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Nonsyndromic orofacial cleft (NSOFC) is a severe birth defect that occurs early in embryonic development and includes the subtypes cleft palate only (CPO), cleft lip only (CLO) and cleft lip with cleft palate (CLP). Given a lack of specific genetic factor analysis for CPO and CLO, the present study aimed to dissect the landscape of genetic factors underlying the pathogenesis of these two subtypes using 6,986 cases and 10,165 controls. By combining a genome-wide association study (GWAS) for specific subtypes of CPO and CLO, as well as functional gene network and ontology pathway analysis, we identified 18 genes/loci that surpassed genome-wide significance (P < 5 × 10−8) responsible for NSOFC, including nine for CPO, seven for CLO, two for both conditions and four that contribute to the CLP subtype. Among these 18 genes/loci, 14 are novel and identified in this study and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. Interestingly, we observed an opposite effect of the genetic variants in the IRF6 gene for CPO and CLO. Moreover, the gene expression dosage effect of IRF6 with two different alleles at the same single-nucleotide polymorphism (SNP) plays important roles in driving CPO or CLO. In addition, PAX9 is a key TF for CPO. Our findings define subtypes of NSOFC using genetic factors and their functional ontologies and provide a clue to improve their diagnosis and treatment in the future. Although GWAS have discovered 43 genes/loci associated with NSOFC, most previous studies used mixed samples of CL/P subtypes rather than CPO or CLO separately. Our findings define the CPO and CLO subtypes using genetic factors and their functional ontologies based on CPO and CLO GWAS data. In this study, we identified 18 genes/loci that contribute to CPO, CLO or CLP. Fourteen of them are novel and identified, and 12 contain developmental transcription factors (TFs), suggesting that TFs are the key factors for the pathogenesis of NSOFC subtypes. We observed an opposite effect in the strongest associated locus IRF6 for CPO and CLO; this information was omitted by previous CL/P GWAS. Furthermore, we reveal that the gene expression dosage of IRF6 plays important roles in driving CPO or CLO. In addition, we found that PAX9 is a strong genetic factor for CPO. These results suggest that transcription factors are the key genetic reason for the pathogenesis of NSOFC subtypes. [ABSTRACT FROM AUTHOR]

Purpose: Human longevity results from a number of factors, including genetic background, favorable environmental, social factors and chance. In this study, we aimed to elucidate the association of human longevity with genetic variations in several major candidate genes in a Han Chinese population. Methods: A case-control association study of 1015 long-lived individuals (aged 90 years or older) and 1725 younger controls (30–70 years old) was undertaken. Rs2075650 in TOMM40 was firstly genotyped using the ABI SNaPshot method in an initial cohort consisted of 597 unrelated long-lived individuals and 1275 younger controls enrolled from Sichuan. Secondly, eighteen tag single-nucleotide polymorphisms (SNPs) in the PVRL2-TOMM40-APOE locus were genotyped for extensive study in the same cohort. Finally, 5 associated SNPs were genotyped in a replication cohort including 418 older individuals and 450 younger controls. The genotype and allele frequencies were evaluated using the χ2 tests. The linkage disequilibrium (LD) block structure was examined using the program Haploview. Results: The case-control study of rs2075650 in TOMM40 showed significant difference in allele frequencies between cases and controls (P = 0.006) in an initial study. Of the 18 SNPs genotyped, rs405509 in APOE and another three SNPs (rs12978931, rs519825 and rs395908) in the PVRL2 gene also showed significant association with human longevity in extensive study in the same cohort. Rs2075650 in TOMM40, rs405509 in APOE and rs519825 in PVRL2 showed a significant association with human longevity in a replication cohort. Conclusion: These results suggested that PVRL2, TOMM40 and APOE might be associated with human longevity. However, further research is needed to identify the causal variants and determine which of these genes are involved in the progress of human longevity. [ABSTRACT FROM AUTHOR]

Stargardt disease is the most common cause of juvenile macular dystrophy. Five subjects from a two-generation Chinese family with Stargardt disease are reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family initiated the disease during childhood, developing progressively impaired central vision and bilateral atrophic macular lesions in the retinal pigmental epithelium (RPE) that resembled a “beaten-bronze” appearance. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. Exome sequencing was used to analyze the exome of two patients II1, II2. A total of 50709 variations shared by the two patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in all family members by PCR and Sanger sequencing. Compound heterozygous variants p.Y808X and p.G607R of the ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) gene, which encodes the ABCA4 protein, a member of the ATP-binding cassette (ABC) transport superfamily, were identified as causative mutations for Stargardt disease of this family. Our findings provide one novel ABCA4 mutation in Chinese patients with Stargardt disease. [ABSTRACT FROM AUTHOR]

Purpose: This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration. Methods: A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons' sequencing was executed to identify mutations. Results: A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide “A” that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain). Conclusion: This report is the first to associate PRPF31 gene's nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel “A” insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD. [ABSTRACT FROM AUTHOR]

Background: Kashin-Beck disease is a kind of degenerative osteoarthropathy. Genetic factors may play an important role in the pathogenesis of KBD. Objective: To investigate the association of the selenoprotein genes GPX1 (rs1050450, rs1800668, and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) with Kashin-Beck disease (KBD) in a Tibetan population and to investigate the association of these SNPs with the serum iodine/selenium concentration in the Tibetan population. Design: Five SNPs including rs1050450, rs1800668, and rs3811699 in the GPX1 gene, rs5748469 in the TrxR2 gene, and rs225014 in the DIO2 gene were analyzed in Tibetan KBD patients and controls using the SNaPshot method. P trend values of the SNPs were calculated using an additive model. Results: None of the five SNPs in the three genes showed a significant association with KBD. Haplotypes TCC, TTC and TTT of rs1050450, rs1800668 and rs3811699 in GPX1 showed a significant association with KBD and controls with P value of 0.0421, 5.0E-4 and 0.0066, respectively. The GPX1 gene (rs1050450) showed a potential significant association with the iodine concentration in the Tibetan study population (P = 0.02726). However, no such association was detected with the selenium concentration (P = 0.2849). Conclusion(s): In this study, we showed that single SNPs in the genes GPX1 (rs1050450, rs1800668 and rs3811699), TrxR2 (rs5748469), and DIO2 (rs225014) may not be significantly associated with KBD in a Tibetan population. However, haplotype analysis of SNPs rs1050450, rs1800668 and rs3811699 in GPX1 gene showed a significant association with KBD. The results suggested that GPX1 gene play a protective role in the susceptivity of KBD in Tibetans. Furthermore, the GPX1 gene (rs1050450) may be significantly associated with the serum iodine concentration in Tibetans. [ABSTRACT FROM AUTHOR]

Objective: To explore the key regulatory genes associated with lung cancer in order to reduce its occurrence and progress through silencing these key genes. Methods: To identify the key regulatory genes involved in lung cancer, we performed a combination of gene array and bioinformatics analyses to compare gene transcription profiles in 3 monoclonal cell strains with high, medium or low metastatic abilities, which were separated from the SPC-A-1sci and SPC-A-1 cell lines by limiting dilution monoclone assay. We then analyzed those genes’ biological activities by knocking down their expression in SPC-A-1sci cells using siRNA and lenti-viral shRNA vectors, followed by determinations of the invasion and migration capabilities of the resulting cell lines in vitro as well as their potential for inducing occurrence and metastasis of lung cancer in vivo. To examine the clinical relevance of these findings, we analyzed the expression levels of the identified genes in human lung cancer tissues (n = 135) and matched adjacent normal tissues by immunohistochemical (IHC) staining. Results: Three monoclonal cell strains characterized with high, medium or low metastatic abilities were successfully selected. Gene array and bioinformatics analyses implied that osteopontin, LAMB3 and ITGB1 were key genes involved in lung cancer. Knockdown of these genes suppressed human lung cancer cell invasion and metastasis in vitro and in vivo. Clinical sample analyses indicated that osteopontin, LAMB3 and ITGB1 protein expression levels were higher in lung cancer patients, compared to non-cancerous adjacent tissues, and correlated with lymphatic metastasis. Conclusions: We confirmed that osteopontin, LAMB3 and ITGB1 played important roles in the occurrence and metastasis of lung cancer, thus provided important clues to understanding the molecular mechanism of metastasis and contributing to the therapeutic treatment of lung cancer. [ABSTRACT FROM AUTHOR]

Although the modulation of Ca2+ channel activity by extremely low-frequency electromagnetic fields (ELF-EMF) has been studied previously, few reports have addressed the effects of such fields on the activity of voltage-activated Na+ channels (Nav). Here, we investigated the effects of ELF-EMF on Nav activity in rat cerebellar granule cells (GCs). Our results reveal that exposing cerebellar GCs to ELF-EMF for 10-60 min significantly increased Nav currents (INa) by 30-125% in a time- and intensity-dependent manner. The Nav channel steady-state activation curve, but not the steady-state inactivation curve, was significantly shifted (by 5.2 mV) towards hyperpolarization by ELF-EMF stimulation. This phenomenon is similar to the effect of intracellular application of arachidonic acid (AA) and prostaglandin E2 (PGE2) on INa in cerebellar GCs. Increases in intracellular AA, PGE2 and phosphorylated PKA levels in cerebellar GCs were observed following ELF-EMF exposure. Western blottings indicated that the NaV 1.2 protein on the cerebellar GCs membrane was increased, the total expression levels of NaV 1.2 protein were not affected after exposure to ELF-EMF. Cyclooxygenase inhibitors and PGE2 receptor (EP) antagonists were able to eliminate this ELF-EMF-induced increase in phosphorylated PKA and INa. In addition, ELF-EMF exposure significantly enhanced the activity of PLA2 in cerebellar GCs but did not affect COX-1 or COX-2 activity. Together, these data demonstrate for the first time that neuronal INa is significantly increased by ELF-EMF exposure via a cPLA2 AA PGE2 EP receptors PKA signaling pathway. [ABSTRACT FROM AUTHOR]

The accessory sex gland (ASG) is an important component of the male reproductive system, which functions to enhance the fertility of spermatozoa during male reproduction. Certain proteins secreted by the ASG are known to bind to the spermatozoa membrane and affect its function. The ASG gene expression profile in Chinese mitten crab (Eriocheir sinensis) has not been extensively studied, and limited genetic research has been conducted on this species. The advent of high-throughput sequencing technologies enables the generation of genomic resources within a short period of time and at minimal cost. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for the ASG of E. sinensis using Illumina sequencing technology. This analysis yielded a total of 33,221,284 sequencing reads, including 2.6 Gb of total nucleotides. Reads were assembled into 85,913 contigs (average 218 bp), or 58,567 scaffold sequences (average 292 bp), that identified 37,955 unigenes (average 385 bp). We assembled all unigenes and compared them with the published testis transcriptome from E. sinensis. In order to identify which genes may be involved in ASG function, as it pertains to modification of spermatozoa, we compared the ASG and testis transcriptome of E. sinensis. Our analysis identified specific genes with both higher and lower tissue expression levels in the two tissues, and the functions of these genes were analyzed to elucidate their potential roles during maturation of spermatozoa. Availability of detailed transcriptome data from ASG and testis in E. sinensis can assist our understanding of the molecular mechanisms involved with spermatozoa conservation, transport, maturation and capacitation and potentially acrosome activation. [ABSTRACT FROM AUTHOR]

The MYO7A encodes a protein classified as an unconventional myosin. Here, we present a family with non-syndromic autosomal dominant hearing impairment that clinically resembles other previously published DFNA11 families. Affected members of the family present with an ascending audiogram affecting low and middle frequencies at young ages and then affecting all frequencies with increasing age. Genome-wide linkage analysis using Illumina Cyto-12 Chip mapped the disease locus to the DFNA11 interval in the family. A c.2003G→A (p.R668H) mutation of the MYO7A, is heterozygous in all affected family members and absent in 100 healthy individuals. Arg668His is located in a region of the myosin VIIA motor domain that is highly conserved among different species. Molecular modeling predicts that the conserved R668 residue plays important structural role in linking different lobes of motor domain together. In the actin-activated ATPase activity assay, the rate of NADH oxidation was higher in the wild-type myosin VIIA, indicating that the ATPase activity in the p.R668H mutant myosin VIIA was significantly destroyed. [ABSTRACT FROM AUTHOR]

Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule- to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB). Far-UV circular dichroism (CD), intrinsic fluorescence, and nuclear magnetic resonance (NMR) spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL) of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins. [ABSTRACT FROM AUTHOR]

Invertebrates rely solely on the innate immune system for defense against pathogens and other stimuli. Fatty acid binding proteins (FABP), members of the lipid binding proteins superfamily, play a crucial role in fatty acid transport and lipid metabolism and are also involved in gene expression induced by fatty acids. In the vertebrate immune system, FABP is involved in inflammation regulated by fatty acids through its interaction with peroxidase proliferator activate receptors (PPARs). However, the immune functions of FABP in invertebrates are not well characterized. For this reason, we investigated the immune functionality of two fatty acid binding proteins, Es-FABP9 and Es-FABP10, following lipopolysaccharide (LPS) challenge in the Chinese mitten crab (Eriocheir sinensis). An obvious variation in the expression of Es-FABP9 and Es-FABP10 mRNA in E. sinensis was observed in hepatopancreas, gills, and hemocytes post-LPS challenge. Recombinant proteins rEs-FABP9 and rEs-FABP10 exhibited distinct bacterial binding activity and bacterial agglutination activity against Escherichia coli and Staphylococcus aureus. Furthermore, bacterial growth inhibition assays demonstrated that rEs-FABP9 responds positively to the growth inhibition of Vibrio parahaemolyticuss and S. aureus, while rEs-FABP10 responds positively to the growth inhibition of Aeromonas hydrophila and Bacillus subtilis. Coating of agarose beads with recombinant rEs-FABP9 and rEs-FABP10 dramatically enhanced encapsulation of the beads by crab hemocytes in vitro. In conclusion, the data presented here demonstrate the participation of these two lipid metabolism-related proteins in the innate immune system of E. sinensis. [ABSTRACT FROM AUTHOR]

Autoimmune polyendocrine syndrome type 1 (APS-1) is a rare autosomal recessive disease defined by the presence of two of the three conditions: mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease. Loss-of-function mutations of the autoimmune regulator (AIRE) gene have been linked to APS-1. Here we report mutational analysis and functional characterization of an AIRE mutation in a consanguineous Chinese family with APS-1. All exons of the AIRE gene and adjacent exon-intron sequences were amplified by PCR and subsequently sequenced. We identified a homozygous missense AIRE mutation c.463G.A (p.Gly155Ser) in two siblings with different clinical features of APS-1. In silico splice-site prediction and minigene analysis were carried out to study the potential pathological consequence. Minigene splicing analysis and subsequent cDNA sequencing revealed that the AIRE mutation potentially compromised the recognition of the splice donor of intron 3, causing alternative pre-mRNA splicing by intron 3 retention. Furthermore, the aberrant AIRE transcript was identified in a heterozygous carrier of the c.463G.A mutation. The aberrant intron 3-retaining transcript generated a truncated protein (p.G155fsX203) containing the first 154 AIRE amino acids and followed by 48 aberrant amino acids. Therefore, our study represents the first functional characterization of the alternatively spliced AIRE mutation that may explain the pathogenetic role in APS-1. [ABSTRACT FROM AUTHOR]

Considering the two-class classification problem in brain imaging data analysis, we propose a sparse representation-based multi-variate pattern analysis (MVPA) algorithm to localize brain activation patterns corresponding to different stimulus classes/brain states respectively. Feature selection can be modeled as a sparse representation (or sparse regression) problem. Such technique has been successfully applied to voxel selection in fMRI data analysis. However, single selection based on sparse representation or other methods is prone to obtain a subset of the most informative features rather than all. Herein, our proposed algorithm recursively eliminates informative features selected by a sparse regression method until the decoding accuracy based on the remaining features drops to a threshold close to chance level. In this way, the resultant feature set including all the identified features is expected to involve all the informative features for discrimination. According to the signs of the sparse regression weights, these selected features are separated into two sets corresponding to two stimulus classes/brain states. Next, in order to remove irrelevant/noisy features in the two selected feature sets, we perform a nonparametric permutation test at the individual subject level or the group level. In data analysis, we verified our algorithm with a toy data set and an intrinsic signal optical imaging data set. The results show that our algorithm has accurately localized two class-related patterns. As an application example, we used our algorithm on a functional magnetic resonance imaging (fMRI) data set. Two sets of informative voxels, corresponding to two semantic categories (i.e., "old people" and "young people"), respectively, are obtained in the human brain. [ABSTRACT FROM AUTHOR]

(+)-SKF 10047 (N-allyl-normetazocine) is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+)-SKF 10047 inhibits K+, Na+and Ca2+channels via sigma-1 receptor activation. We found that (+)-SKF 10047 inhibited Na1.2 and Na1.4 channels independently of sigma-1 receptor activation. (VV+)-SKF 10047 equally inhibited NaV1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+)-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+)-SKF 10047 inhibition of Nav1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM) and1,3-di-o-tolyl-guanidine (DTG) also inhibited Nav1.2 currents through a sigma-1 receptor-independent pathway. The (+)-SKF 10047 inhibition of NaV1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe1764 and Tyr1771 in the IV-segment 6 domain of the Nav1.2 channel and Phe1579 in the Nav1.4 channel for (+)-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit NaV1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies. [ABSTRACT FROM AUTHOR]

Target site insensitivity resulting from point mutations within the voltage-gated sodium channel of the insect nervous system is known to be of primary importance in the development of resistance to pyrethroid insecticides. This study shifts current research paradigms by conducting, for the first time, a global analysis of all the naturally occurring mutations, both nonsynonymous and synonymous mutations, as well as mutation combinations in the entire mosquito sodium channel of Culex quinquefasciatus and analyzing their evolutionary and heritable feature and roles in insecticide resistance. Through a systematic analysis of comparing nucleotide polymorphisms in the entire sodium channel cDNAs of individuals between susceptible and resistant mosquito strains, between field parental mosquitoes and their permethrin selected offspring, and among different mosquito groups categorized by their levels of tolerance to specific permethrin concentrations within and among the mosquito strains of the field parental strains and their permethrin selected offspring, 3 nonsynonymous (A109S, L982F, and W1573R) and 6 synonymous (L852, G891, A1241, D1245, P1249, and G1733) mutations were identified. The co-existence of all 9 mutations, both nonsynonymous and synonymous, and their homozygousity were found to be important factors for high levels of resistance. Our study, for the first time, provide a strong case demonstrating the co-existence of both nonsynonymous and synonymous mutations in the sodium channel of resistant mosquitoes in response to insecticide resistance and the inheritance of these mutations in the offspring of field mosquito strains following insecticide selection. [ABSTRACT FROM AUTHOR]

Wnt proteins are secreted molecules that play multiple roles during hair follicle development and postnatal hair cycling. Wntless (Wls) is a cargo protein required for the secretion of various Wnt ligands. However, its role during hair follicle development and hair cycling remains unclear. Here, we examined the expression of Wls during hair follicle induction and postnatal hair cycling. We also conditionally deleted Wls with K14-cre to investigate its role in hair follicle induction. K14-cre;Wlsc/c mice exhibited abnormal hair follicle development, which is possibly caused by impaired canonical Wnt signaling. Meanwhile, Wnt5a is also expressed in embryonic epidermis, but Wnt5a null mice showed no significant defect in embryonic hair follicle morphogenesis. Therefore, Wls may regulate hair follicle induction by mediating the Wnt/β-catenin pathway. [ABSTRACT FROM AUTHOR]

Background: The DNA demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation. Methods and Principal Findings: Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type-specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5' flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners. Conclusions: The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR. [ABSTRACT FROM AUTHOR]

Cyproheptadine (CPH) is a histamine- and serotonin-receptor antagonist, and its effects are observed recently in the modulation of multiple intracellular signals. In this study, we used cortical neurons and HEK-293 cells transfected with Kv2.1 a-subunit to address whether CPH modify neural voltage-gated K+ channels by a mechanism independent of its serotonergic and histaminergic properties. Our results demonstrate that intracellularly delivered CPH increased the IK by reducing the activity of protein kinas A (PKA). Inhibition of Gi eliminated the CPH-induced effect on both the IK and PKA. Blocking of 5-HT-, M-, D2-, H1- or H2- type GPCR receptors with relevant antagonists did not eliminate the CPH-induced effect on the IK. Antagonists of the sigma-1 receptor, however, blocked the effect of CPH. Moreover, the inhibition of sigma-1 by siRNA knockdown significantly reduced the CPH-induced effect on the IK. On the contrary, sigma-1 receptor agonist mimicked the effects of CPH on the induction of IK. A ligand-receptor binding assay indicated that CPH bound to the sigma- 1 receptor. Similar effect of CPH were obtained from HEK-293 cells transfected with the α-subunit of Kv2.1. In overall, we reveal for the first time that CPH enhances the IK by modulating activity of PKA, and that the associated activation of the sigma-1 receptor/Gi-protein pathway might be involved. Our findings illustrate an uncharacterized effect of CPH on neuron excitability through the IK, which is independent of histamine H1 and serotonin receptors. [ABSTRACT FROM AUTHOR]

CYP2E1 promoter polymorphisms can lead to significant interindividual differences in expression of CYP2E1. Using a database of CYP2E1 gene polymorphisms established in 2010, our study aimed to functionally characterize the single nucleotide polymorphisms (SNPs) of the promoter region and corresponding haplotypes in the Chinese Han population. Six novel SNPs and seven haplotypes with a frequency equal to or greater than 0.01 were constructed on a luciferase reporter system on the basis of site-directed mutagenesis. Dual luciferase reporter systems were used to analyze regulatory activity. The constructs including single novel SNP mutations exhibited insignificant change in luciferase activity, whereas, the activity produced by Haplo1(GTTGCTATAT), Haplo2 (CTTGCTATAT) and Haplo7 (GAGCTCACAT), containing a -333T>A polymorphism was significantly greater than for the wild type in Hep G2 cells (p<0.05), being 1.5-, 2.0- and 1.4- times greater respectively. These findings suggest the possibility of significant clinical prediction of adverse drug reaction and the facilitation of personalized medicine. [ABSTRACT FROM AUTHOR]

Background: CYP2E1 is a member of the cytochrome P450 superfamily, which is involved in the metabolism and activation of both endobiotics and xenobiotics. The genetic polymorphisms of CYP2E1 gene (Chromosome 10q26.3, Accession Number NC_000010.10) are reported to be related to the development of several mental diseases and to be involved in the clinical efficacy of some psychiatric medications. We investigated the possible association of CYP2E1 polymorphisms with susceptibility to schizophrenia in the Chinese Han Population as well as the relationship with response to risperidone in schizophrenia patients. Methods: In a case-control study, we identified 11 polymorphisms in the 5' flanking region of CYP2E1 in 228 schizophrenia patients and 384 healthy controls of Chinese Han origin. From among the cases, we chose 130 patients who had undergone 8 weeks of risperidone monotherapy to examine the relationship between their response to risperidone and CYP2E1 polymorphisms. Clinical efficacy was assessed using the Brief Psychiatric Rating Scale (BPRS). Results: Statistically significant differences in allele or genotype frequencies were found between cases and controls at rs8192766 (genotype p = 0.0048, permutation p = 0.0483) and rs2070673 (allele: p = 0.0018, permutation p = 0.0199, OR = 1.4528 95%CI = 1.1487-1.8374; genotype: p = 0.0020, permutation p = 0.0225). In addition, a GTCAC haplotype containing 5 SNPs (rs3813867, rs2031920, rs2031921, rs3813870 and rs2031922) was observed to be significantly associated with schizophrenia (p = 7.47E-12, permutation p<0.0001). However, no association was found between CYP2E1 polymorphisms/haplotypes and risperidone response. Conclusions: Our results suggest that CYP2E1 may be a potential risk gene for schizophrenia in the Chinese Han population. However, polymorphisms of the CYP2E1 gene may not contribute significantly to individual differences in the therapeutic efficacy of risperidone. Further studies in larger groups are warranted to confirm our results. [ABSTRACT FROM AUTHOR]

Recent research has revealed various molecular markers in lung cancer. However, the organizational principles underlying their genetic regulatory networks still await investigation. Here we performed Network Component Analysis (NCA) and Pathway Crosstalk Analysis (PCA) to construct a regulatory network in human lung cancer (A549) cells which were treated with 50 uM motexafin gadolinium (MGd), a metal cation-containing chemotherapeutic drug for 4, 12, and 24 hours. We identified a set of key TFs, known target genes for these TFs, and signaling pathways involved in regulatory networks. Our work showed that putative interactions between these TFs (such as ESR1/Sp1, E2F1/Sp1, c-MYC-ESR, Smad3/c-Myc, and NFKB1/RELA), between TFs and their target genes (such as BMP41/Est1, TSC2/Myc, APE1/Sp1/p53, RARA/HOXA1, and SP1/ USF2), and between signaling pathways (such as PPAR signaling pathway and Adipocytokines signaling pathway). These results will provide insights into the regulatory mechanism of MGd-treated human lung cancer cells. [ABSTRACT FROM AUTHOR]

The testis is a highly specialized tissue that plays dual roles in ensuring fertility by producing spermatozoa and hormones. Spermatogenesis is a complex process, resulting in the production of mature sperm from primordial germ cells. Significant structural and biochemical changes take place in the seminiferous epithelium of the adult testis during spermatogenesis. The gene expression pattern of testis in Chinese mitten crab (Eriocheir sinensis) has not been extensively studied, and limited genetic research has been performed on this species. The advent of high-throughput sequencing technologies enables the generation of genomic resources within a short period of time and at minimal cost. In the present study, we performed de novo transcriptome sequencing to produce a comprehensive transcript dataset for testis of E. sinensis. In two runs, we produced 25,698,778 sequencing reads corresponding with 2.31 Gb total nucleotides. These reads were assembled into 342,753 contigs or 141,861 scaffold sequences, which identified 96,311 unigenes. Based on similarity searches with known proteins, 39,995 unigenes were annotated based on having a Blast hit in the non-redundant database or ESTscan results with a cut-off E-value above 10-5 . This is the first report of a mitten crab transcriptome using high-throughput sequencing technology, and all these testes transcripts can help us understand the molecular mechanisms involved in spermatogenesis and testis maturation. [ABSTRACT FROM AUTHOR]

Programmed cell death 5 (PDCD5) is a cytosolic protein suppressing growth of multiple types of cancer cells through activating p53. We hypothesized that PDCD5 plays an essential role in cardiac remodeling and function. PDCD5 was significantly up-regulated in the hearts from mice subjected to angiotensin II treatment or transverse aortic constriction. Thus, we generated transgenic mice over-expressing human PDCD5 under the control of alpha myosin heavy chain promoter to examine the role of PDCD5 in cardiac remodeling. Transgenic founder died spontaneously displayed enlarged heart. The high PDCD5 over-expressing line (10-fold) showed reduced survival rate, increase in heart weight normalized to body weight. Real-Time RT-PCR analysis revealed fetal gene program was up-regulated. Echocardiography and histopathological examination showed characteristics of dilated cardiomyopathy and heart failure in transgenic mice. Western blot and immunohistochemistry analysis showed autophagy was dramatically increased in transgenic mice as compared to WT littermates control mice, while apoptosis remained unchanged. The enhanced autophagy in high overexpressing line was associated with significant increase in p53 activity and its downstream target damage-regulated autophagy modulator expression. The low over-expressing line (3.5-fold) appeared normal, but was more susceptible to angiotensin II-induced cardiac hypertrophy. This study is the first providing evidence that PDCD5 plays an important role in cardiac remodeling. [ABSTRACT FROM AUTHOR]

Background: Recent genome-wide association (GWA) studies have identified a number of novel genetic determinants of blood lipid concentrations in Europeans. However, it is still unclear whether these loci identified in the Caucasian GWA studies also exert the same effect on lipid concentrations in the Chinese population. Methods and Results: We conducted a replication study assessing associations between SNPs at 15 loci and blood lipid and lipoprotein concentrations in two Chinese cohorts, comprising 2533 and 2105 individuals respectively. SNPs in APO(A1/C3/ A4/A5), TIMD4-HAVCR1, DOCK7, TRIB1, ABCA1, and TOMM40-APOE showed strong associations with at least one lipids trait, and rs174546 in FADS1/2/3 showed modest association with triglyceride in the Chinese population. Conclusions: We successfully replicated 7 loci associated plasma lipid concentrations in the Chinese population. Our study confirmed the implication of APO(A1/C3/A4/A5), TOMM40-APOE, ABCA1, DOCK7, TIMD4-HAVCR1, TRIB1 and FADS1/2 in plasma lipid and lipoprotein concentrations in Chinese population. [ABSTRACT FROM AUTHOR]

In the era of personalized medical practice, understanding the genetic basis of patient specific adverse drug reaction (ADR) s a major challenge. Clozapine provides effective treatments for schizophrenia but its usage is limited because of life threatening agranulocytosis. A recent high impact study showed the necessity of moving clozapine to a first line drug, thus identifying the biomarkers for drug-induced agranulocytosis has become important. Here we report a methodology termed as antithesis chemical-protein interactome (CPI), which utilizes the docking method to mimic the differences in the drug protein interactions across a panel of human proteins. Using this method, we identified HSPA1A, a known susceptibility gene for CIA, to be the off-target of clozapine. Furthermore, the mRNA expression of HSPA1A-related genes (off-target associated systems) was also found to be differentially expressed in clozapine treated leukemia cell line. Apart from identifying the CIA causal genes we identified several novel candidate genes which could be responsible for agranulocytosis. Proteins related to reactive oxygen clearance system, such as oxidoreductases and glutathione metabolite enzymes, were significantly enriched in the antithesis CPI. This methodology conducted a multi-dimensional analysis of drugs' perturbation to the biological system, investigating both the off-targets and the associated off-systems to explore the molecular basis of an adverse event or the new uses for old drugs. [ABSTRACT FROM AUTHOR]

Background: Myelin transcription factor 1-like (MYT1L) is a member of the myelin transcription factor 1 (MYT1) gene family, and the neural specific, zinc-finger-containing, DNA-binding protein that it encodes plays a role in the development of the nervous system. On the basis of a recent copy number variation (CNV) study showing that this gene is disrupted in mental disorder patients, we investigated whether MYT1L also plays a role in MDD. Methods: In this study, 8 SNPs were analyzed in 1139 MDD patients and 1140 controls of Chinese Han origin. Results: Statistically significant differences were noted between cases and controls for rs3748989 (allele: permutated p = 0.0079, corrected p = 0.0048, genotype: corrected p = 0.0204). A haplotype of rs1617213 and rs6759709 G-C was also significant (permutated p = 0.00007). Conclusion: Our results indicate that MYT1L may be a potential risk gene for MDD in the Chinese Han population. [ABSTRACT FROM AUTHOR]

Background: Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals. Methodology/Principal Findings: In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab (Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5′-untranslated region (5′ UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3′ UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs. Conclusions/Significance: We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs. [ABSTRACT FROM AUTHOR]

Drug medications inevitably affect not only their intended protein targets but also other proteins as well. In this study we examined the hypothesis that drugs that share the same therapeutic effect also share a common therapeutic mechanism by targeting not only known drug targets, but also by interacting unexpectedly on the same cryptic targets. By constructing and mining an Alzheimer's disease (AD) drug-oriented chemical-protein interactome (CPI) using a matrix of 10 drug molecules known to treat AD towards 401 human protein pockets, we found that such cryptic targets exist. We recovered from CPI the only validated therapeutic target of AD, acetylcholinesterase (ACHE), and highlighted several other putative targets. For example, we discovered that estrogen receptor (ER) and histone deacetylase (HDAC), which have recently been identified as two new therapeutic targets of AD, might already have been targeted by the marketed AD drugs. We further established that the CPI profile of a drug can reflect its interacting character towards multi-protein sets, and that drugs with the same therapeutic attribute will share a similar interacting profile. These findings indicate that the CPI could represent the landscape of chemical-protein interactions and uncover ''behind-the-scenes'' aspects of the therapeutic mechanisms of existing drugs, providing testable hypotheses of the key nodes for network pharmacology or brand new drug targets for one-target pharmacology paradigm. [ABSTRACT FROM AUTHOR]

Background: Apoptosis has been speculated to be involved in schizophrenia. In a previously study, we reported the association of the MEGF10 gene with the disease. In this study, we followed the apoptotic engulfment pathway involving the MEGF10, GULP1, ABCA1 and ABCA7 genes and tested their association with the disease. Methodology/Principal Findings: Ten, eleven and five SNPs were genotyped in the GULP1, ABCA1 and ABCA7 genes respectively for the ISHDSF and ICCSS samples. In all 3 genes, we observed nominally significant associations. Rs2004888 at GULP1 was significant in both ISHDSF and ICCSS samples (p = 0.0083 and 0.0437 respectively). We sought replication in independent samples for this marker and found highly significant association (p = 0.0003) in 3 Caucasian replication samples. But it was not significant in the 2 Chinese replication samples. In addition, we found a significant 2-marker (rs2242436 * rs3858075) interaction between the ABCA1 and ABCA7 genes in the ISHDSF sample (p = 0.0022) and a 3-marker interaction (rs246896 * rs4522565 * rs3858075) amongst the MEGF10, GULP1 and ABCA1 genes in the ICCSS sample (p = 0.0120). Rs3858075 in the ABCA1 gene was involved in both 2- and 3-marker interactions in the two samples. Conclusions/Significance: From these data, we concluded that the GULP1 gene and the apoptotic engulfment pathway are involved in schizophrenia in subjects of European ancestry and multiple genes in the pathway may interactively increase the risks to the disease. [ABSTRACT FROM AUTHOR]

Identifying genetic factors responsible for serious adverse drug reaction (SADR) is of critical importance to personalized medicine. However, genome-wide association studies are hampered due to the lack of case-control samples, and the selection of candidate genes is limited by the lack of understanding of the underlying mechanisms of SADRs. We hypothesize that drugs causing the same type of SADR might share a common mechanism by targeting unexpectedly the same SADR-mediating protein. Hence we propose an approach of identifying the common SADR-targets through constructing and mining an in silico chemical-protein interactome (CPI), a matrix of binding strengths among 162 drug molecules known to cause at least one type of SADR and 845 proteins. Drugs sharing the same SADR outcome were also found to possess similarities in their CPI profiles towards this 845 protein set. This methodology identified the candidate gene of sulfonamide-induced toxic epidermal necrolysis (TEN): all nine sulfonamides that cause TEN were found to bind strongly to MHC I (Cw*4), whereas none of the 17 control drugs that do not cause TEN were found to bind to it. Through an insight into the CPI, we found the Y116S substitution of MHC I (B*5703) enhances the unexpected binding of abacavir to its antigen presentation groove, which explains why B*5701, not B*5703, is the risk allele of abacavir-induced hypersensitivity. In conclusion, SADR targets and the patient-specific off-targets could be identified through a systematic investigation of the CPI, generating important hypotheses for prospective experimental validation of the candidate genes. [ABSTRACT FROM AUTHOR]

The Sp4 transcription factor plays a critical role for both development and function of mouse hippocampus. Reduced expression of the mouse Sp4 gene results in a variety of behavioral abnormalities relevant to human psychiatric disorders. The human SP4 gene is therefore examined for its association with both bipolar disorder and schizophrenia in European Caucasian and Chinese populations respectively. Out of ten SNPs selected from human SP4 genomic locus, four displayed significant association with bipolar disorder in European Caucasian families (rs12668354, p = 0.022; rs12673091, p = 0.0005; rs3735440, p = 0.019; rs11974306, p = 0.018). To replicate the genetic association, the same set of SNPs was examined in a Chinese bipolar case control sample. Four SNPs displayed significant association (rs40245, p = 0.009; rs12673091, p = 0.002; rs1018954, p = 0.001; rs3735440, p = 0.029), and two of them (rs12673091, rs3735440) were shared with positive SNPs from European Caucasian families. Considering the genetic overlap between bipolar disorder and schizophrenia, we extended our studies in Chinese trios families for schizophrenia. The SNP7 (rs12673091, p = 0.012) also displayed a significant association. The SNP7 (rs12673091) was therefore significantly associated in all three samples, and shared the same susceptibility allele (A) across all three samples. On the other hand, we found a gene dosage effect for mouse Sp4 gene in the modulation of sensorimotor gating, a putative endophenotype for both schizophrenia and bipolar disorder. The deficient sensorimotor gating in Sp4 hypomorphic mice was partially reversed by the administration of dopamine D2 antagonist or mood stabilizers. Both human genetic and mouse pharmacogenetic studies support Sp4 gene as a susceptibility gene for bipolar disorder or schizophrenia. The studies on the role of Sp4 gene in hippocampal development may provide novel insights for the contribution of hippocampal abnormalities in these psychiatric disorders. [ABSTRACT FROM AUTHOR]

Genetic affinities between aboriginal Taiwanese and populations from Oceania and Southeast Asia have previously been explored through analyses of mitochondrial DNA (mtDNA), Y chromosomal DNA, and human leukocyte antigen loci. Recent genetic studies have supported the ‘slow boat’ and ‘entangled bank’ models according to which the Polynesian migration can be seen as an expansion from Melanesia without any major direct genetic thread leading back to its initiation from Taiwan. We assessed mtDNA variation in 640 individuals from nine tribes of the central mountain ranges and east coast regions of Taiwan. In contrast to the Han populations, the tribes showed a low frequency of haplogroups D4 and G, and an absence of haplogroups A, C, Z, M9, and M10. Also, more than 85% of the maternal lineages were nested within haplogroups B4, B5a, F1a, F3b, E, and M7. Although indicating a common origin of the populations of insular Southeast Asia and Oceania, most mtDNA lineages in Taiwanese aboriginal populations are grouped separately from those found in China and the Taiwan general (Han) population, suggesting a prevalence in the Taiwanese aboriginal gene pool of its initial late Pleistocene settlers. Interestingly, from complete mtDNA sequencing information, most B4a lineages were associated with three coding region substitutions, defining a new subclade, B4a1a, that endorses the origin of Polynesian migration from Taiwan. Coalescence times of B4a1a were 13.2 ± 3.8 thousand years (or 9.3 ± 2.5 thousand years in Papuans and Polynesians). Considering the lack of a common specific Y chromosomal element shared by the Taiwanese aboriginals and Polynesians, the mtDNA evidence provided here is also consistent with the suggestion that the proto-Oceanic societies would have been mainly matrilocal. [ABSTRACT FROM AUTHOR]

Presents a correction to the article "Traces of Archaic Mitochondrial Lineages Persist in Austronesian-Speaking Formosan Populations," published previously in the journal "PLoS Biology."

Purpose: Human longevity results from a number of factors, including genetic background, favorable environmental, social factors and chance. In this study, we aimed to elucidate the association of human longevity with genetic variations in several major candidate genes in a Han Chinese population., Methods: A case-control association study of 1015 long-lived individuals (aged 90 years or older) and 1725 younger controls (30-70 years old) was undertaken. Rs2075650 in TOMM40 was firstly genotyped using the ABI SNaPshot method in an initial cohort consisted of 597 unrelated long-lived individuals and 1275 younger controls enrolled from Sichuan. Secondly, eighteen tag single-nucleotide polymorphisms (SNPs) in the PVRL2-TOMM40-APOE locus were genotyped for extensive study in the same cohort. Finally, 5 associated SNPs were genotyped in a replication cohort including 418 older individuals and 450 younger controls. The genotype and allele frequencies were evaluated using the χ2 tests. The linkage disequilibrium (LD) block structure was examined using the program Haploview., Results: The case-control study of rs2075650 in TOMM40 showed significant difference in allele frequencies between cases and controls (P = 0.006) in an initial study. Of the 18 SNPs genotyped, rs405509 in APOE and another three SNPs (rs12978931, rs519825 and rs395908) in the PVRL2 gene also showed significant association with human longevity in extensive study in the same cohort. Rs2075650 in TOMM40, rs405509 in APOE and rs519825 in PVRL2 showed a significant association with human longevity in a replication cohort., Conclusion: These results suggested that PVRL2, TOMM40 and APOE might be associated with human longevity. However, further research is needed to identify the causal variants and determine which of these genes are involved in the progress of human longevity.

Stargardt disease is the most common cause of juvenile macular dystrophy. Five subjects from a two-generation Chinese family with Stargardt disease are reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family initiated the disease during childhood, developing progressively impaired central vision and bilateral atrophic macular lesions in the retinal pigmental epithelium (RPE) that resembled a "beaten-bronze" appearance. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. Exome sequencing was used to analyze the exome of two patients II1, II2. A total of 50709 variations shared by the two patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in all family members by PCR and Sanger sequencing. Compound heterozygous variants p.Y808X and p.G607R of the ATP-binding cassette, sub-family A (ABC1), member 4 (ABCA4) gene, which encodes the ABCA4 protein, a member of the ATP-binding cassette (ABC) transport superfamily, were identified as causative mutations for Stargardt disease of this family. Our findings provide one novel ABCA4 mutation in Chinese patients with Stargardt disease.

Purpose: This study was intended to identify the disease causing genes in a large Chinese family with autosomal dominant retinitis pigmentosa and macular degeneration., Methods: A genome scan analysis was conducted in this family for disease gene preliminary mapping. Snapshot analysis of selected SNPs for two-point LOD score analysis for candidate gene filter. Candidate gene PRPF31 whole exons' sequencing was executed to identify mutations., Results: A novel nonsense mutation caused by an insertion was found in PRPF31 gene. All the 19 RP patients in 1085 family are carrying this heterozygous nonsense mutation. The nonsense mutation is in PRPF31 gene exon9 at chr19:54629961-54629961, inserting nucleotide "A" that generates the coding protein frame shift from p.307 and early termination at p.322 in the snoRNA binding domain (NOP domain)., Conclusion: This report is the first to associate PRPF31 gene's nonsense mutation and adRP and JMD. Our findings revealed that PRPF31 can lead to different clinical phenotypes in the same family, resulting either in adRP or syndrome of adRP and JMD. We believe our identification of the novel "A" insertion mutation in exon9 at chr19:54629961-54629961 in PRPF31 can provide further genetic evidence for clinical test for adRP and JMD.

Background: The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines., Methodology/principal Findings: In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested., Conclusions/significance: A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.

Background: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored., Methodology/principal Findings: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer., Conclusions/significance: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level., (© 2012 Lin et al.)

Myopia is the most common ocular disorder worldwide, and high myopia in particular is one of the leading causes of blindness. Genetic factors play a critical role in the development of myopia, especially high myopia. Recently, the exome sequencing approach has been successfully used for the disease gene identification of Mendelian disorders. Here we show a successful application of exome sequencing to identify a gene for an autosomal dominant disorder, and we have identified a gene potentially responsible for high myopia in a monogenic form. We captured exomes of two affected individuals from a Han Chinese family with high myopia and performed sequencing analysis by a second-generation sequencer with a mean coverage of 30× and sufficient depth to call variants at ∼97% of each targeted exome. The shared genetic variants of these two affected individuals in the family being studied were filtered against the 1000 Genomes Project and the dbSNP131 database. A mutation A672G in zinc finger protein 644 isoform 1 (ZNF644) was identified as being related to the phenotype of this family. After we performed sequencing analysis of the exons in the ZNF644 gene in 300 sporadic cases of high myopia, we identified an additional five mutations (I587V, R680G, C699Y, 3'UTR+12 C>G, and 3'UTR+592 G>A) in 11 different patients. All these mutations were absent in 600 normal controls. The ZNF644 gene was expressed in human retinal and retinal pigment epithelium (RPE). Given that ZNF644 is predicted to be a transcription factor that may regulate genes involved in eye development, mutation may cause the axial elongation of eyeball found in high myopia patients. Our results suggest that ZNF644 might be a causal gene for high myopia in a monogenic form., Competing Interests: The authors have declared that no competing interests exist.