is located in the central region of the mouse chromosome 8, which is syntenic with human chromosomes 4q28-31 where the human glycophorin A gene has been mapped. The mouse gene consists of 8 exons, while the human gene consists of 7 exons and the length of each exon is quite short except for the last exon. The last 4 exons showed extensive homology between the mouse and human genes but divergence in the 5'-exons of the two genes was high. The results suggest that glycophorin genes of mouse and human may have been generated from the same ancestor, but diverged greatly during evolution. The upstream regulatory region of the mouse gene consists of multiple motifs for DNA binding factors that may be required for its erythroid-specific expression.

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pers syndrome, juvenile spinocerebellar ataxia-epilepsy syndrome, and progressive external ophthalmoplegia, each with vastly different clinical presentations, tissue specificities, and ages of onset. The A467T mutant enzyme possesses only 4% of wild-type DNA polymerase activity, and the catalytic defect is manifest primarily through a 6-fold reduction in kcat with minimal effect on exonuclease function. Human DNA polymerase gamma (pol gamma) requires association of a 55-kDa accessory subunit for enhanced DNA binding and highly processive DNA synthesis. However, the A467T mutant enzyme failed to interact with and was not stimulated by the accessory subunit, as judged by processivity, heat inactivation, and N-ethylmaleimide protection assays in vitro. Thermolysin digestion and immunoprecipitation experiments further indicate weak association of the subunits for A467T pol gamma. This is the first example of a mutation in POLG that disrupts physical association of the pol gamma subunits. We propose that reduced polymerase activity and loss of accessory subunit interaction are responsible for the depletion and deletion of mitochondrial DNA observed in patients with this POLG mutation.

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he localization of the mouse genes (Grik4 and Grik5) was established by interspecific backcross mapping. GRIK4 and GRIK5 are located on separate chromosomes (Chrs) in all species. GRIK4 mapped to human Chr 11q22.3, mouse Chr 9, and rat Chr 8. GRIK5 mapped to human Chr 19q13.2, mouse Chr 7, and rat Chr 1. The genes encoding the (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-preferring subunit GluR4, or GluRD (GRIA4), the neural cell adhesion molecule (NCAM), the D2 dopamine receptor (DRD2), and the Thy-1 cell surface antigen (THY1) have all been previously mapped to the human Chr 11q22 region. The mapping of the human GRIK4 and GRIK5 genes confirms and extends the relationship between human Chr 11 and mouse Chr 9 and also human Chr 19 and mouse Chr 7. GRIK4 is the fifth gene shared by human Chr 11 and rat Chr 8, whereas GRIK5 is 1 out of the 12 genes that are located on both human Chr 19 and rat Chr 1. Our data extend the conserved synteny established between certain human, mouse, and rat Chrs.

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BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium. Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice. Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation. Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth.

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ue-specific expression. Furthermore, lymphocytes have a unique T beta 4 transcript relative to the ubiquitous transcript found in many other tissues and cells. To determine how T beta 4 gene expression is regulated and how the alternative transcripts are derived, we cloned the mouse T beta 4 gene. We established that there is a single mouse T beta 4 gene and found that the lymphoid-specific transcript is generated by extending the ubiquitous exon 1 with an alternate downstream splice site. The transcription start site is defined by primer extension analysis, and the 5'-flanking region has many of the characteristics of a promoter. It is pyrimidine-rich and contains typical promoter elements, including a GC box, an initiator site, and consensus transcription factor binding sites. The mouse T beta 4 gene locus (Ptmb4) is located by interspecific backcross mapping to the distal region of the mouse X chromosome, linked to Btk and Gja6.

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in HCC.
METHODS: HCC cell death, and natural killer (NK) cell activation and cytotoxicity were assessed in vitro after treatment with the TLR3 ligand poly(I:C). The effect of TLR3 on the tumor parenchyma and infiltrating immune cells was investigated in a spontaneous liver tumor mouse model and a transplanted tumor mouse model (n = 3-9 mice per group). Immunohistochemistry and quantitative polymerase chain reaction were used to analyze tumor samples from 172 HCC patients. Paired t-tests and analysis of variance tests were used to calculate P-values. The relationship between TLR3 expression and survival was determined by the Kaplan-Meier univariate survival analysis and a log-rank test. All statistical tests were two-sided.
RESULTS: TLR3 activation increased cell death in the TLR3(+) SNU182 HCC cell line (30.5% vs 8.5%, P = .03) and promoted NK-cell activation (32.6% vs 19.4%, P < .001) and cytotoxicity (relative fourfold increase, P = .03) in vitro. In vivo, poly(I:C) treatment increased intratumoral chemokine expression, NK-cell activation and tumor infiltration, and proliferation of tumor-infiltrating T and NK cells. Proliferation of tumor parenchyma cells was decreased. Also, expression of chemokines or treatment with poly(I:C) decreased tumor growth. TLR3 expression in patient samples correlated with NK-cell activation, NK- and T-cell tumor infiltration, and inversely correlated with tumor parenchyma cell viability. TLR3 expression was also associated with longer survival in HCC patients (hazard ratio of survival = 2.1, 95% confidence interval = 1.3 to 3.4, P = .002).
CONCLUSIONS: TLR3 is an important modulator of HCC progression and is a potential target for novel immunotherapy.

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is overexpressed in TNBC and in metastatic TNBC in lymph nodes (LN) compared to normal breast tissue and normal lymphatic tissue, respectively. We studied expression of TEM8 in cases of primary (n=17) and metastatic (n=2) TNBC using immunohistochemical analyses. RESULTS: All cases demonstrated increased expression of TEM8 in tumor tissue compared to non-cancerous breast tissue. TEM8 was expressed at a higher level in the stroma adjacent to the TNBC in all cases, with focal immunoreactive areas within the tumor. TEM8 was not expressed in normal lymphoid tissue, but showed expression at sites of LN metastases. CONCLUSION: TEM8 would appear to represent a new biologic target for designing novel diagnostic or therapeutic approaches for TNBC.

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olymerase gamma (POLG) is commonly involved. A splice-site mutation in POLG, c.3104+3A > T, was previously identified in three families with findings of PEO, and studies demonstrated this variant to result in skipping of exon 19. Here, we report a 57-year-old female who presented with ophthalmoplegia, ptosis, muscle weakness, and exercise intolerance with a subsequent muscle biopsy demonstrating mitochondrial myopathy on histopathologic evaluation and multiple mtDNA deletions by southern blot analysis. Whole-exome sequencing identified the previously characterized c. 3104+3A > T splice-site mutation in compound heterozygosity with a novel frameshift variant, p.Gly23Serfs ∗ 236 (c.67_88del). mtDNA copy number analysis performed on the patient's muscle showed mtDNA depletion, as expected in a patient with biallelic pathogenic mutations in POLG. This is the first reported case with POLG p.Gly23Serfs ∗ 236, discovered in a patient presenting with features of PEO.

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nt cytokine production and target cell death. Nevertheless, Yersinia infection induces inflammatory responses in vivo. Moreover, increasing the extent of YopJ-dependent cytotoxicity induced by Yersinia pestis and Yersinia pseudotuberculosis paradoxically leads to decreased virulence in vivo, suggesting that cell death promotes anti-Yersinia host defense. However, the specific pathways responsible for YopJ-induced cell death and how this cell death mediates immune defense against Yersinia remain poorly defined. YopJ activity induces processing of multiple caspases, including caspase-1, independently of inflammasome components or the adaptor protein ASC. Unexpectedly, caspase-1 activation in response to the activity of YopJ required caspase-8, receptor-interacting serine/threonine kinase 1 (RIPK1), and Fas-associated death domain (FADD), but not RIPK3. Furthermore, whereas RIPK3 deficiency did not affect YopJ-induced cell death or caspase-1 activation, deficiency of both RIPK3 and caspase-8 or FADD completely abrogated Yersinia-induced cell death and caspase-1 activation. Mice lacking RIPK3 and caspase-8 in their hematopoietic compartment showed extreme susceptibility to Yersinia and were deficient in monocyte and neutrophil-derived production of proinflammatory cytokines. Our data demonstrate for the first time to our knowledge that RIPK1, FADD, and caspase-8 are required for YopJ-induced cell death and caspase-1 activation and suggest that caspase-8-mediated cell death overrides blockade of immune signaling by YopJ to promote anti-Yersinia immune defense.

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identified an orphan protein related to a vesicular neurotransmitter transporter as system N. Functional analysis shows that this protein (SN1) involves H+ exchange as well as Na+ cotransport and, under physiological conditions, mediates glutamine efflux as well as uptake. Together with the pattern of SN1 expression, these unusual properties suggest novel physiological roles for system N in nitrogen metabolism and synaptic transmission.

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axonal processes as well as lysosomes. In axons LYAAT1 fails to colocalize with synaptic markers. Rather, axonal LYAAT1 colocalizes with the exocyst, suggesting a role for membranes expressing LYAAT1 in specifying sites for exocytosis. A protease protection assay and measurements of intracellular pH further indicate abundant expression at the plasma membrane, raising the possibility of physiological roles for LYAAT1 on the cell surface as well as in lysosomes.

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ncluding autistic behaviors. Most male patients with MeCP2 mutations exhibit moderate to severe developmental delay/mental retardation. Ninety nine patients from the South Carolina autism project (SCAP) were screened for MeCP2 mutations, including all 41 female patients from whom DNA samples were available plus the 58 male patients with the lowest scores on standard IQ tests and/or the Vineland Adaptive Behavior Scale. No pathogenic mutations were observed in these patients. One patient had the C582T variant, previously reported in the unaffected father of an RS patient. Two other patients had single nucleotide polymorphisms in the 3' UTR of the gene, G1470A and C1516G. These variants were seen in 12/82 and 1/178 phenotypically normal male controls, respectively. The findings from this and other studies suggest that mutations in the coding sequence of the MeCP2 gene are not a significant etiological factor in autism.

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the pleural ADA (P-ADA) level and histopathological findings of CNPB specimens in patients with pleural tuberculosis. METHODS: This prospective study consisted of two groups of examinations with a proven diagnosis of pleural effusion. The P-ADA level was measured in 218 patients with pleural effusion due to a number of causes, and 157 CNPB specimens underwent histopathological analysis. RESULTS: CNPBs were performed in patients with tuberculosis (n=122) and other diseases: adenocarcinoma (n=23), lymphoma (n=5), systemic lupus erythematosus (n=4), squamous cell carcinoma (n=2), and small cell lung cancer (n=1). According to the ROC curve, the optimal cut-off value of the P-ADA level (Giusti and Galanti colorimetric method) was equal to or greater than 40.0 U/L. The diagnostic accuracy of the P-ADA test was 83.0%, and that of histopathological examination of the CNPB tissue, was 78.8% (AUC=0.293, P=0.7695). The association between the P-ADA assay and pleural histopathology was 24.41 (P<0.0001). The tetrachoric correlation coefficient was 0.563 (high correlation). CONCLUSION: In Brazil and other countries with a high incidence of tuberculosis, P-ADA activity is an accurate test for the diagnosis of tuberculous pleural effusions, and its use should be encouraged. The high diagnostic performance of the P-ADA test could to aid the diagnosis of pleural tuberculosis and render CNPB unnecessary.

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style='font-weight:700;'>copeptin, a robust marker of arginine vasopressin secretion. METHODS: This was a prospective emergency-setting cross-sectional study of 161 children between six months and five years of age. Of these, 83 were diagnosed with febrile seizures, 69 had a febrile infection without seizures and nine had epileptic seizures not triggered by infection. Serum copeptin and prolactin levels were measured in addition to standard clinical, neurophysiological, and laboratory assessment. CLINICAL TRIAL REGISTRATION: NCT01884766. RESULTS: Circulating copeptin was significantly higher in children with febrile seizures (median [interquartile range] 18.9 pmol/L [8.5-36.6]) compared to febrile controls (5.6 pmol/L [4.1-9.4]; p < 0.001), with no differences between febrile and epileptic seizures (21.4 pmol/L [16.1-46.6]; p = 0.728). In a multivariable regression model, seizures were the major determinant of serum copeptin (beta 0.509; p < 0.001), independently of clinical and baseline laboratory indices. The area under the receiver operating curve for copeptin was 0.824 (95% CI 0.753-0.881), significantly higher compared to prolactin (0.667 [0.585-0.742]; p < 0.001). The diagnostic accuracy of copeptin increased with decreasing time elapsed since the convulsive event (at 120 min: 0.879 [0.806-0.932] and at <60 min: 0.975 [0.913-0.997]). CONCLUSIONS: Circulating copeptin has high diagnostic accuracy in febrile seizures and may be a useful adjunct for accurately diagnosing postictal states in the emergency setting.

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ntial injection of four hypothalamic releasing hormones before and after a seven-day course of fluoxetine, which inhibits serotonin re-uptake by presynaptic neurons and acts specifically in the brain. Ten obese women (body mass index (BMI) 35.6 +/- 1.0 kg/m2) and nine women of normal weight (BMI 22.9 +/- 0.9 kg/m2) were studied in the early and mid-follicular phases of the menstrual cycle. Basal concentrations of pituitary hormones were measured at 09.00. Subsequently 200 micrograms of TRH and 100 micrograms of CRH, GnRH and GHRH were injected intravenously. The pituitary hormone response was measured at regular intervals until 180 min after the four injections. The experiment was repeated after a seven-day course of 60 mg fluoxetine orally. We found the basal concentrations of prolactin (PRL) and growth hormone to be significantly lower in obese subjects than in the normal controls. Basal concentrations of ACTH, beta-endorphin, TSH, LH and FSH in the two groups were comparable. Releasing hormone-induced responses in the two groups were not significantly different. Administration of fluoxetine "restored" the basal PRL concentrations in obese subjects. It did not affect the other basal hormone concentrations. Furthermore, fluoxetine treatment reduced TRH-induced TSH release in both normal and obese subjects. It did not influence the other releasing hormone-induced responses.(ABSTRACT TRUNCATED AT 250 WORDS)

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ks at the beginning of experiments: the animals had reached the ontogenic stage when the thyroxine-binding globulin had declined, after its high postnatal surge, to undetectable levels. Short-term administration (seven days) of one or the other restricted diet similarly induced resynthesis of the protein. Its serum concentrations reached 26-46% of those measured in eight-day pups (peak of the neonatal surge) and its liver mRNAs showed corresponding enhanced signals. Serum T4 binding activities were increased, although concomitantly transthyretin, second specific T4 carrier of the rat serum, decreased markedly (65-75% of controls) in response to the dietary restrictions. Longer-term diet administration (14 or 28 days) resulted in the further increase of the thyroxine-binding globulin in the protein-restricted rats, in contrast to its decline and eventual disappearance in the energy-restricted animals. Protein restriction was associated with increased total and free T3 serum concentrations, in contrast to energy restriction which little affected these parameters. These studies reveal rat thyroxine-binding globulin as a positive (increasing), highly sensitive reactant of malnutrition, able to discriminate between energy deficiency and composition dysequilibrium of diets. They suggest that up-regulation of its synthesis in the two dietary models involves differential mechanisms.

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increased during positive tilt tests resulting in syncope. Copeptin is a stable cleavage product of vasopressin formation. We conducted a pilot study to assess the utility of endothelin-1 and copeptin as circulating biomarkers of vasovagal syncope. METHODS: Three populations were studied: syncope patients, epilepsy patients and controls. Vasovagal syncope diagnosis was ascertained with the Calgary Syncope Score and epilepsy diagnosis was confirmed with EEG. Plasma levels of endothelin-1 were measured using by ELISA and copeptin levels were determined using an EIA kit. RESULTS: Asymptomatic control subjects had mean age 35 +/- 11 years (7/22 male); epileptic subjects had mean age 32 +/- 7 years (4/15 male); and syncope subjects had mean age 33 +/- 16 years (4 of 21 male). Circulating plasma levels of endothelin-1 and copeptin were no different among the three groups. Mean concentrations of endothelin-1 were as follows: syncope, 23 +/- 32 pg/mL; controls, 21 +/- 17 pg/mL; and epileptics, 18 +/- 12 pg/mL. Mean concentrations of copeptin were as follows: syncope, 1.29 +/- 0.79 ng/mL; controls, 1.25 +/- 0.79 ng/mL; and seizures, 1.23 +/- 0.45 ng/mL. There were no significant correlations between syncope frequency and copeptin or endothelin-1 levels. CONCLUSION: Circulating plasma endothelin-1 and copeptin levels are not significantly different among populations of controls, syncope patients and seizure patients.

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ic, and population genetic studies. Here, after having refined the human mtDNA phylogeny to an unprecedented level by adding information from 8,216 modern mitogenomes, we propose switching the reference to a Reconstructed Sapiens Reference Sequence, which was identified by considering all available mitogenomes from Homo neanderthalensis. This "Copernican" reassessment of the human mtDNA tree from its deepest root should resolve previous problems and will have a substantial practical and educational influence on the scientific and public perception of human evolution by clarifying the core principles of common ancestry for extant descendants.

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the Kryptor Compact Plus platform (BRAHMS, Hennigsdorf, Germany). The study included 107 consecutive patients with diagnosed acute PE (47 males, 60 females), with median age of 65 years (range 20-88). High risk PE was diagnosed in 3 patients (2.8 %), intermediate risk in 69 (64.5 %), and low risk PE in 35 (32.7 %) patients. Control group included 64 subjects (25 males, 39 females; median age 52.5 year, range 17-87). Four patients (3.7 %) died during 30-day observation. Complicated clinical course (CCC) was experienced by 10 (9.3 %) patients. COP level was higher in PE patients than in controls [11.55 pmol/L (5.16-87.97), and 19.00 pmol/L (5.51-351.90), respectively, p < 0.0001], and reflected PE severity. COP plasma concentration in low risk PE was 14.67 nmol/L (5.51-59.61) and in intermediate/high risk PE 19.84 mol/L (5.64-351.90) p < 0.05. Median COP levels in nonsurvivors was higher than in survivors, 84.6 (28.48-351.9) pmol/L and 18.68 (5.512-210.1) pmol/L, respectively, p = 0.009. Subjects with CCC presented higher COP levels than patients with benign clinical course 53.1 (17.95-351.9) pmol/L and 18.16 (5.51-210.1) pmol/L, respectively, p = 0.001. Log-transformed plasma COP was the significant predictor of CCC, OR 16.5 95 % CI 23.2-111.9, p < 0.001. AUC-for prediction of CCC using plasma COP was 0.811 (95 % CI 0.676-0.927). The COP cut off value of 17.95 nmol/l had sensitivity of 100 %, specificity 49.5 %, positive predictive value of 16.9 % and negative predictive value of 100 %. We conclude that plasma COP levels can be regarded for promising marker of severity of acute PE and show potential in risk stratification of these patients.

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ciated with elevated blood pressure. METHODS: We genotyped 9185 individuals from the adult Danish general population. RESULTS: Allele frequencies of 16Arg, 27Glu, and 164Ile were 0.38, 0.44, and 0.01, respectively. Among women never treated with antihypertensive medication those heterozygous for Thr164Ile versus non-carriers had increased diastolic blood pressure (P=0.02). Women heterozygous for Thr164Ile versus non-carriers had an odds ratio for elevated blood pressure of 1.93 (95% CI: 1.30-2.86). Finally, women double heterozygous for Thr164Ile and Gln27Glu or Gly16Arg versus non-carriers at all 3 loci had an odds ratio for elevated blood pressure of 2.49 (1.28-4.85) or 3.19 (1.46-6.97). In men, blood pressure was not influenced by this genetic variation. CONCLUSION: In women Thr164Ile heterozygosity is associated with increased diastolic blood pressure, and represent a risk factor for elevated blood pressure in women in the general population. This was most pronounced in those women also heterozygous for Gln27Glu or Gly16Arg.

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344), August x Copenhagen Irish (ACI), Buffalo/N (Buf/N), Wistar-Furth (WF) strains and F1 (Cop x F344) progeny (F1). Gene expression profiles in mammary tissue within each rat strain were remarkably similar, indicating that gene expression was determined by genetic background. We next identified the subset of genes that were differentially expressed in all susceptible strains relative to the resistant Cop strain. Among these, the messenger RNAs encoding prolactin (Prl) and its cell surface receptor were significantly elevated in all susceptible strains. The expression levels of several Prl-regulated genes were also significantly elevated, indicating the presence of increased Prl signaling in mammary glands of all susceptible strains. Pathway analysis of gene expression profiles further identified the Prl-activated Jak/STAT-signaling pathway among the pathways that most distinguished sensitive rat strains from the resistant Cop rat. To test the hypothesis that reduced levels of the Prl signaling in mammary tissue partially contributed to the genetic resistance to mammary carcinogenesis, we used the neuroleptic drug, perphenazine, to transiently elevate serum Prl levels in the Cop strain. Whereas Cop rats are resistant to N-nitroso-N-methylurea (NMU)-induced mammary carcinogenesis, approximately 5% of pubescent Cop females treated with perphenazine and NMU exposure developed mammary adenocarcinomas with latencies comparable with those of sensitive strains. Together, these finding indicated that in the rat, the molecular mechanisms underlying genetic susceptibility to mammary carcinogenesis include de-regulation of Prl signaling.

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g group, and 30 healthy individuals as the control group. The levels of troponin I (cTnI), IMA, and copeptin were detected. Based on the presence of complications, the patients were assigned to the complication (26 patients) or non-complication (84 patients) group. Levels of cTnI, IMA, and copeptin were compared among the control, complication, and non-complication groups. Compared with the control group, in the 2 h after admission, the IMA levels decreased and copeptin levels increased in the poisoning group; these changes were more significant in patients with severe ACOP than in those with mild ACOP, and the difference was statistically significant (P < 0.05). There were no differences in the IMA and copeptin levels between the groups 7 days after admission; the cTnI levels increased more significantly in patients with severe ACOP than in patients with mild and moderate ACOP, and the differences were statistically significant (P < 0.05). In the complication group, at 7 days after admission, the IMA levels decreased whereas the copeptin and cTnI levels were significantly higher than in the non-complication group, with a statistically significant difference (P < 0.05). IMA was negatively correlated with copeptin. IMA and copeptin detection is clinically useful in the early diagnosis and prognosis of ACOP-related myocardial injury and in guiding early clinical drug application.

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tor in the etiology of estrogen-induced mammary cancer in rats. Therefore, we have examined the ability of administered estrogens to induce development of PRL-producing pituitary tumors and mammary carcinomas in COP rats. Diethylstilbestrol (DES), administered to male COP rats for 12 weeks, beginning when the animals were 9 weeks of age, induced development of PRL-producing pituitary tumors, defined as grossly enlarged pituitary masses displaying lactotroph hyperplasia and associated hyperprolactinemia. When treated with 17beta-estradiol (E2), female COP rats developed pituitary tumors and hyperprolactinemia, but displayed a high degree of resistance to development of mammary carcinomas. These data indicate that E2-induced hyperprolactinemia is insufficient to induce development of mammary carcinomas in the female COP rat.

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gen-induced pituitary tumorigenesis in reciprocal intercrosses between the genetically related ACI and Copenhagen (COP) rat strains. Following 12 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), pituitary mass, an accurate surrogate marker of absolute lactotroph number, was increased 10.6-fold in ACI rats and 4.5-fold in COP rats. Composite interval mapping analyses of the phenotypically defined F2 progeny from the reciprocal crosses identified six quantitative trait loci (QTL) that determine the pituitary growth response to DES. These loci reside on chromosome 6, Ept1 (Estrogen-induced pituitary tumor); chromosome 3, Ept2 and Ept6; chromosome 10, Ept9; and chromosome 1, Ept10 and Ept13. Together, these 6 Ept loci and one additional suggestive locus on chromosome 4 account for an estimated 40% of the phenotypic variance exhibited by the combined F2 population, while 34% of the phenotypic variance was estimated to result from environmental factors. These data indicate that DES-induced pituitary mass behaves as a quantitative trait and provide information that will facilitate identification of genes that determine the tumorigenic response of the pituitary gland to estrogens.

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echol metabolites. Alterations in the expression of antioxidant and xenobiotic metabolizing enzymes upon administration of pharmacological doses of E2 have been previously identified, but the effect of chronic exposure to low concentrations of E2 on activities of those enzymes in liver is unclear. The August-Copenhagen Irish (ACI) rat is more sensitive to estrogen-induced carcinogenesis than the Sprague-Dawley rat. Accordingly, the effect of treatment of female ACI and Sprague-Dawley rats for 6 weeks with E2 on activities of NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione peroxidase, glutathione S-transferase (GST), phenol sulfotransferase (SULT1A1), cytochrome P450 (CYP450) and UDP-glucuronosyltransferase (UGT) was studied. Basal expression of these enzymes was similar in livers from both strains prior to exposure to E2. However, only NQO1 and GST activity was increased (3- and 2.5-fold, respectively) in liver cytosol of ACI rats treated with E2. In contrast, only NQO1 activity was increased modestly in livers of Sprague-Dawley rats. Other enzymes were not significantly affected in the livers of ACI or Sprague-Dawley rats following chronic treatment with E2. The selective induction of NQO1 and GST activity suggests that under physiological conditions, E2 may protect against oxidative stress via elevation of these antioxidant enzymes. The marked induction of NQO1 and GST in the ACI rat indicates a potential for this strain to be used as a model to study the E2-mediated modulation of these enzymes in tissues that are either sensitive to E2 carcinogenesis or to its protective effects.

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nd coenzyme Q10 on copeptin and adrenomedullin as oxidative stress biomarkers. Therefore 437 elderly individuals were included and given intervention for 4 years. Clinical examination and blood samples were undertaken at start and after 18 and 48 months. Evaluations of copeptin and MR-proADM changes were performed using repeated measures of variance. Cardiovascular mortality was evaluated using a 10-year-period of follow-up, and presented in Kaplan-Meier plots. A significant increase in copeptin level could be seen in the placebo group during the intervention period (from 9.4 pmol/L to 15.3 pmol/L), compared to the active treatment group. The difference between the groups was confirmed in the repeated measurement of variance analyses (P = 0.031) with less copeptin increase in the active treatment group. Furthermore, active treatment appeared to protect against cardiovascular death both in those with high and with low copeptin levels at inclusion. Less increase of MR-proADM could also be seen during the intervention in the active treatment group compared to controls (P = 0.026). Both in those having an MR-proADM level above or below median level, significantly less cardiovascular mortality could be seen in the active treatment group (P = 0.0001, and P = 0.04 respectively). In conclusion supplementation with selenium and coenzyme Q10 during four years resulted in less concentration of both copeptin and MR-proADM. A cardioprotective effect of the supplementation was registered, irrespective of the initial levels of these biomarkers, and this protection was recognized also after 10 years of observation.

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with an acute seizure, in four EDs in France and the United Kingdom. Participants were followed up for 28 days. The primary endpoint was a composite of seizure recurrence, all-cause mortality, hospitalization or rehospitalisation, or return visit in the ED within seven days. RESULTS: Among the 389 participants included in the analysis, 156 (40%) experienced the primary endpoint within seven days and 195 (54%) at 28 days. Mean levels of both S100-B (0.11 mug/l [95% CI 0.07-0.20] vs 0.09 mug/l [0.07-0.14]) and copeptin (23 pmol/l [9-104] vs 17 pmol/l [8-43]) were higher in participants meeting the primary endpoint. However, both biomarkers were poorly predictive of the primary outcome with a respective area under the receiving operator characteristic curve of 0.57 [0.51-0.64] and 0.59 [0.54-0.64]. Multivariable logistic regression analysis identified higher age (odds ratio [OR] 1.3 per decade [1.1-1.5]), provoked seizure (OR 4.93 [2.5-9.8]), complex partial seizure (OR 4.09 [1.8-9.1]) and first seizure (OR 1.83 [1.1-3.0]) as independent predictors of the primary outcome. A second regression analysis including the biomarkers showed no additional predictive benefit (S100-B OR 3.89 [0.80-18.9] copeptin OR 1 [1.00-1.00]). CONCLUSION: The plasma biomarkers S100-B and copeptin did not improve prediction of poor outcome following seizure. Higher age, a first seizure, a provoked seizure and a partial complex seizure are independently associated with adverse outcomes.

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pendent and exhibit genomic instability. In contrast, the genetically related Copenhagen (COP) rat strain is relatively resistant to E2-induced mammary cancers. In this study we evaluated susceptibility to E2-induced mammary cancers in first filial (F(1)), second filial (F(2)), and backcross (BC) progeny generated from reciprocal intercrosses between the ACI and COP strains. F(1) progeny resembled the parental ACI strain with respect to incidence of E2-induced mammary cancers. However, latency was significantly prolonged in the F(1) populations. These data indicate that susceptibility behaves as an incompletely dominant phenotype in these crosses. Analysis of phenotypes exhibited by the F(1), F(2), and BC populations suggests that mammary cancer susceptibility is modified by one or two genetic loci in the reciprocal intercrosses between the ACI and COP strains. Susceptibility to E2-induced mammary cancers did not correlate with E2-induced pituitary growth in the genetically diverse F(2) and BC populations, suggesting that the genetic bases for susceptibility to E2-induced mammary cancers differ from those for E2-induced lactotroph hyperplasia.