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While rapid demographic changes in Asia are driving the incidence of chronic aging-related diseases, the limited availability of high-quality in vivo data hampers our ability to understand complex multi-factorial contributions, including gut microbial, to healthy aging. Leveraging a well-phenotyped cohort of community-living octogenarians in Singapore, we used deep shotgun-metagenomic sequencing for high-resolution taxonomic and functional characterization of their gut microbiomes (n = 234). Joint species-level analysis with other Asian cohorts identified distinct age-associated shifts characterized by reduction in microbial richness, and specific Alistipes and Bacteroides species enrichment (e.g., Alistipes shahii and Bacteroides xylanisolvens). Functional analysis confirmed these changes correspond to metabolic potential expansion in aging towards alternate pathways synthesizing and utilizing amino-acid precursors, vis-à-vis dominant microbial guilds producing butyrate in gut from pyruvate (e.g., Faecalibacterium prausnitzii, Roseburia inulinivorans). Extending these observations to key clinical markers helped identify >10 robust microbial associations to inflammation, cardiometabolic and liver health, including potential probiotic species (e.g., Parabacteroides goldsteinii) and pathobionts (e.g., Klebsiella pneumoniae), highlighting the microbiome's role as biomarkers and potential targets for promoting healthy aging., (© 2024. The Author(s).)

Purpose: Transglutaminase (TG)-2 is a ubiquitous multi-functional protein expressed in all living cells. The purpose of the current study was to investigate the role of TG-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors., Methods: Corneal barrier function to ions in TG-2 -/- and TG-2 +/+ mice was assessed by Ussing chamber assay. Hypo-osmolar water or FITC-dextran was applied on top of mouse eyes to evaluate the corneal barrier function to water and macromolecules. Western blots, qPCR and immunofluorescent staining were used to investigate the expression of tight junction proteins in TG-2 -/- and TG-2 +/+ mouse corneas, and also in TG-2 knockdown human corneal epithelial cells., Results: Corneal explants from TG-2 -/- mice had a lower trans-epithelial electrical resistance compared to TG-2 +/+ mice. When challenged by hypo-osmolar water, the central corneal thickness of TG-2 -/- mice increased faster, and these mice had a faster rise of fluorescence in the anterior chamber after ocular exposure to FITC-dextran, compared to TG-2 +/+ . Claudin-1 protein and transcript levels were reduced in the cornea of TG-2 -/- mice and in TG-2 knockdown human corneal epithelial cells. Slug which previously reported suppressing Claudin-1 transcription, was increased at both protein and transcript level in TG-2 knockdown cells. TG-2 and Claudin-1 protein levels were unchanged in shRNA and shTG cells after MG132 treatment, while Slug accumulated in treated cells., Conclusion: TG-2 may positively regulate Claudin-1 through repressing Slug at transcript level, and thus it is critical for normal corneal barrier function., Competing Interests: Declaration of competing interest None., (Copyright © 2023. Published by Elsevier Inc.)

Hepatitis B virus (HBV) infection persists as a major global health problem despite the availability of HBV vaccines for disease prevention. However, vaccination rates remains low in some regions of the world, driving the need for novel strategies to minimise infections and prevent disease progression. Thus, understanding of perturbed molecular signaling events during early phases of HBV infection is required. Phosphosignaling is known to be involved in the HBV infection processes, yet systems-level changes in phosphosignaling pathways in the host during infection remain unclear. To this end, we performed phosphoproteome profiling on HBV-infected HepG2-NTCP cells. Our results showed that HBV infection drastically altered the host phosphoproteome and its associated proteins, including kinases. Computational analysis of this phosphoproteome revealed dysregulation of the pathways involved in immune responses, cell cycle processes, and RNA processing during HBV infection. Kinase Substrate Enrichment Analysis (KSEA) identified the dysregulated activities of important kinases, including those from CMGC (CDK, MAPK, GSK, and CLK), AGC (protein kinase A, G, and C), and TK (Tyrosine Kinase) families. Of note, the inhibition of CLKs significantly reduced HBV infection in HepG2-NTCP cells. In all, our study unravelled the aberrated phosphosignaling pathways and the associated kinases, presenting potential entry points for developing novel therapeutic strategies for HBV treatment.

We present a global atlas of 4,728 metagenomic samples from mass-transit systems in 60 cities over 3 years, representing the first systematic, worldwide catalog of the urban microbial ecosystem. This atlas provides an annotated, geospatial profile of microbial strains, functional characteristics, antimicrobial resistance (AMR) markers, and genetic elements, including 10,928 viruses, 1,302 bacteria, 2 archaea, and 838,532 CRISPR arrays not found in reference databases. We identified 4,246 known species of urban microorganisms and a consistent set of 31 species found in 97% of samples that were distinct from human commensal organisms. Profiles of AMR genes varied widely in type and density across cities. Cities showed distinct microbial taxonomic signatures that were driven by climate and geographic differences. These results constitute a high-resolution global metagenomic atlas that enables discovery of organisms and genes, highlights potential public health and forensic applications, and provides a culture-independent view of AMR burden in cities., Competing Interests: Declaration of interests C.E.M. is co-founder of Biotia and Onegevity Health. D.B. is co-founder and CSO of Poppy Health Inc. The other authors declare they have no competing interests that impacted this study., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mammography is extensively used for breast cancer screening but has high false-positive rates. Here, prospectively collected blood samples were used to identify circulating microRNA (miRNA) biomarkers to discriminate between malignant and benign breast lesions among women with abnormal mammograms. The Discovery cohort comprised 72 patients with breast cancer and 197 patients with benign breast lesions, while the Validation cohort had 73 and 196 cancer and benign cases, respectively. Absolute expression levels of 324 miRNAs were determined using RT-qPCR. miRNA biomarker panels were identified by: (1) determining differential expression between malignant and benign breast lesions, (2) focusing on top differentially expressed miRNAs, and (3) building panels from an unbiased search among all expressed miRNAs. Two-fold cross-validation incorporating a feature selection algorithm and logistic regression was performed. A six-miRNA biomarker panel identified by the third strategy, had an area under the curve (AUC) of 0.785 and 0.774 in the Discovery and Validation cohorts, respectively, and an AUC of 0.881 when differentiating between cases versus those with benign lesions or healthy individuals with normal mammograms. Biomarker panel scores increased with tumor size, stage and number of lymph nodes involved. Our work demonstrates that circulating miRNA signatures can potentially be used with mammography to differentiate between patients with malignant and benign breast lesions.

Background: This study aims to characterize, the gut and oral microbiome in Asian subjects with Crohn's disease (CD) using whole genome shotgun sequencing, thereby allowing for strain-level comparison., Methods: A case-control study with age, sex and ethnicity matched healthy controls was conducted. CD subjects were limited to well-controlled patients without oral manifestations. Fecal and saliva samples were collected for characterization of gut and oral microbiome respectively. Microbial DNA were extracted, libraries prepared and sequenced reads profiled. Taxonomic diversity, taxonomic association, strain typing and microbial gene pathway analyses were conducted., Results: The study recruited 25 subjects with CD and 25 healthy controls. The oral microbe Streptococcus salivarius was found to be enriched and of concordant strains in the gut and oral microbiome of Crohn's disease subjects. This was more likely in CD subjects with higher Crohn's Disease Activity Index (184.3 ± 2.9 vs 67.1 ± 82.5, p = 0.012) and active disease status (Diarrhoea/abdominal pain/blood-in-stool/fever and fatigue) (p = 0.016). Gut species found to be significantly depleted in CD compared to control (Relative abundance: Median[Range]) include: Faecalibacterium prausnitzii (0.03[0.00-4.56] vs 13.69[5.32-18.71], p = 0.010), Roseburia inulinivorans (0.00[0.00-0.03] vs 0.21[0.01-0.53], p = 0.010) and Alistipes senegalensis (0.00[0.00-0.00] vs 0.00[0.00-0.02], p = 0.029). While Clostridium nexile (0.00[0.00-0.12] vs 0.00[0.00-0.00], p = 0.038) and Ruminococcus gnavus (0.43[0.02-0.33] vs 0.00[0.00-0.13], p = 0.043) were found to be enriched. C. nexile enrichment was not found in CD subjects of European descent. Microbial arginine (Linear-discriminant-analysis: 3.162, p = 0.001) and isoprene (Linear-discriminant-analysis: 3.058, p < 0.001) pathways were found at a higher relative abundance level in gut microbiome of Crohn's disease., Conclusions: There was evidence of ectopic gut colonization by oral bacteria, especially during the active phase of CD. Previously studied gut microbial differences were detected, in addition to novel associations which could have resulted from geographical/ethnic differences to subjects of European descent. Differences in microbial pathways provide possible targets for microbiome modification.

Vaccines have revolutionized public health, preventing millions of deaths each year, particularly in childhood. Yet, there is considerable variability in the magnitude and persistence of vaccine-induced immunity. Maintenance of specific antibody is essential for continuity of vaccine-induced serological protection. We conducted a genome-wide association study into the persistence of immunity to three childhood vaccines: capsular group C meningococcal (MenC), Haemophilus influenzae type b, and tetanus toxoid (TT) vaccines. We detail associations between variants in a locus containing a family of signal-regulatory proteins and the persistence MenC immunity. We postulate a regulatory role for the lead SNP, with supporting epigenetic and expression quantitative trait loci data. Furthermore, we define associations between SNPs in the human leukocyte antigen (HLA) locus and the persistence of TT-specific immunity. Moreover, we describe four classical HLA alleles, HLA DRB1 ∗ 0301, HLA DQB1 ∗ 0201, HLA DQB1 ∗ 0602, and HLA DRB1 ∗ 1501, associated with TT-specific immunity, independent of the lead SNP association., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA - a NF-kB subunit, master regulator of the response to infection - under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes.

The human Fc-gamma receptors (FcγRs) link adaptive and innate immunity by binding immunoglobulin G (IgG). All human low-affinity FcγRs are encoded by the FCGR2/3 locus containing functional single nucleotide polymorphisms (SNPs) and gene copy number variants. This locus is notoriously difficult to genotype and high-throughput methods commonly used focus on only a few SNPs. We performed multiplex ligation-dependent probe amplification for all relevant genetic variations at the FCGR2/3 locus in >4,000 individuals to define linkage disequilibrium (LD) and allele frequencies in different populations. Strong LD and extensive ethnic variation in allele frequencies was found across the locus. LD was strongest for the FCGR2C -ORF haplotype (rs759550223+rs76277413), which leads to expression of FcγRIIc. In Europeans, the FCGR2C -ORF haplotype showed strong LD with, among others, rs201218628 ( FCGR2A -Q27W, r 2 = 0.63). LD between these two variants was weaker ( r 2 = 0.17) in Africans, whereas the FCGR2C -ORF haplotype was nearly absent in Asians (minor allele frequency <0.005%). The FCGR2C -ORF haplotype and rs1801274 ( FCGR2A -H131R) were in weak LD ( r 2 = 0.08) in Europeans. We evaluated the importance of ethnic variation and LD in Kawasaki Disease (KD), an acute vasculitis in children with increased incidence in Asians. An association of rs1801274 with KD was previously shown in ethnically diverse genome-wide association studies. Now, we show in 1,028 European KD patients that the FCGR2C -ORF haplotype, although nearly absent in Asians, was more strongly associated with susceptibility to KD than rs1801274 in Europeans. Our data illustrate the importance of interpreting findings of association studies concerning the FCGR2/3 locus with knowledge of LD and ethnic variation.

Breast cancer patients commonly present with comorbidities which are known to influence treatment decisions and survival. We aim to examine agreement between self-reported and register-based medical records (National Patient Register [NPR]). Ascertainment of nine conditions, using individually-linked data from 64,961 women enrolled in the Swedish KARolinska MAmmography Project for Risk Prediction of Breast Cancer (KARMA) study. Agreement was assessed using observed proportion of agreement (overall agreement), expected proportion of agreement, and Cohen's Kappa statistic. Two-stage logistic regression models taking into account chance agreement were used to identify potential predictors of overall agreement. High levels of overall agreement (i.e. ≥86.6%) were observed for all conditions. Substantial agreement (Cohen's Kappa) was observed for myocardial infarction (0.74), diabetes (0.71) and stroke (0.64) between self-reported and NPR data. Moderate agreement was observed for preeclampsia (0.51) and hypertension (0.46). Fair agreement was observed for heart failure (0.40) and polycystic ovaries or ovarian cysts (0.27). For hyperlipidemia (0.14) and angina (0.10), slight agreement was observed. In most subgroups we observed negative specific agreement of >90%. There is no clear reference data source for ascertainment of conditions. Negative specific agreement between NPR and self-reported data is consistently high across all conditions.

Background: We previously described the association of genetic variants in calcium channel genes and susceptibility to Kawasaki disease (KD), an acute, self-limited vasculitis, and the most common cause of acquired cardiac disease in children. Abnormal repolarization of cardiomyocytes and changes in T wave morphology have been reported in KD but have not been studied systematically., Methods: We analyzed acute and convalescent ECG T wave morphology in two independent cohorts of KD subjects and studied the association between bifid T waves and genetic variants in previously reported genes with SNVs associated with cardiac repolarization., Results: Bifid T waves in limb leads were identified in 24% and 27% of two independent cohorts of acute KD subjects. Calcium channel voltage-dependent beta 2 subunit gene (CACNB2) (rs1409207) showed association with bifid T waves in both cohorts (nominal P = .04 and P = .0003, respectively). This CACNB2 polymorphism also showed association with KD susceptibility in a previously published KD genome wide association study data (nominal P = .009)., Conclusion: This genotype/phenotype association study uncovered a variant in CACNB2 that may be associated with both KD susceptibility and bifid T waves, a novel signature of altered myocardial repolarization. The present study combined with published reports suggests that genetic variants in calcium channels and intracellular calcium signaling play a prominent role in shaping susceptibility to KD., (© 2018 Wiley Periodicals, Inc.)

Transglutaminase (TG)-2 interacts with matrix proteins and integrins, forming focal adhesions (FA) to initiate cell migration, thus playing a vital role in wound healing. Previously we showed that TG-2 influenced phosphorylation of paxillin and other FA proteins. Here, we aimed to investigate the molecular mechanism of TG-2 regulation of paxillin. Human corneal epithelial cells expressing shRNA against TG-2 (shTG) and scrambled sequence control (shRNA) were cultured. TG-2 was pulled down by anti-paxillin antibody, but not MAP3K12. Cell-free interaction assay with immobilized paxillin shows that TG-2 bind to paxillin directly. JNK was the strongest kinase for paxillin phosphorylation in the in-vitro kinase screen, but TG-2 could not phosphorylate paxillin directly. Increasing TG-2 concentrations did not increase the amount of JNK in the TG-2/paxillin complex. Immunofluoresent staining shows that TG-2 colocalises with vinculin and paxillin in FA of migrating cells. TG-2 binds to paxillin and JNK-containing FA but does not recruit JNK directly. Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate paxillin, these processes critically regulate corneal epithelial adhesion and migration.

Motivation: We introduce PRINCESS, a privacy-preserving international collaboration framework for analyzing rare disease genetic data that are distributed across different continents. PRINCESS leverages Software Guard Extensions (SGX) and hardware for trustworthy computation. Unlike a traditional international collaboration model, where individual-level patient DNA are physically centralized at a single site, PRINCESS performs a secure and distributed computation over encrypted data, fulfilling institutional policies and regulations for protected health information., Results: To demonstrate PRINCESS' performance and feasibility, we conducted a family-based allelic association study for Kawasaki Disease, with data hosted in three different continents. The experimental results show that PRINCESS provides secure and accurate analyses much faster than alternative solutions, such as homomorphic encryption and garbled circuits (over 40 000× faster)., Availability and Implementation: https://github.com/achenfengb/PRINCESS&#95;opensource., Contact: shw070@ucsd.edu., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

Meningococcal disease (MD) remains an important infectious cause of life threatening infection in both industrialized and resource poor countries. Genetic factors influence both occurrence and severity of presentation, but the genes responsible are largely unknown. We performed a genome-wide association study (GWAS) examining 5,440,063 SNPs in 422 Spanish MD patients and 910 controls. We then performed a meta-analysis of the Spanish GWAS with GWAS data from the United Kingdom (combined cohorts: 897 cases and 5,613 controls; 4,898,259 SNPs). The meta-analysis identified strong evidence of association (P-value ≤ 5 × 10 -8 ) in 20 variants located at the CFH gene. SNP rs193053835 showed the most significant protective effect (Odds Ratio (OR) = 0.62, 95% confidence interval (C.I.) = 0.52-0.73; P-value = 9.62 × 10 -9 ). Five other variants had been previously reported to be associated with susceptibility to MD, including the missense SNP rs1065489 (OR = 0.64, 95% C.I.) = 0.55-0.76, P-value = 3.25 × 10 -8 ). Theoretical predictions point to a functional effect of rs1065489, which may be directly responsible for protection against MD. Our study confirms the association of CFH with susceptibility to MD and strengthens the importance of this link in understanding pathogenesis of the disease.

Primary angle closure glaucoma (PACG) is a major cause of blindness worldwide. We conducted a genome-wide association study (GWAS) followed by replication in a combined total of 10,503 PACG cases and 29,567 controls drawn from 24 countries across Asia, Australia, Europe, North America, and South America. We observed significant evidence of disease association at five new genetic loci upon meta-analysis of all patient collections. These loci are at EPDR1 rs3816415 (odds ratio (OR) = 1.24, P = 5.94 × 10(-15)), CHAT rs1258267 (OR = 1.22, P = 2.85 × 10(-16)), GLIS3 rs736893 (OR = 1.18, P = 1.43 × 10(-14)), FERMT2 rs7494379 (OR = 1.14, P = 3.43 × 10(-11)), and DPM2-FAM102A rs3739821 (OR = 1.15, P = 8.32 × 10(-12)). We also confirmed significant association at three previously described loci (P < 5 × 10(-8) for each sentinel SNP at PLEKHA7, COL11A1, and PCMTD1-ST18), providing new insights into the biology of PACG.

Most disease associations detected by genome-wide association studies (GWAS) lie outside coding genes, but very few have been mapped to causal regulatory variants. Here, we present a method for detecting regulatory quantitative trait loci (QTLs) that does not require genotyping or whole-genome sequencing. The method combines deep, long-read chromatin immunoprecipitation-sequencing (ChIP-seq) with a statistical test that simultaneously scores peak height correlation and allelic imbalance: the genotype-independent signal correlation and imbalance (G-SCI) test. We performed histone acetylation ChIP-seq on 57 human lymphoblastoid cell lines and used the resulting reads to call 500,066 single-nucleotide polymorphisms de novo within regulatory elements. The G-SCI test annotated 8,764 of these as histone acetylation QTLs (haQTLs)—an order of magnitude larger than the set of candidates detected by expression QTL analysis. Lymphoblastoid haQTLs were highly predictive of autoimmune disease mechanisms. Thus, our method facilitates large-scale regulatory variant detection in any moderately sized cohort for which functional profiling data can be generated, thereby simplifying identification of causal variants within GWAS loci.

Cell-matrix adhesion is a fundamental biological process that governs survival, migration, and proliferation of living eukaryotic cells. Paxillin is an important central player in a network of adhesome proteins that form focal adhesion complexes. Phosphorylation of tyrosine and serine residues in paxillin is critical for the coordinated sequential recruitment of other adaptor and kinase proteins to adhesion complexes. Recently, the phosphorylation of serine178 in paxillin has been shown to be vital for epithelial cell adhesion and migration. In vivo and in vitro evidence have shown that transglutaminase (TG)-2 positively regulates this phosphorylation. Here, we propose three possible mechanisms that may explain these observations. First, TG-2 itself may be an adhesome member directly interacting with paxillin in a non-covalent way. Second, TG-2 may cross link a mitogen-activated protein kinase kinase kinase (MAP3K), which eventually activates c-Jun N-terminal kinase (JNK), and the latter phosphorylates paxillin. Lastly, TG-2 may have intrinsic kinase activity that phosphorylates paxillin. Future studies investigating these hypotheses on TG-2-paxillin relationships are necessary in order to address this fundamental process in cell matrix adhesion signaling.

Migration of cells in the ocular surface underpins physiological wound healing as well as many human diseases. Transglutaminase (TG)-2 is a multifunctional cross-linking enzyme involved in the migration of skin fibroblasts and wound healing, however, its functional role in epithelial migration has not been evaluated. This study investigated the importance of TG-2 in a murine corneal wound healing model as well as the mechanistic role of TG-2 in the regulation of related biological processes such as cell adhesion and migration of cultured human corneal epithelial (HCE-T) cells. Corneal wound closure was delayed in homozygous TG-2 deleted mice compared to wild type mice. HCE-T cells that were knocked-down for TG-2 expression through stable expression of a short-hairpin (sh) RNA targeting TG-2, were delayed in closure of scratch wounds (48 compared to 12h in control cells expressing scrambled shRNA). TG-2 knockdown did not influence epithelial cell cycle progression or proliferation, rather, it led to reduced epithelial cell adhesion, spreading and velocity of migration. At the molecular level, TG-2 knockdown reduced phosphorylation of β-3 integrin at Tyr747, paxillin at Ser178, vinculin at Tyr822 and focal adhesion kinase at Tyr925 simultaneous with reduced activation of Rac and CDC42. Phosphorylation of paxillin at Ser178A has been shown to be indispensable for the migration of corneal epithelial cells (Kimura et al., 2008) [18]. TG-2 dependent β-3 integrin activation, serine-phosphorylation of paxillin, and Rac and CDC42 activation may thus play a key functional role in enhancing corneal epithelial cell adhesion and migration during wound healing., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Despite being high transmissible, Mycobacterium tuberculosis (M. tuberculosis) infection causes active disease in only 5-10% of disease-susceptible individuals. This has instigated interest in studying potentially underlying genetic host factors and mechanisms in tuberculosis (TB). The recent identification of the Intracellular pathogen resistance 1 (Ipr1) gene, which plays a major role in controlling M. tuberculosis susceptibility and infection severity in mice (Pan et al., 2005), has prompted studies on its human homolog; SP110 in humans. Association of SP110 SNPs with pulmonary TB were first reported in a study on West African families (Tosh et al., 2006). Subsequent attempts to replicate these findings in other populations, including another West African (Ghanaian) cohort (Thye et al., 2006), however, were unsuccessful. Here we have genotyped 20 SNPs located in the SP110 gene, including the previously TB associated variants; rs2114592 and rs3948464, for the first time in a South East Asian cohort from Indonesia. Our study did not reveal any statistically significant associations between SP110 SNPs and pulmonary TB. In addition, a meta-analysis of the two previously TB associated SNPs revealed that these are not associated with TB, further confirming the lack of convincing evidence for SP110 to be implicated in TB susceptibility, as yet in humans., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: There is reason to expect strong genetic influences on the risk of developing active pulmonary tuberculosis (TB) among latently infected individuals. Many of the genome wide linkage and association studies (GWAS) to date have been conducted on African populations. In order to identify additional targets in genetically dissimilar populations, and to enhance our understanding of this disease, we performed a multi-stage GWAS in a Southeast Asian cohort from Indonesia., Methods: In stage 1, we used the Affymetrix 100 K SNP GeneChip marker set to genotype 259 Indonesian samples. After quality control filtering, 108 cases and 115 controls were analyzed for association of 95,207 SNPs. In stage 2, we attempted validation of 2,453 SNPs with promising associations from the first stage, in 1,189 individuals from the same Indonesian cohort, and finally in stage 3 we selected 251 SNPs from this stage to test TB association in an independent Caucasian cohort (n = 3,760) from Russia., Results: Our study suggests evidence of association (P = 0.0004-0.0067) for 8 independent loci (nominal significance P < 0.05), which are located within or near the following genes involved in immune signaling: JAG1, DYNLRB2, EBF1, TMEFF2, CCL17, HAUS6, PENK and TXNDC4., Conclusions: Mechanisms of immune defense suggested by some of the identified genes exhibit biological plausibility and may suggest novel pathways involved in the host containment of infection with TB.

Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.

Hyperosmolar-induced ocular surface cell death is a key mitochondria-mediated event in inflammatory eye diseases. Transglutaminase (TGM)-2, a cross-linking enzyme, is purported to mediate cell death, but its link to mitochondria is unclear. In the cornea, the integrity of the epithelial cells is important for maintaining transparency of the cornea and therefore functional vision. We evaluated the role of TGM-2 and its involvement in hyperosmolarity-stimulated mitochondrial cell death in human corneal epithelial (HCE-T) cells. HCE-T cell lines stably expressing either shRNA targeting TGM-2 (shTG) or scrambled shRNA (shRNA) were constructed. Hyperosmolar conditions reduced viability and increased mitochondrial depolarization in shRNA cells. However, hyperosmolarity failed to induce mitochondrial depolarization to the same extent in shTG cells. Transient overexpression of TGM-2 resulted in very high levels of TGM-2 expression in shTG and shRNA cells. In the case of shTG cells after overexpression of TGM-2, hyperosmolarity induced the same extent of mitochondrial depolarization as similarly treated shRNA cells. Overexpression of TGM-2 also elevated transamidase activity and reduced viability. It also induced mitochondrial depolarization, increased caspase-3/7 and -9 activity, and these increases were partially suppressed by pan-caspase inhibitor Z-VAD-FMK. Corneal epithelial apoptosis via mitochondrial dysfunction after hyperosmolar stimulation is partially dependent on TGM-2. This TGM-2-dependent mechanism occurs in part via caspase-3/7 and -9. Protection against mitochondrial stress in the ocular surface targeting TGM-2 may have important implications in the survival of cells in hyperosmolar stress., (Copyright © 2010 Wiley-Liss, Inc.)

Asia harbors substantial cultural and linguistic diversity, but the geographic structure of genetic variation across the continent remains enigmatic. Here we report a large-scale survey of autosomal variation from a broad geographic sample of Asian human populations. Our results show that genetic ancestry is strongly correlated with linguistic affiliations as well as geography. Most populations show relatedness within ethnic/linguistic groups, despite prevalent gene flow among populations. More than 90% of East Asian (EA) haplotypes could be found in either Southeast Asian (SEA) or Central-South Asian (CSA) populations and show clinal structure with haplotype diversity decreasing from south to north. Furthermore, 50% of EA haplotypes were found in SEA only and 5% were found in CSA only, indicating that SEA was a major geographic source of EA populations.