The genomic basis of circadian and circalunar timing adaptations in a midge.

Organisms use endogenous clocks to anticipate regular environmental cycles, such as days and tides. Natural variants resulting in differently timed behaviour or physiology, known as chronotypes in humans, have not been well characterized at the molecular level. We sequenced the genome of Clunio marinus, a marine midge whose reproduction is timed by circadian and circalunar clocks. Midges from different locations show strain-specific genetic timing adaptations. We examined genetic variation in five C. marinus strains from different locations and mapped quantitative trait loci for circalunar and circadian chronotypes. The region most strongly associated with circadian chronotypes generates strain-specific differences in the abundance of calcium/calmodulin-dependent kinase II.1 (CaMKII.1) splice variants. As equivalent variants were shown to alter CaMKII activity in Drosophila melanogaster, and C. marinus (Cma)-CaMKII.1 increases the transcriptional activity of the dimer of the circadian proteins Cma-CLOCK and Cma-CYCLE, we suggest that modulation of alternative splicing is a mechanism for natural adaptation in circadian timing.

Serum Helicobacter pylori antibody reactivity in seven Asian countries using an automated latex aggregation turbidity assay.

BACKGROUND AND AIM: To determine the application range of diagnostic kits utilizing anti-Helicobacter pylori antibody, we tested a newly developed latex aggregation turbidity assay (latex) and a conventional enzyme-linked immunosorbent assay (E-plate), both containing Japanese H. pylori protein lysates as antigens, using sera from seven Asian countries. METHODS: Serum samples (1797) were obtained, and standard H. pylori infection status and atrophy status were determined by culture and histology (immunohistochemistry) using gastric biopsy samples from the same individuals. The two tests (enzyme-linked immunosorbent assay and latex) were applied, and receiver operating characteristics analysis was performed. RESULTS: Area under the curve (AUC) from the receiver operating characteristic of E-plate and latex curves were almost the same and the highest in Vietnam. The latex AUC was slightly lower than the E-plate AUC in other countries, and the difference became statistically significant in Myanmar and then Bangladesh as the lowest. To consider past infection cases, atrophy was additionally evaluated. Most of the AUCs decreased using this atrophy-evaluated status; however, the difference between the two kits was not significant in each country, but the latex AUC was better using all samples. Practical cut-off values were 3.0 U/mL in the E-test and 3.5 U/mL in the latex test, to avoid missing gastric cancer patients to the greatest extent possible. CONCLUSIONS: The kits were applicable in all countries, but new kits using regional H. pylori strains are recommended for Myanmar and Bangladesh. Use of a cut-off value lower than the best cut-off value is essential for screening gastric cancer patients.

Complex Models of Sequence Evolution Require Accurate Estimators as Exemplified with the Invariable Site Plus Gamma Model.

The invariable site plus $\Gamma$ model (I$+\Gamma)$ is widely used to model rate heterogeneity among alignment sites in maximum likelihood and Bayesian phylogenetic analyses. The proof that the I$+$ continuous $\Gamma$ model is identifiable (model parameters can be inferred correctly given enough data) has increased the creditability of its application to phylogeny reconstruction. However, most phylogenetic software implement the I$+$ discrete $\Gamma$ model, whose identifiability is likely but unproven. How well the parameters of the I$+$ discrete $\Gamma$ model are estimated is still disputed. Especially the correlation between the fraction of invariable sites and the fractions of sites with a slow evolutionary rate is discussed as being problematic. We show that optimization heuristics as implemented in frequently used phylogenetic software (PhyML, RAxML, IQ-TREE, and MrBayes) cannot always reliably estimate the shape parameter, the proportion of invariable sites, and the tree length. Here, we propose an improved optimization heuristic that accurately estimates the three parameters. While research efforts mainly focus on tree search methods, our results signify the equal importance of verifying and developing effective estimation methods for complex models of sequence evolution.

W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis.

This article presents W-IQ-TREE, an intuitive and user-friendly web interface and server for IQ-TREE, an efficient phylogenetic software for maximum likelihood analysis. W-IQ-TREE supports multiple sequence types (DNA, protein, codon, binary and morphology) in common alignment formats and a wide range of evolutionary models including mixture and partition models. W-IQ-TREE performs fast model selection, partition scheme finding, efficient tree reconstruction, ultrafast bootstrapping, branch tests, and tree topology tests. All computations are conducted on a dedicated computer cluster and the users receive the results via URL or email. W-IQ-TREE is available at http://iqtree.cibiv.univie.ac.at It is free and open to all users and there is no login requirement.

IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies.

Large phylogenomics data sets require fast tree inference methods, especially for maximum-likelihood (ML) phylogenies. Fast programs exist, but due to inherent heuristics to find optimal trees, it is not clear whether the best tree is found. Thus, there is need for additional approaches that employ different search strategies to find ML trees and that are at the same time as fast as currently available ML programs. We show that a combination of hill-climbing approaches and a stochastic perturbation method can be time-efficiently implemented. If we allow the same CPU time as RAxML and PhyML, then our software IQ-TREE found higher likelihoods between 62.2% and 87.1% of the studied alignments, thus efficiently exploring the tree-space. If we use the IQ-TREE stopping rule, RAxML and PhyML are faster in 75.7% and 47.1% of the DNA alignments and 42.2% and 100% of the protein alignments, respectively. However, the range of obtaining higher likelihoods with IQ-TREE improves to 73.3-97.1%. IQ-TREE is freely available at http://www.cibiv.at/software/iqtree.

Genomic profiling of renal cell carcinoma in patients with end-stage renal disease.

The purpose of the present study was to determine the genomic profile of renal cell carcinoma (RCC) in end-stage renal disease (ESRD) by analyzing genomic copy number aberrations. Seventy-nine tumor samples from 63 patients with RCC-ESRD were analyzed by array comparative genomic hybridization using the Agilent Whole Human Genome 4 × 44K Oligo Micro Array (Agilent Technologies Inc., Palo Alto, CA, USA). Unsupervised hierarchical clustering analysis revealed that the 63 cases could be divided into two groups, Clusters A and B. Cluster A was comprised mainly of clear cell RCC (CCRCC), whereas Cluster B was comprised mainly of papillary RCC (PRCC), acquired cystic disease (ACD)-associated RCC, and clear cell papillary RCC. Analysis of the averaged frequencies revealed that the genomic profiles of Clusters A and B resembled those of sporadic CCRCC and sporadic PRCC, respectively. Although it has been proposed on the basis of histopathology that ACD-associated RCC, clear cell papillary RCC and PRCC-ESRD are distinct subtypes, the present data reveal that the genomic profiles of these types, categorized as Cluster B, resemble one another. Furthermore, the genomic profiles of PRCC, ACD-associated RCC and clear cell papillary RCC admixed in one tissue tended to resemble one another. On the basis of genomic profiling of RCC-ESRD, we conclude that the molecular pathogenesis of CCRCC-ESRD resembles that of sporadic CCRCC. Although various histologic subtypes of non-clear cell RCC-ESRD have been proposed, their genomic profiles resemble those of sporadic PRCC, suggesting that the molecular pathogenesis of non-CCRCC-ESRD may be related to that of sporadic PRCC.

Association of helicobacter pylori dupA with the failure of primary eradication.

GOALS: To determine whether the presence of dupA Helicobacter pylori (H. pylori) influences the cure rate of primary eradication therapy. BACKGROUND: Several virulence factors of H. pylori have been reported to affect the efficacy of the eradication rate. However, no study has investigated whether the presence of dupA affects eradication failure. STUDY: The presence of dupA was evaluated in 142 H. pylori strains isolated from 142 patients with gastrointestinal diseases. Of these patients, 104 received primary eradication therapy for 1 week. The risk factors for eradication failure were determined using univariate and multivariate analyses. RESULTS: Among 142 strains, 44 (31.0%) were dupA positive. There was no association between dupA status and gastroduodenal diseases (P>0.05). The clarithromycin (CLR) resistance rate was generally lower in the dupA-positive than in the dupA-negative group (20.4% vs. 35.7%, P=0.06). However, dupA prevalence was higher in the eradication failure group than in the success group (36.3% vs. 21.9%). Among the CLR-resistant H. pylori infected group, the successful eradication rate was significantly lower in patients infected with dupA-positive H. pylori than dupA-negative H. pylori (P=0.04). In multivariate analysis adjusted for age, sex, and type of disease, not only CLR resistance but also dupA presence was independent risk factors for eradication failure (adjusted odds ratio=3.71; 95% confidence interval,1.07-12.83). CONCLUSIONS: Although CLR resistant was more reliable predictor, the presence of dupA may also be an independent risk factor for eradication failure.

Genomic profiling of gastric carcinoma in situ and adenomas by array-based comparative genomic hybridization.

Although genomic copy number aberrations (CNAs) of gastric carcinoma at the advanced stage have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis, those of gastric carcinoma in situ (CIS) are still poorly understood. Furthermore, no reports have demonstrated correlations between CNAs and histopathological features of gastric adenoma. In this study, we investigated CNAs of 20 gastric CISs (Vienna category 4.2) and 20 adenomas including seven low-grade adenomas (LGA; Vienna category 3) and 13 high-grade adenomas (HGA; Vienna category 4.1), using oligonucleotide-based array CGH. The most frequent aberrations in CIS were gains at 8q (85%) and 20q (50%), and losses at 5q (50%) and 17p (50%), suggesting that these CNAs are involved in the development of CIS. We found that the pattern of CNAs in HGA was quite different from that in LGA. The most frequent CNAs in HGA were gains at 8q (62%) and 7pq (54%), whereas those in LGA were gain at 7q21.3-q22.1 (57%) and loss at 5q (43%). Interestingly, gains at 8q and 7pq, both of which occurred most frequently in HGA, were not detected in any cases of LGA. Of note, 8q gain was detected most frequently in both HGA and CIS but was undetected in LGA. Since HGA is believed to have a higher risk of progression to invasive carcinoma than LGA, these data suggest that 8q gain is important for the malignant transformation of gastric adenoma.

A genomic perspective on the potential of termite-associated Cellulosimicrobium cellulans MP1 as producer of plant biomass-acting enzymes and exopolysaccharides.

BACKGROUND: Lignocellulose is a renewable and enormous biomass resource, which can be degraded efficiently by a range of cocktails of carbohydrate-active enzymes secreted by termite gut symbiotic bacteria. There is an urgent need to find enzymes with novel characteristics for improving the conversion processes in the production of lignocellulosic-based products. Although various studies dedicated to the genus Cellulosimicrobium as gut symbiont, genetic potential related to plant biomass-acting enzymes and exopolysaccharides production has been fully untapped to date. METHODS: The cellulolytic bacterial strain MP1 was isolated from termite guts and identified to the species level by phenotypic, phylogenetic, and genomic analysis. To further explore genes related to cellulose and hemicellulose degradation, the draft genome of strain MP1 was obtained by using whole-genome sequencing, assembly, and annotation through the Illumina platform. Lignocellulose degrading enzymes and levan production in the liquid medium were also examined to shed light on bacterial activities. RESULTS: Among 65 isolates obtained, the strain MP1 was the most efficient cellulase producer with cellulase activity of 0.65 ± 0.02 IU/ml. The whole genome analysis depicted that strain MP1 consists of a circular chromosome that contained 4,580,223 bp with an average GC content of 73.9%. The genome comprises 23 contigs including 67 rRNA genes, three tRNA genes, a single tmRNA gene, and 4,046 protein-coding sequences. In support of the phenotypic identification, the 16S rRNA gene sequence, average nucleotide identity, and whole-genome-based taxonomic analysis demonstrated that the strain MP1 belongs to the species Cellulosimicrobium cellulans. A total of 30 genes related to the degradation of cellulases and hemicellulases were identified in the C. cellulans MP1 genome. Of note, the presence of sacC1-levB-sacC2-ls operon responsible for levan and levan-type fructooligosaccharides biosynthesis was detected in strain MP1 genome, but not with closely related C. cellulans strains, proving this strain to be a potential candidate for further studies. Endoglucanases, exoglucanases, and xylanase were achieved by using cheaply available agro-residues such as rice bran and sugar cane bagasse. The maximum levan production by C. cellulans MP1 was 14.8 ± 1.2 g/l after 20 h of cultivation in media containing 200 g/l sucrose. To the best of our knowledge, the present study is the first genome-based analysis of a Cellulosimicrobium species which focuses on lignocellulosic enzymes and levan biosynthesis, illustrating that the C. cellulans MP1 has a great potential to be an efficient platform for basic research and industrial exploitation.

A bacterial small RNA regulates the adaptation of Helicobacter pylori to the host environment.

Long-term infection of the stomach with Helicobacter pylori can cause gastric cancer. However, the mechanisms by which the bacteria adapt to the stomach environment are poorly understood. Here, we show that a small non-coding RNA of H. pylori (HPnc4160, also known as IsoB or NikS) regulates the pathogen's adaptation to the host environment as well as bacterial oncoprotein production. In a rodent model of H. pylori infection, the genomes of bacteria isolated from the stomach possess an increased number of T-repeats upstream of the HPnc4160-coding region, and this leads to reduced HPnc4160 expression. We use RNA-seq and iTRAQ analyses to identify eight targets of HPnc4160, including genes encoding outer membrane proteins and oncoprotein CagA. Mutant strains with HPnc4160 deficiency display increased colonization ability of the mouse stomach, in comparison with the wild-type strain. Furthermore, HPnc4160 expression is lower in clinical isolates from gastric cancer patients than in isolates derived from non-cancer patients, while the expression of HPnc4160's targets is higher in the isolates from gastric cancer patients. Therefore, the small RNA HPnc4160 regulates H. pylori adaptation to the host environment and, potentially, gastric carcinogenesis.

Evaluation of rapid urine test for the detection of Helicobacter pylori infection in the Vietnamese population.

PURPOSE: This study attempted to assess the value of the RAPIRUN test for the diagnosis of Helicobacter pylori infection in the Vietnamese population. METHODS: We recruited 148 Vietnamese patients undergoing upper gastrointestinal endoscopy during which five gastric biopsies were taken; blood and urine samples were collected from each patient. Helicobacter pylori infection status was determined by a combination of three different methods, including culture, immunohistochemistry, and serum ELISA. RAPIRUN tests were performed using urine samples. RESULTS: The sensitivity, specificity, and accuracy of the RAPIRUN test in these Vietnamese patients were 79.5, 90.7, and 84.5%, respectively. CONCLUSIONS: The RAPIRUN test is useful for the diagnosis of H. pylori infection in the Vietnamese population, showing high specificity, acceptable sensitivity, non-invasiveness, convenience, and rapidity.

Analysis of virulence factors of Helicobacter pylori isolated from a Vietnamese population.

BACKGROUND: The incidence of gastric cancer differs among countries in Asia, and it has been suggested that virulence factors associated with Helicobacter pylori are partly responsible. The aim of this study was to investigate several genetic factors regarded as virulence or molecular epidemiologic markers in H. pylori isolates from Vietnamese subjects. RESULTS: The cagA, vacA and cag right-end junction genotypes of 103 H. pylori strains from Vietnam (54 from Hanoi and 49 from Ho Chi Minh) were determined by PCR and sequencing. Three types of deletion in the region located upstream of the cagA Glu-Pro-Ile-Tyr-Ala (EPIYA) repeat region were identified: the 39-bp deletion type, the 18-bp deletion type, and the no-deletion type. The majority of strains studied (77%; 80/103) had the 18-bp deletion irrespective of geographical location in the country or clinical outcome. All of the 39-bp and 18-bp deletion-type strains possessed the East Asian type cagA repeat region. The type II cag right-end junction genotype was predominant (84%). The vacA m1 genotype was significantly more common in strains isolated in Hanoi, where the incidence of gastric cancer is higher, than in strains from Ho Chi Minh. CONCLUSION: Pre-EPIYA-region typing of the cagA gene could provide a new genetic marker of H. pylori genomic diversity. Our data support the hypothesis that vacA m1 is closely associated with gastric carcinogenesis.

Helicobacter pylori virulence and the diversity of gastric cancer in Asia.

Infection with cagPAI positive strains of Helicobacter pylori is recognized as being associated with an increased risk of gastric cancer. This article reviews the current knowledge on the structures and pathological functions of cagPAI and the CagA protein, focusing especially on the molecular mechanism through which CagA may be involved in gastric carcinogenesis. The possible link between the geographical distribution of cagPAI and cagA variations and gastric cancer diversity in Asia is also discussed.

Genotyping of the cagA gene of Helicobacter pylori on immunohistochemistry with East Asian CagA-specific antibody.

The cytotoxin-associated antigen A (CagA) of Helicobacter pylori prevalent in East Asian countries, where the mortality rate due to gastric cancer is high, has been reported to be structurally different from that in Western countries, where the gastric cancer mortality rate is relatively low. Based on the structural features of the EPIYA motifs located at the carboxyl terminal of the protein, CagA was subdivided into two types: East Asian CagA and Western CagA. A recent study suggested that immunohistochemistry with anti-East Asian-specific antibody (alpha-EAS Ab), which was specifically immunoreactive with East Asian CagA but not with Western CagA, may be useful for diagnosis of the cagA genotype. To further evaluate the value of this diagnostic method in terms of sensitivity, specificity, and accuracy, 143 gastric biopsy specimens with alpha-EAS Ab were analyzed on immunohistochemistry and compared with the sequencing of the cagA gene. It was found that diagnosis of the cagA genotype of H. pylori on immunohistochemistry using the alpha-EAS Ab was highly sensitive (sensitivity 93.2%) and specific (specificity 72.7%), suggesting that immunohistochemical diagnosis of the cagA genotype is useful for diagnosis of the cagA genotype.

Genomic profiling of submucosal-invasive gastric cancer by array-based comparative genomic hybridization.

Genomic copy number aberrations (CNAs) in gastric cancer have already been extensively characterized by array comparative genomic hybridization (array CGH) analysis. However, involvement of genomic CNAs in the process of submucosal invasion and lymph node metastasis in early gastric cancer is still poorly understood. In this study, to address this issue, we collected a total of 59 tumor samples from 27 patients with submucosal-invasive gastric cancers (SMGC), analyzed their genomic profiles by array CGH, and compared them between paired samples of mucosal (MU) and submucosal (SM) invasion (23 pairs), and SM invasion and lymph node (LN) metastasis (9 pairs). Initially, we hypothesized that acquisition of specific CNA(s) is important for these processes. However, we observed no significant difference in the number of genomic CNAs between paired MU and SM, and between paired SM and LN. Furthermore, we were unable to find any CNAs specifically associated with SM invasion or LN metastasis. Among the 23 cases analyzed, 15 had some similar pattern of genomic profiling between SM and MU. Interestingly, 13 of the 15 cases also showed some differences in genomic profiles. These results suggest that the majority of SMGCs are composed of heterogeneous subpopulations derived from the same clonal origin. Comparison of genomic CNAs between SMGCs with and without LN metastasis revealed that gain of 11q13, 11q14, 11q22, 14q32 and amplification of 17q21 were more frequent in metastatic SMGCs, suggesting that these CNAs are related to LN metastasis of early gastric cancer. In conclusion, our data suggest that generation of genetically distinct subclones, rather than acquisition of specific CNA at MU, is integral to the process of submucosal invasion, and that subclones that acquire gain of 11q13, 11q14, 11q22, 14q32 or amplification of 17q21 are likely to become metastatic.

MicroRNA-375 is downregulated in gastric carcinomas and regulates cell survival by targeting PDK1 and 14-3-3zeta.

We investigated expression profiles of microRNA (miRNA) in gastric carcinomas by use of a miRNA microarray platform covering a total of 470 human miRNAs. We identified 39 differentially expressed miRNAs in gastric carcinoma, of which six were significantly downregulated and the other 33 were upregulated. We found that miRNA-375 (miR-375) was the most downregulated and that its ectopic expression in gastric carcinoma cells markedly reduced cell viability via the caspase-mediated apoptosis pathway. Interestingly, we found that expression of miR-375 inhibited expression of PDK1, which is a direct target of miR-375, followed by suppression of Akt phosphorylation. Further analysis by gene expression microarray revealed that 14-3-3zeta, a potent antiapoptotic gene, was significantly downregulated at both the mRNA and protein levels in cells transfected with miR-375. The activity of a luciferase reporter containing the miR-375 binding sequence at the 3' untranslated region (UTR) of 14-3-3zeta mRNA was repressed by the ectopic expression of miR-375, suggesting that miR-375 targets the 3' UTR of 14-3-3zeta. In addition, knockdown of either PDK1 or 14-3-3zeta in gastric carcinoma cells induced caspase activation, which was also observed in miR-375-transfected cells, suggesting that miR-375 may exert its proapoptotic function, at least in part, through the downregulation of PDK1 and 14-3-3zeta. Taken together, we propose that miR-375 is a candidate tumor suppressor miRNA in gastric carcinoma.

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type.

Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA-positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori-infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up-regulated by adhesion to epithelial cells.

Evaluation of the anti-East Asian CagA-specific antibody for CagA phenotyping.

The determination of the cagA genotype is generally based on sequencing the variable 3' region of the cagA gene. In a previous study, we successfully generated an anti-East Asian CagA-specific antibody (anti-EAS Ab) immunoreactive only with the East Asian CagA and not with the Western CagA. In a small number of Japanese patients, anti-EAS Ab appeared to be a useful tool for phenotyping CagA immunohistochemically. The present study was conducted to validate the anti-EAS Ab immunohistochemistry method in a larger number of patients from Vietnam and Thailand. A total of 385 Vietnamese and Thais were recruited. Helicobacter pylori status was determined by a combination of three methods, including culture, histology, and immunohistochemistry with anti-H. pylori antibody. The sensitivity, specificity, and accuracy of the anti-EAS Ab immunohistochemistry method for the diagnosis of CagA phenotype were calculated based on the results of the cagA sequencing as the gold standard. The sensitivity, specificity, and accuracy of our immunohistochemistry method were 96.7%, 97.9%, and 97.1%, respectively. Moreover, anti-EAS Ab was not cross-reactive with noninfected gastric mucosa. In conclusion, immunohistochemistry with anti-EAS Ab appears to be a good method for determination of CagA phenotype.