Genetic exchange with an outcrossing sister species causes severe genome-wide dysregulation in a selfing Caenorhabditis nematode.

Different modes of reproduction evolve rapidly, with important consequences for genome composition. Selfing species often occupy a similar niche as their outcrossing sister species with which they are able to mate and produce viable hybrid progeny, raising the question of how they maintain genomic identity. Here, we investigate this issue by using the nematode Caenorhabditis briggsae, which reproduces as a hermaphrodite, and its outcrossing sister species Caenorhabditis nigoni We hypothesize that selfing species might develop some barriers to prevent gene intrusions through gene regulation. We therefore examined gene regulation in the hybrid F2 embryos resulting from reciprocal backcrosses between F1 hybrid progeny and C. nigoni or C. briggsae F2 hybrid embryos with ∼75% of their genome derived from C. briggsae (termed as bB2) were inviable, whereas those with ∼75% of their genome derived from C. nigoni (termed as nB2) were viable. Misregulation of transposable elements, coding genes, and small regulatory RNAs was more widespread in the bB2 compared with the nB2 hybrids, which is a plausible explanation for the differential phenotypes between the two hybrids. Our results show that regulation of the C. briggsae genome is strongly affected by genetic exchanges with its outcrossing sister species, C. nigoni, whereas regulation of the C. nigoni genome is more robust on genetic exchange with C. briggsae The results provide new insights into how selfing species might maintain their identity despite genetic exchanges with closely related outcrossing species.

Technical challenges in defining RNA modifications.

It is well established that DNA base modifications play a key role in gene regulation during development and in response to environmental stress. This type of epigenetic control of development and environmental responses has been intensively studied over the past few decades. Similar to DNA, various RNA species also undergo modifications that play important roles in, for example, RNA splicing, protein translation, and the avoidance of immune surveillance by host. More than 160 different types of RNA modifications have been identified. In addition to base modifications, RNA modification also involves splicing of pre-mRNAs, leading to as many as tens of transcript isoforms from a single pre-RNA, especially in higher organisms. However, the function, prevalence and distribution of RNA modifications are poorly understood. The lack of a suitable method for the reliable identification of RNA modifications constitutes a significant challenge to studying their functions. This review focuses on the technologies that enable de novo identification of RNA base modifications and the alternatively spliced mRNA transcripts.

Interferon-γ+ Th1 activates intrahepatic resident memory T cells to promote HBsAg loss by inducing M1 macrophage polarization.

The immune mechanism underlying hepatitis B surface antigen (HBsAg) loss, particularly type I inflammatory response, during pegylated interferon-α (PEG-IFN) therapy remains unclear. In this study, we aimed to elucidate such immune mechanisms. Overall, 82 patients with chronic hepatitis B (CHB), including 41 with HBsAg loss (cured group) and 41 uncured patients, received nucleos(t)ide analogue and PEG-IFN treatments. Blood samples from all patients, liver tissues from 14 patients with CHB, and hepatic perfusate from 8 liver donors were collected for immune analysis. Jurkat, THP-1 and HepG2.2.15 cell lines were used in cell experiments. The proportion of IFN-γ+ Th1 cells was higher in the cured group than in the uncured group, which was linearly correlated with HBsAg decline and alanine aminotransferase (ALT) levels during treatment. However, CD8+ T cells were weakly associated with HBsAg loss. Serum and intrahepatic levels of Th1 cell-associated chemokines (C-X-C motif chemokine ligand [CXCL] 9, CXCL10, CXCL11, IFN-γ) were significantly lower in the cured patients than in patients with a higher HBsAg quantification during therapy. Serum from cured patients induced more M1 (CD68+CD86+ macrophage) cells than that from uncured patients. Patients with chronic HBV infection had significantly lower proportions of CD86+ M1 and CD206+ M2 macrophages in their livers than healthy controls. M1 polarization of intrahepatic Kupffer cells promoted HBsAg loss by upregulating the effector function of tissue-resident memory T cells with increased ALT levels. IFN-γ+ Th1 activates intrahepatic resident memory T cells to promote HBsAg loss by inducing M1 macrophage polarization.

Efficient targeted recombination with CRISPR/Cas9 in hybrids of Caenorhabditis nematodes with suppressed recombination.

BACKGROUND: Homology-based recombination (HR) is the cornerstone of genetic mapping. However, a lack of sufficient sequence homology or the presence of a genomic rearrangement prevents HR through crossing, which inhibits genetic mapping in relevant genomic regions. This is particularly true in species hybrids whose genomic sequences are highly divergent along with various genome arrangements, making the mapping of genetic loci, such as hybrid incompatibility (HI) loci, through crossing impractical. We previously mapped tens of HI loci between two nematodes, Caenorhabditis briggsae and C. nigoni, through the repeated backcrossing of GFP-linked C. briggsae fragments into C. nigoni. However, the median introgression size was over 7 Mb, indicating apparent HR suppression and preventing the subsequent cloning of the causative gene underlying a given HI phenotype. Therefore, a robust method that permits recombination independent of sequence homology is desperately desired. RESULTS: Here, we report a method of highly efficient targeted recombination (TR) induced by CRISPR/Cas9 with dual guide RNAs (gRNAs), which circumvents the HR suppression in hybrids between the two species. We demonstrated that a single gRNA was able to induce efficient TR between highly homologous sequences only in the F1 hybrids but not in the hybrids that carry a GFP-linked C. briggsae fragment in an otherwise C. nigoni background. We achieved highly efficient TR, regardless of sequence homology or genetic background, when dual gRNAs were used that each specifically targeted one parental chromosome. We further showed that dual gRNAs were able to induce efficient TR within genomic regions that had undergone inversion, in which HR-based recombination was expected to be suppressed, supporting the idea that dual-gRNA-induced TR can be achieved through nonhomology-based end joining between two parental chromosomes. CONCLUSIONS: Recombination suppression can be circumvented through CRISPR/Cas9 with dual gRNAs, regardless of sequence homology or the genetic background of the species hybrid. This method is expected to be applicable to other situations in which recombination is suppressed in interspecies or intrapopulation hybrids.

Novel, high accuracy models for hepatocellular carcinoma prediction based on longitudinal data and cell-free DNA signatures.

BACKGROUND & AIMS: Current hepatocellular carcinoma (HCC) risk scores do not reflect changes in HCC risk resulting from liver disease progression/regression over time. We aimed to develop and validate two novel prediction models using multivariate longitudinal data, with or without cell-free DNA (cfDNA) signatures. METHODS: A total of 13,728 patients from two nationwide multicenter prospective observational cohorts, the majority of whom had chronic hepatitis B, were enrolled. aMAP score, as one of the most promising HCC prediction models, was evaluated for each patient. Low-pass whole-genome sequencing was used to derive multi-modal cfDNA fragmentomics features. A longitudinal discriminant analysis algorithm was used to model longitudinal profiles of patient biomarkers and estimate the risk of HCC development. RESULTS: We developed and externally validated two novel HCC prediction models with a greater accuracy, termed aMAP-2 and aMAP-2 Plus scores. The aMAP-2 score, calculated with longitudinal data on the aMAP score and alpha-fetoprotein values during an up to 8-year follow-up, performed superbly in the training and external validation cohorts (AUC 0.83-0.84). The aMAP-2 score showed further improvement and accurately divided aMAP-defined high-risk patients into two groups with 5-year cumulative HCC incidences of 23.4% and 4.1%, respectively (p = 0.0065). The aMAP-2 Plus score, which incorporates cfDNA signatures (nucleosome, fragment and motif scores), optimized the prediction of HCC development, especially for patients with cirrhosis (AUC 0.85-0.89). Importantly, the stepwise approach (aMAP -> aMAP-2 -> aMAP-2 Plus) stratified patients with cirrhosis into two groups, comprising 90% and 10% of the cohort, with an annual HCC incidence of 0.8% and 12.5%, respectively (p <0.0001). CONCLUSIONS: aMAP-2 and aMAP-2 Plus scores are highly accurate in predicting HCC. The stepwise application of aMAP scores provides an improved enrichment strategy, identifying patients at a high risk of HCC, which could effectively guide individualized HCC surveillance. IMPACT AND IMPLICATIONS: In this multicenter nationwide cohort study, we developed and externally validated two novel hepatocellular carcinoma (HCC) risk prediction models (called aMAP-2 and aMAP-2 Plus scores), using longitudinal discriminant analysis algorithm and longitudinal data (i.e., aMAP and alpha-fetoprotein) with or without the addition of cell-free DNA signatures, based on 13,728 patients from 61 centers across mainland China. Our findings demonstrated that the performance of aMAP-2 and aMAP-2 Plus scores was markedly better than the original aMAP score, and any other existing HCC risk scores across all subsets, especially for patients with cirrhosis. More importantly, the stepwise application of aMAP scores (aMAP -> aMAP-2 -> aMAP-2 Plus) provides an improved enrichment strategy, identifying patients at high risk of HCC, which could effectively guide individualized HCC surveillance.

Direct full-length RNA sequencing reveals unexpected transcriptome complexity during Caenorhabditis elegans development.

Massively parallel sequencing of the polyadenylated RNAs has played a key role in delineating transcriptome complexity, including alternative use of an exon, promoter, 5' or 3' splice site or polyadenylation site, and RNA modification. However, reads derived from the current RNA-seq technologies are usually short and deprived of information on modification, compromising their potential in defining transcriptome complexity. Here, we applied a direct RNA sequencing method with ultralong reads using Oxford Nanopore Technologies to study the transcriptome complexity in Caenorhabditis elegans We generated approximately six million reads using native poly(A)-tailed mRNAs from three developmental stages, with average read lengths ranging from 900 to 1100 nt. Around half of the reads represent full-length transcripts. To utilize the full-length transcripts in defining transcriptome complexity, we devised a method to classify the long reads as the same as existing transcripts or as a novel transcript using sequence mapping tracks rather than existing intron/exon structures, which allowed us to identify roughly 57,000 novel isoforms and recover at least 26,000 out of the 33,500 existing isoforms. The sets of genes with differential expression versus differential isoform usage over development are largely different, implying a fine-tuned regulation at isoform level. We also observed an unexpected increase in putative RNA modification in all bases in the coding region relative to the UTR, suggesting their possible roles in translation. The RNA reads and the method for read classification are expected to deliver new insights into RNA processing and modification and their underlying biology in the future.

Genomic architecture of 5S rDNA cluster and its variations within and between species.

BACKGROUND: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution. RESULTS: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae. CONCLUSIONS: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies.

Real-world study on HBsAg loss of combination therapy in HBeAg-negative chronic hepatitis B patients.

Combination therapy with pegylated interferon (PEG-IFN) and nucleos(t)ide analogues (NAs) can enhance hepatitis B surface antigen (HBsAg) clearance. However, the specific treatment strategy and the patients who would benefit the most are unclear. Therefore, we assessed the HBsAg loss rate of add-on PEG-IFN and explored the factors associated with HBsAg loss in chronic hepatitis B (CHB) patients. This was a real-world cohort study of adults with CHB. Hepatitis B e antigen (HBeAg)-negative NAs-treated patients with baseline HBsAg ≤1500 IU/ml and HBV DNA < the lower limit of detection, or 100 IU/ml, received 48 weeks of add-on PEG-IFN. The primary outcome of the study was the rate of HBsAg loss at 48 weeks of combination treatment. Using multivariable logistic regression analysis, we determined factors associated with HBsAg loss. HBsAg loss in 2579 patients (mean age: 41.2 years; 80.9% male) was 36.7% (947 patients) at 48 weeks. HBsAg loss was highest in patients from south-central and southwestern China (40.0%). Factors independently associated with HBsAg loss included: increasing age (odds ratio = 0.961); being male (0.543); baseline HBsAg level (0.216); HBsAg decrease at 12 weeks (between 0.5 and 1.0 log10 IU/ml [2.405] and >1.0 log10 IU/ml [7.370]); alanine aminotransferase (ALT) increase at 12 weeks (1.365); haemoglobin (HGB) decrease at 12 weeks (1.558). There was no difference in the primary outcomes associated with the combination regimen. In conclusion, HBsAg loss by combination therapy was higher in patients from southern China than those from the north. An increased chance of HBsAg loss was associated with baseline characteristics and dynamic changes in clinical indicators.

Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.

Mortality from hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is high due to limited treatment options. Preclinical and clinical investigations have proved that treatment with mesenchymal stromal cells (MSCs) is beneficial for recovery from liver injury. We hypothesized that the outcome of HBV-related ACLF would be improved by MSC treatment. From 2010 to 2013, 110 patients with HBV-related ACLF were enrolled in this open-label, nonblinded randomized controlled study. The control group (n = 54) was treated with standard medical therapy (SMT) only. The experimental group (n = 56) was infused weekly for 4 weeks with 1.0 to 10 × 105 cells/kg allogeneic bone marrow-derived MSCs and then followed for 24 weeks. The cumulated survival rate of the MSC group was 73.2% (95% confidence interval 61.6%-84.8%) versus 55.6% (95% confidence interval 42.3%-68.9%) for the SMT group (P = 0.03). There were no infusion-related side effects, but fever was more frequent in MSC compared to SMT patients during weeks 5-24 of follow-up. No carcinoma occurred in any trial patient in either group. Compared with the control group, allogeneic bone marrow-derived MSC treatment markedly improved clinical laboratory measurements, including serum total bilirubin and Model for End-Stage Liver Disease scores. The incidence of severe infection in the MSC group was much lower than that in the SMT group (16.1% versus 33.3%, P = 0.04). Mortality from multiple organ failure and severe infection was higher in the SMT group than in the MSC group (37.0% versus 17.9%, P = 0.02). CONCLUSION: Peripheral infusion of allogeneic bone marrow-derived MSCs is safe and convenient for patients with HBV-related ACLF and significantly increases the 24-week survival rate by improving liver function and decreasing the incidence of severe infections. (Hepatology 2017;66:209-219).

IFI16 is involved in HBV-associated acute-on-chronic liver failure inflammation.

BACKGROUND: Hepatitis B virus (HBV) is a hepatotropic DNA virus, and its DNA may be a potent inflammatory molecule. Interferon-inducible protein 16 (IFI16), a newly discovered DNA sensor, plays an important role in the process of inflammation in viral infections. Our study sought to identify a correlation between IFI16 expression and inflammation in patients with chronic hepatitis B (CHB) and HBV-associated acute-on-chronic liver failure (HBV-ACLF). METHODS: We performed flow cytometry to measure IFI16 levels in peripheral blood mononuclear cells (PBMC) and used immunohistochemistry and western blotting to measure IFI16 protein levels in liver tissues. The cellular source of IFI16 was detected using double immunofluorescence. All datum were analyzed using SPSS 13.0 and GraphPad Prism 6. RESULTS: The number of IFI16+ cells was significantly associated with the degree of inflammation. In detail, the number of IFI16+ cells was higher in livers but lower in PBMCs in HBV-ACLF patients than those in CHB patients and healthy controls. There was no significant difference between CHB patients and healthy controls in numbers of IFI6+ cells in livers and PBMCs. There was no significant relationship between IFI16 expression levels and HBV parameters. Furthermore, IFI16 was expressed in the nucleus of Kupffer cells (KCs), endothelial cells, natural killer cells, dendritic cells, and hepatic stellate cells in healthy donors and CHB patients, but only in the cytoplasm of KCs in the livers of HBV-ACLF patients. CONCLUSIONS: IFI16 was closely related to the degree of inflammation in CHB and HBV-ACLF patients and may serve as a vital contributor to the pathogeneses of liver damage in HBV-ACLF.

A Genome-wide hybrid incompatibility landscape between Caenorhabditis briggsae and C. nigoni.

Systematic characterization of hybrid incompatibility (HI) between related species remains the key to understanding speciation. The genetic basis of HI has been intensively studied in Drosophila species, but remains largely unknown in other species, including nematodes, which is mainly due to the lack of a sister species with which C. elegans can mate and produce viable progeny. The recent discovery of a C. briggsae sister species, C. nigoni, has opened up the possibility of dissecting the genetic basis of HI in nematode species. However, the paucity of dominant and visible marker prevents the efficient mapping of HI loci between the two species. To elucidate the genetic basis of speciation in nematode species, we first generated 96 chromosomally integrated GFP markers in the C. briggsae genome and mapped them into the defined locations by PCR and Next-Generation Sequencing (NGS). Aided by the marker, we backcrossed the GFP-associated C. briggsae genomic fragments into C. nigoni for at least 15 generations and produced 111 independent introgressions. The introgression fragments cover most of the C. briggsae genome. We finally dissected the patterns of HI by scoring the embryonic lethality, larval arrest, sex ratio and male sterility for each introgression line, through which we identified pervasive HI loci and produced a genome-wide landscape of HI between the two nematode species, the first of its type for any non-Drosophila species. The HI data not only provided insights into the genetic basis of speciation, but also established a framework for the possible cloning of HI loci between the two nematode species. Furthermore, the data on hybrids confirmed Haldane's rule and suggested the presence of a large X effect in terms of fertility between the two species. Importantly, this work opens a new avenue for studying speciation genetics between nematode species and allows parallel comparison of the HI with that in Drosophila and other species.

Entecavir Combined With Adefovir Ameliorates Patients With Chronic Hepatitis B Who Fail to Respond to Nucleotide (Acid) Analog Monotherapy.

The aim of this study was to evaluate the efficacy and safety of entecavir (ETV) combined treatment with adefovir (ADV) on chronic hepatitic B (CHB) patients who failed to respond to nucleotide (acid) analog (NA) treatment. On this basis, the possible factors in the combined treatment of these patients will be analyzed. The safety, biochemical index, and the possible factors that might affect the ETV and ADV combined treatment at different points in time were retrospectively analyzed. The biochemical index included the following: virological response, hepatitis B virus (HBV) DNA decline, primary nonresponse, biochemical response, and the hepatitis B virus E antigen/hepatitis B virus E antibody seroconversion rate. There were 94 CHB patients and compensated liver cirrhosis patients who received ETV plus ADV treatment for over 12 weeks after failure of treatment with NAs. The authors have also investigated 76 CHB patients (80.9%) and 18 hepatitis B cirrhosis patients (19.1%) in this study. The HBV DNA baseline was 4.4 ± 1.4 log10 IU/mL, and the positive rate of HBeAg before salvage treatment was 78.7% (74/94). The sample sizes were 94, 78, 42, 10, 6, and 1 for follow-up of 24, 48, 96, 144, 192, and 240 weeks, respectively. The virological responses (HBV DNA < 2 log10 IU/mL) and biochemical responses were 52.1%, 74.3%, and 90.4% and 63.1%, 61.6%, and 81.1%, respectively, at 24, 48, and 96 weeks, which showed significant differences (P < 0.001 and P < 0.005, respectively). The HBV DNA decline was presented as mean ± SEM, which were 1.53 ± 1.23, 1.75 ± 1.37, 2.07 ± 1.54, and 2.39 ± 1.77 log10 IU/mL at 12, 24, 48, and 96 weeks, respectively. They showed significant differences compared with the baseline (χ = 8.084, P < 0.05). The rate of primary nonresponse was 30.9% (29/94), and the primary treatment failure rates were 26.6% (25/94), 24.4% (19/78), and 4.8% (2/42) at 24, 48, and 96 weeks, respectively. They all have statistical difference (P = 0.011 < 0.05). There were 23 patients who experienced virological breakthrough after the HBV DNA levels were undetectable, whereas after follow-up for 12-24 weeks, the HBV DNA levels were back to undetectable again. ETV plus ADV treatment is an efficient and safe treatment for CHB and compensated liver cirrhosis patients who experienced NA treatment failure. The high quantity of baseline HBV DNA level is a risk factor for poor efficacy of salvage treatment.

Decreases in activated CD8+ T cells in patients with severe hepatitis B are related to outcomes.

BACKGROUND: Many studies on T helper (Th)1, Th2, T regulatory and Th17 cells have been carried out in acute-on-chronic liver failure (ACLF). However, CD8(+) T cell, as a main participant in immune-mediated injuries and defense against microorganisms, has seldom been studied in ACLF. AIMS: The purpose of this study was to investigate the CD8(+) T cell function, and the outcomes of patients with severe hepatitis [SH; serum bilirubin (SB) ≥ 10 mg/dl and prothrombin activity (PTA) < 60 %]. METHODS: Thirty-six patients with chronic HBV-associated SH were included. Twenty normal chronic hepatitis B (CHB) patients (2 < SB < 10 (mg/dl) and PTA ≥ 60 %) and 28 healthy volunteers were enrolled as control groups. RESULTS: Twenty-six patients with SH were diagnosed with ACLF (SB ≥ 10 mg/dl and PTA ≤ 40 %). The non-recovered ACLFs (NR-ACLF) had higher HBV DNA loads than recovered ACLFs (R-ACLF) (6.03 ± 1.79 vs. 4.36 ± 1.61 (log10, IU/L)). The NR-ACLFs had the highest neutrophil:lymphocyte ratios (5.10 ± 2.37) (all P < 0.001; a = 0.05). The CHBs had higher perforin(+) and TCM (CD45RA(-)CD62L(hi)CCR7(+)) proportions [31.28 ± 19.51, 5.32 ± 3.57 (%)] compared to R-ACLFs (11.75 ± 15.35, 0.78 ± 0.76 (%); P = 0.004, 0.001, respectively), or NR-ACLFs (11.61 ± 5.79, 1.14 ± 0.67 (%); P = 0.006, 0.003). The non-ACLF SHs had higher CD38(+) proportions than R-ACLFs or NR-ACLFs (25.46 ± 8.02 vs. 16.24 ± 7.77 or 16.81 ± 6.30 (%), P = 0.039, 0.023). CONCLUSIONS: High neutrophil:lymphocyte ratios and a decrease in activated CD8(+) T cells may be related to poor outcomes in patients with SH.

Soluble ST2 plasma concentrations predict mortality in HBV-related acute-on-chronic liver failure.

Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a rapidly progressing and frequently fatal condition. The aim of this study was to determine whether interleukin- (IL-) 33 and soluble ST2 (sST2) were associated with disease severity and mortality in HBV-ACLF. We found that plasma levels of sST2 but not IL-33 were higher in HBV-ACLF patients compared with chronic hepatitis B (CHB) patients and healthy controls. However, plasma levels of IL-33, TNF-α, IFN-γ, and IL-10 did not correlate with sST2 levels. Similarly, immunohistochemistry revealed low IL-33 expression and high ST2 expression in liver sections of patients with HBV-ACLF. Evaluation of dynamic changes of sST2 in HBV-ACLF showed that plasma sST2 levels increased over time in patients who died during the 180-day follow-up but decreased in those who survived. In addition, plasma sST2 level after week 1 correlated with disease severity, as assessed by total bilirubin, prothrombin time, and model for end-stage liver disease score. Results of Kaplan-Meier survival analysis showed that higher sST2 concentration (≥87 ng/mL) at week 3 was associated with poor survival. These findings indicate the potential usefulness of sST2 as a predictor of disease severity and in making treatment decisions for patients with HBV-ACLF.

Collaborative regulation of development but independent control of metabolism by two epidermis-specific transcription factors in Caenorhabditis elegans.

Cell fate specification is typically initiated by a master regulator, which is relayed by tissue-specific regulatory proteins (usually transcription factors) for further enforcement of cell identities, but how the factors are coordinated among each other to "finish up" the specification remains poorly understood. Caenorhabditis elegans epidermis specification is initiated by a master regulator, ELT-1, that activates its targets, NHR-25 and ELT-3, two epidermis-specific transcription factors that are important for development but not for initial specification of epidermis, thus providing a unique paradigm for illustrating how the tissue-specific regulatory proteins work together to enforce cell fate specification. Here we addressed the question through contrasting genome-wide in vivo binding targets between NHR-25 and ELT-3. We demonstrate that the two factors bind discrete but conserved DNA motifs, most of which remain in proximity, suggesting formation of a complex between the two. In agreement with this, gene ontology analysis of putative target genes suggested differential regulation of metabolism but coordinated control of epidermal development between the two factors, which is supported by quantitative analysis of expression of their specific or common targets in the presence or absence of either protein. Functional validation of a subset of the target genes showed both activating and inhibitory roles of NHR-25 and ELT-3 in regulating their targets. We further demonstrated differential control of specification of AB and C lineage-derived epidermis. The results allow us to assemble a comprehensive gene network underlying C. elegans epidermis development that is likely to be widely used across species and provides insights into how tissue-specific transcription factors coordinate with one another to enforce cell fate specification initiated by its master regulator.

High level of IL-27 positively correlated with Th17 cells may indicate liver injury in patients infected with HBV.

BACKGROUND: Interleukin-6/IL-12 family cytokines play a key role in inflammatory diseases via their effects on the differentiation or regulation of T helper cells. AIMS: The aim of this study was to determine the role of interleukin-27 (IL-27) and its association with helper T cells in hepatitis B virus (HBV)-infected patients. METHODS: Samples were assessed from 51 HBV-infected patients [28 chronic hepatitis B (CHB) subjects and 23 acute-on-chronic liver failure (ACLF) subjects] and 18 normal controls (NC). Serum IL-27 levels were examined by enzyme-linked immunosorbent assay. Circulating helper T cells were determined using flow cytometry and associations between IL-27 expression and helper T cells were analysed. RESULTS: Serum IL-27 levels rose in HBV-infected patients (502.88 ± 23.35 pg/ml) compared to (NC, 277.14 ± 23.96 pg/ml, P < 0.0001). Furthermore, it significantly increased in patients with ACLF (587.90 ± 33.08 pg/ml) when compared with CHB (433.04 ± 26.57 pg/ml, P = 0.001). However, no statistically significant differences were observed between IL-27 and the presence of HBeAg. High levels of IL-27 then positively correlated with Tbil levels (r = 0.401, P = 0.004), but negatively associated with prothrombin time activity levels (r = -0.496, P < 0.001), and a slightly negative correlation trend with HBV-DNA loads (r = -0.228, P = 0.107) existed in these HBV-infected subjects. Additionally, frequency of circulating interleukin-17-producing CD4(+) T cells (Th17 cells) increased in HBV-infected patients (ACLF, mean, 5.39%; CHB, median, 3.12%) as compared to NC subjects (median, 2.22%, P < 0.0001). Moreover, correlation analysis showed that serum IL-27 level was positively associated with circulating Th17 cells (r = 0.342, P = 0.036). CONCLUSION: These results provided evidence that IL-27 was positively correlated with Th17 cells commitment, and may exerted a proinflammatory effect in the development of liver injury in HBV-infected patients.

Severe Organ Impairment Was Common in Elderly Individuals with Dengue in Guangdong, China.

Guangdong, China, has experienced several dengue epidemics involving thousands of confirmed cases in recent decades, and elderly individuals suffered severe dengue (SD) most seriously. However, the clinical characteristics and risk factors for SD among elderly patients in Guangdong have not been investigated. Patients older than 65 years were recruited and divided into a dengue fever (DF) group and an SD group according to the 2009 Dengue Guidelines of the WHO. We analyzed the clinical manifestations of the elderly patients with dengue and then assessed the risk factors for SD. Of a total of 1,027 patients, 868 patients were diagnosed as having DF and 159 as having SD. Of the 159 elderly patients with SD, 129 (81%) had comorbidities, with hypertension being the most common. Severe organ impairment (SOI) (115, 54%) was the most common presentation in SD patients, followed by severe plasma leakage (52, 24.4%) and severe hemorrhage (46, 21.6%). The most common symptom of SOI was kidney injury, followed by heart injury and central nervous system injury. Furthermore, multivariate regression revealed that the presence of chronic obstructive pulmonary disease (COPD), a lower red blood cell (RBC) count (≤3.5 × 1012/L; odds ratio [OR], 0.35; 95% CI, 0.17-0.55; P <0.001), lower serum albumin (ALB) (≤35 U/L; OR, 0.18; 95% CI, 0.09-0.32; P <0.001), and hyperpyrexia (body temperature ≥39°C; OR, 1.8; 95% CI, 1.2-2.6, P <0.001) were risk factors for SD. Severe organ impairment was the predominant manifestation in elderly individuals with SD characterized by kidney injury. The potential risk factors of SD such as presence of COPD and hyperpyrexia and lower RBC and ALB levels might help clinicians identify patients with SD early.

LARP1 predict the prognosis for early-stage and AFP-normal hepatocellular carcinoma.

BACKGROUND: The La-related protein 1 (LARP1) has been found to be a RNA binding protein and was related to spermatogenesis, embryogenesis and cell-cycle progression. The aim of this study was to investigate the prognostic value of LARP1 in hepatocellular carcinoma (HCC). METHODS: LARP1 expression was examined in 15 HCC cell lines and 272 clinical specimens using real-time PCR, immunohistochemistry (IHC) and western blot analysis (WB). LARP1 expression was also studied in 6 paired HCC lesions and the adjacent non-cancerous tissue samples. Statistical analyses were applied to derive association between LARP1 expression scores and clinical characters as well as patient survival. RESULTS: mRNA and protein levels of LARP1 were higher in HCC cell lines and HCC lesions than in normal liver epithelial cells and the paired adjacent noncancerous tissues. LARP1 expression was correlated to survival time, vital status, tumor size and Child-Pugh score. Overall survival analysis showed HCC patients with high LARP1 expression level had lower survival rate (P<0.01). Importantly, this correlation remained significant in patients with early-stage HCC or with normal serum AFP level. CONCLUSIONS: LARP1 protein may represent a promising biomarker for predicting the prognosis of HCC, including in early-stage and AFP-normal patients.

Imbalance of interleukin-17-producing CD4 T cells/regulatory T cells axis occurs in remission stage of patients with hepatitis B virus-related acute-on-chronic liver failure.

BACKGROUND AND AIM: Although regulatory T cells (Treg) and interleukin-17-producing CD4 T cells (Th17) have been demonstrated to play opposing roles in inflammation-associated diseases, their frequency and balance in different stages of hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) remain unknown. METHODS: Fourteen patients with HBV-associated ACLF were studied and defined into different stages according to disease activity. Circulating Th17 cells and Treg cells were analyzed by flow cytometry, and the cytokines were quantitated by enzyme-linked immunosorbent assay. Results were correlated with temporal changes in viral load, disease progression and compared with 30 chronic hepatitis B (CHB) subjects and 18 healthy subjects. RESULTS: We showed a significantly higher frequency of circulating Th17 cells in the remission stage of ACLF when compared with the progression stage, the CHB group, or normal controls. However, the frequency of circulating Treg cells was significantly lower in the remission stage of ACLF when compared with the progression stage or the CHB group. The increase in Th17 cells and concomitant decrease in Treg cells created an imbalance in the remission stage of ACLF patients, which negatively correlated with disease progression. In addition, we showed that ACLF patients in the remission stage had an altered profile of cytokines that regulated the induction of Th17 cells and Treg cells. CONCLUSIONS: ACLF patients in the remission stage had an imbalance of Th17 to Treg cells, which could be used as a prognostic marker to predict disease progression. This imbalance could play a role in the immunopathogenesis of HBV-related ACLF.

[Up-regulator of cell proliferation predicts poor prognosis in hepatocellular carcinoma].

OBJECTIVE: To explore the potential value of up-regulator of cell proliferation (URGCP) as a biomarker for predicting the prognosis of hepatocellular carcinoma (HCC). METHODS: The expression of URGCP was analyzed in 15 HCC cell lines and in 10 pairs of HCC and adjacent tissues with reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The expression of URGCP in 278 paraffin-embedded, archived clinical HCC samples was analyzed by immunohistochemistry (IHC) and statistic analysis conducted to examine the relationship of prognosis and URGCP expression. RESULTS: IHC analysis revealed a high expression of URGCP in all HCC cell lines and in 122/278 (43.8%) paraffin-embedded archived HCC specimens. The expression level of URGCP was significantly correlated with clinical staging and poor patient survival of HCC in the study cohort and in various clinical subgroups, but not correlated with HCC patient age, tumor size, tumor number or alpha-fetoprotein level. CONCLUSION: URGCP plays an important role in promoting the proliferation and tumorigenesis of HCC and may represent a novel prognostic biomarker and therapeutic target for the disease.