Genome-Wide Association Study of Hepatocellular Carcinoma in Southern Chinese Patients with Chronic Hepatitis B Virus Infection

One of the most relevant risk factors for hepatocellular carcinoma (HCC) development is chronic hepatitis B virus (HBV) infection, but only a fraction of chronic HBV carriers develop HCC, indicating that complex interactions among viral, environmental and genetic factors lead to HCC in HBV-infected patients. So far, host genetic factors have incompletely been characterized. Therefore, we performed a genome-wide association (GWA) study in a Southern Chinese cohort consisting of 95 HBV-infected HCC patients (cases) and 97 HBV-infected patients without HCC (controls) using the Illumina Human610-Quad BeadChips. The top single nucleotide polymorphisms (SNPs) were then validated in an independent cohort of 500 cases and 728 controls. 4 SNPs (rs12682266, rs7821974, rs2275959, rs1573266) at chromosome 8p12 showed consistent association in both the GWA and replication phases (ORcombined = 1.31–1.39; pcombined = 2.71×10−5–5.19×10−4; PARcombined = 26–31%). We found a 2.3-kb expressed sequence tag (EST) in the region using in-silico data mining and verified the existence of the full-length EST experimentally. The expression level of the EST was significantly reduced in human HCC tumors in comparison to the corresponding non-tumorous liver tissues (P<0.001). Results from sequence analysis and in-vitro protein translation study suggest that the transcript might function as a long non-coding RNA. In summary, our study suggests that variations at chromosome 8p12 may promote HCC in patients with HBV. Further functional studies of this region may help understand HBV-associated hepatocarcinogenesis

Chromosome segregation in Archaea : SegA– and SegB–DNA complex structures provide insights into segrosome assembly

Genome segregation is a vital process in all organisms. Chromosome partitioning remains obscure in Archaea, the third domain of life. Here, we investigated the SegAB system from Sulfolobus solfataricus. SegA is a ParA Walker-type ATPase and SegB is a site-specific DNA-binding protein. We determined the structures of both proteins and those of SegA–DNA and SegB–DNA complexes. The SegA structure revealed an atypical, novel non-sandwich dimer that binds DNA either in the presence or in the absence of ATP. The SegB structure disclosed a ribbon–helix–helix motif through which the protein binds DNA site specifically. The association of multiple interacting SegB dimers with the DNA results in a higher order chromatin-like structure. The unstructured SegB N-terminus plays an essential catalytic role in stimulating SegA ATPase activity and an architectural regulatory role in segrosome (SegA–SegB–DNA) formation. Electron microscopy results also provide a compact ring-like segrosome structure related to chromosome organization. These findings contribute a novel mechanistic perspective on archaeal chromosome segregation

Nuclear-Targeted Deleted in Liver Cancer 1 (DLC1) Is Less Efficient in Exerting Its Tumor Suppressive Activity Both In Vitro and In Vivo

BACKGROUND: Deleted in liver cancer 1 (DLC1) serves as an important RhoGTPase activating protein (RhoGAP) protein that terminates active RhoA signaling in human cancers. Increasing evidence has demonstrated that the tumor suppressive activity of DLC1 depends not only on RhoGAP activity, but also relies on proper focal adhesion localization through its interaction with tensin family proteins. Recently, there are reports showing that DLC1 can also be found in the nucleus; however, the existence and the relative tumor suppressive activity of nuclear DLC1 have never been clearly addressed. METHODOLOGY AND PRINCIPAL FINDINGS: We herein provide new evidence that DLC1 protein, which predominantly associated with focal adhesions and localized in cytosol, dynamically shuttled between cytoplasm and nucleus. Treatment of cells with nuclear export blocker, Leptomycin B (LMB), retained DLC1 in the nucleus. To understand the nuclear entry of DLC1, we identified amino acids 600-700 of DLC1 as a novel region that is important for its nuclear localization. The tumor suppressive activity of nuclear DLC1 was directly assessed by employing a nuclear localization signal (NLS) fusion variant of DLC1 (NLS-DLC1) with preferential nuclear localization. In SMMC-7721 HCC cells, expression of NLS-DLC1 failed to suppress colony formation and actin stress fiber formation in vitro. The abrogated tumor suppressive activity of nuclear DLC1 was demonstrated for the first time in vivo by subcutaneously injecting p53(-/-) RasV12 hepatoblasts with stable NLS-DLC1 expression in nude mice. The injected hepatoblasts with NLS-DLC1 expression effectively formed tumors when compared with the non-nuclear targeted DLC1. CONCLUSIONS/SIGNIFICANCE: Our study identified a novel region responsible for the nuclear entry of DLC1 and demonstrated the functional difference of DLC1 in different cellular compartments both in vitro and in vivo

Deleted in Liver Cancer 1 (DLC1) Utilizes a Novel Binding Site for Tensin2 PTB Domain Interaction and Is Required for Tumor-Suppressive Function

Background: Deleted in liver cancer 1 (DLC1) is a Rho GTPase-activating protein (RhoGAP) frequently deleted and underexpressed in hepatocellular carcinoma (HCC) as well as in other cancers. Recent independent studies have shown interaction of DLC1 with members of the tensin focal adhesion protein family in a Src Homology 2 (SH2) domain-dependent mechanism. DLC1 and tensins interact and co-localize to punctate structures at focal adhesions. However, the mechanisms underlying the interaction between DLC1 and various tensins remain controversial. Methodology/Principal Findings: We used a co-immunoprecipitation assay to identify a previously undocumented binding site at 375-385 of DLC1 that predominantly interacted with the phosphotyrosine binding (PTB) domain of tensin2. DLC1-tensin2 interaction is completely abolished in a DLC1 mutant lacking this novel PTB binding site (DLC1ΔPTB). However, as demonstrated by immunofluorescence and co-immunoprecipitation, neither the focal adhesion localization nor the interaction with tensin1 and C-terminal tensin-like (cten) were affected. Interestingly, the functional significance of this novel site was exhibited by the partial reduction of the RhoGAP activity, which, in turn, attenuated the growth-suppressive activity of DLC1 upon its removal from DLC1. Conclusions/Significance: This study has provided new evidence that DLC1 also interacts with tensin2 in a PTB domain-dependent manner. In addition to properly localizing focal adhesions and preserving RhoGAP activity, DLC1 interaction with tensin2 through this novel focal adhesion binding site contributes to the growth-suppressive activity of DLC1. © 2009 Chan et al.published_or_final_versio

Integrin-Linked Kinase Overexpression and Its Oncogenic Role in Promoting Tumorigenicity of Hepatocellular Carcinoma

Background: Integrin-linked kinase (ILK) was first discovered as an integrin β1-subunit binding protein. It localizes at the focal adhesions and is involved in cytoskeleton remodeling. ILK overexpression and its dysregulated signaling cascades have been reported in many human cancers. Aberrant expression of ILK influenced a wide range of signaling pathways and cellular functions. Although ILK has been well characterized in many malignancies, its role in hepatocellular carcinoma (HCC) is still largely unknown. Methodology/Principal Findings: Quantitative PCR analysis was used to examine ILK mRNA expression in HCC clinical samples. It was shown that ILK was overexpressed in 36.9% (21/57) of HCC tissues when compared to the corresponding non-tumorous livers. The overall ILK expression level was significantly higher in tumorous tissues (P = 0.004), with a significant stepwise increase in expression level along tumor progression from tumor stage I to IV (P = 0.045). ILK knockdown stable clones were established in two HCC cell lines, BEL7402 and HLE, and were subjected to different functional assays. Knockdown of ILK significantly suppressed HCC cell growth, motility and invasion in vitro and inhibited tumorigenicity in vivo. Western blot analysis revealed a reduced phosphorylated-Akt (pAkt) at Serine-473 expression in ILK knockdown stable clones when compared to control clones. Conclusion/Significance: This study provides evidence about the clinical relevance of ILK in hepatocarcinogenesis. ILK was found to be progressively elevated along HCC progression. Here our findings also provide the first validation about the oncogenic capacity of ILK in vivo by suppressing its expression in HCC cells. The oncogenic role of ILK is implicated to be mediated by Akt pathway. © 2011 Chan et al.published_or_final_versio

Deleted in Liver Cancer 1 (DLC1) Negatively Regulates Rho/ROCK/MLC Pathway in Hepatocellular Carcinoma

Aims: Deleted in liver cancer 1 (DLC1), a member of RhoGTPase activating protein (GAP) family, is known to have suppressive activities in tumorigenicity and cancer metastasis. However, the underlying molecular mechanisms of how DLC1 suppresses cell motility have not been fully elucidated. Rho-kinase (ROCK) is an immediate down-stream effector of RhoA in mediating cellular cytoskeletal events and cell motility. In the present study, we aimed to investigate the effects of DLC1 on Rho/ROCK signaling pathway in hepatocellular carcinoma (HCC). Methodology/Principal Findings: We demonstrated that DLC1 negatively regulated ROCK-dependent actomyosin contractility. From immumofluorescence study, we found that ectopic expression of DLC1 abrogated Rho/ROCK-mediated cytoskeletal reorganization including formation of stress fibers and focal adhesions. It also downregulated cortical phosphorylation of myosin light chain 2 (MLC2). These inhibitory events by DLC1 were RhoGAP-dependent, as RhoGAP-deficient mutant of DLC1 (DLC1 K714E) abolished these inhibitory events. In addition, from western study, DLC1 inhibited ROCK-related myosin light chain phosphatase targeting unit 1 (MYPT1) phosphorylation at Threonine 853. By examining cell morphology under microscope, we found that ectopic expression of dominant-active ROCK released cells from DLC1-induced cytoskeletal collapse and cell shrinkage. Conclusion: Our data suggest that DLC1 negatively regulates Rho/ROCK/ MLC2. This implicates a ROCK-mediated pathway of DLC1 in suppressing metastasis of HCC cells and enriches our understanding in the molecular mechanisms involved in the progression of hepatocellular carcinoma. © 2008 Wong et al.published_or_final_versio

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Meta-analysis Followed by Replication Identifies Loci in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as Associated with Systemic Lupus Erythematosus in Asians

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease with a strong genetic involvement and ethnic differences. Susceptibility genes identified so far only explain a small portion of the genetic heritability of SLE, suggesting that many more loci are yet to be uncovered for this disease. In this study, we performed a meta-analysis of genome-wide association studies on SLE in Chinese Han populations and followed up the findings by replication in four additional Asian cohorts with a total of 5,365 cases and 10,054 corresponding controls. We identified genetic variants in or near CDKN1B, TET3, CD80, DRAM1, and ARID5B as associated with the disease. These findings point to potential roles of cell-cycle regulation, autophagy, and DNA demethylation in SLE pathogenesis. For the region involving TET3 and that involving CDKN1B, multiple independent SNPs were identified, highlighting a phenomenon that might partially explain the missing heritability of complex diseases

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data