Time Required for Nanopore Whole-Genome Sequencing of Neisseria gonorrhoeae for Identification of Phylogenetic Relationships.

BACKGROUND: Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a global health challenge. Limitations to AMR surveillance reporting, alongside reduction in culture-based susceptibility testing, has resulted in a need for rapid diagnostics and strain detection. We investigated Nanopore sequencing time, and depth, to accurately identify closely related N. gonorrhoeae isolates, compared to Illumina sequencing. METHODS: N. gonorrhoeae strains collected from a London sexual health clinic were cultured and sequenced with MiSeq and MinION sequencing platforms. Accuracy was determined by comparing variant calls at 68 nucleotide positions (37 resistance-associated markers). Accuracy at varying MinION sequencing depths was determined through retrospective time-stamped read analysis. RESULTS: Of 22 MinION-MiSeq pairs reaching sufficient sequencing depth, agreement of variant call positions passing quality control criteria was 185/185 (100%; 95% confidence interval [CI], 98.0%-100.0%), 502/503 (99.8%; 95% CI, 98.9%-99.9%), and 564/565 (99.8%; 95% CI, 99.0%-100.0%) at 10x, 30x, and 40x MinION depth, respectively. Isolates identified as closely related by MiSeq, within one yearly evolutionary distance of ≤5 single nucleotide polymorphisms, were accurately identified via MinION. CONCLUSIONS: Nanopore sequencing shows utility as a rapid surveillance tool, identifying closely related N. gonorrhoeae strains, with just 10x sequencing depth, taking a median time of 29 minutes. This highlights its potential for tracking local transmission and AMR markers.

Comprehensive analysis on the regulation of differentially expressed of mRNA and ncRNA in different ovarian stages of ark shell Scapharca broughtonii.

BACKGROUND: Ovarian development is an important prerequisite and basis for animal reproduction. In many vertebrates, it is regulated by multiple genes and influenced by sex steroid hormones and environmental factors. However, relative information is limited in shellfish. To explore the biological functions and molecular mechanisms of mRNA and non-coding RNA that regulate ovarian development in Scapharca broughtonii, we performed whole transcriptome sequencing analysis on ovaries at three developmental stages. Furthermore, the biological processes involved in the differential expression of mRNA and ncRNA were analyzed. RESULTS: A total of 11,342 mRNAs, 6897 lncRNAs, 135 circRNAs, and 275 miRNAs were differentially expressed. By mapping the differentially expressed RNAs from the three developmental stages of Venn diagram, multiple groups of shared mRNAs and lncRNAs were found to be associated with ovarian development, with some mRNA and ncRNA functions associated with steroid hormone. In addition, we constructed and visualized the lncRNA/circRNA-miRNA-mRNA network based on ceRNA targeting relationships. CONCLUSIONS: These findings may facilitate our further understanding the mRNA and ncRNAs roles in the regulation of shellfish reproduction.

Diagnostic markers and potential therapeutic agents for Sjögren's syndrome screened through multiple machine learning and molecular docking.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune disease, which mainly damages patients' exocrine glands. Sensitive early diagnostic indicators and effective treatments for pSS are lacking. Using machine learning methods to find diagnostic markers and effective therapeutic ways for pSS is of great significance. In our study, first, 1643 differentially expressed genes (DEGs; 737 were upregulated and 906 were downregulated) were ultimately screened out and analyzed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes based on the datasets from the Gene Expression Omnibus. Then, support vector machine, least absolute shrinkage and selection operator regression, random forest, and weighted correlation network analysis were used to screen out feature genes from DEGs. Subsequently, the intersection of the feature genes was taken to screen 10 genes as hub genes. Meanwhile, the analysis of the diagnostic efficiency of 10 hub genes showed their good diagnostic value for pSS, which was validated through immunohistochemistry on the paraffin sections of the labial gland. Subsequently, a multi-factor regulatory network and correlation analysis of hub genes were performed, and the results showed that ELAVL1 and IGF1R were positively correlated with each other but both negatively correlated with the other seven hub genes. Moreover, several meaningful results were detected through the immune infiltration landscape. Finally, we used molecular docking to screen potential therapeutic compounds of pSS based on the hub genes. We found that the small molecules DB08006, DB08036, and DB15308 had good docking scores with ELAVL1 and IGF1R simultaneously. Our study might provide effective diagnostic biomarkers and new therapeutic ideas for pSS.

MicroRNA expression signature in the striated and smooth adductor muscles of Yesso scallop Patinopecten yessoensis.

Increasing evidences point to the potential role of microRNAs (miRNAs) in muscle growth and development in animals. However, knowledge on the identity of miRNAs and their targets in molluscs remains largely unknown. Scallops have one large adductor muscle, composed of fast (striated) and slow (smooth) muscle types, which display great differences in muscle fibers, meat quality, cell types and molecular components. In the present study, we performed a comprehensive investigation of miRNA transcriptomes in fast and slow adductor muscles of Yesso scallop Patinopecten yessoensis. As a result, 47 differentially expressed miRNAs representing ten miRNA families were identified between the striated and smooth adductor muscles. The KEGG enrichment analysis of their target genes were mainly associated with amino acid metabolism, energy metabolism and glycan biosynthesis. The target genes of miR-133 and miR-71 were validated by the dual-luciferase reporter assays and miRNA antagomir treatment in vivo. The identification and functional validation of these different miRNAs in scallops will greatly help our understanding of miRNA regulatory mechanism that achieves the unique muscle phenotypes in scallops. The present findings provide the direct evidences for muscle-specific miRNAs involved in muscle growth and differentiation in molluscs.

Vaginal microbiota in ethnically diverse young women who did or did not develop pelvic inflammatory disease: community-based prospective study.

OBJECTIVES: A lactobacilli-dominated vaginal microbiome may protect against pelvic inflammatory disease (PID), but one dominated by Gardnerella species might increase susceptibility. Not all lactobacilli are equally protective. Recent research suggests that D(-) isomer lactic acid producing lactobacilli (Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus gasseri) may protect against infection with Chlamydia trachomatis, an important cause of PID. Lactobacillus iners , which produces L(+) isomer lactic acid, may be less protective. We investigated the microbiome in stored vaginal samples from participants who did or did not develop PID during the prevention of pelvic infection (POPI) chlamydia screening trial. METHODS: Long-read 16S rRNA gene nanopore sequencing was used on baseline vaginal samples (one per participant) from all 37 women who subsequently developed clinically diagnosed PID during 12-month follow-up, and 111 frequency matched controls who did not, matched on four possible risk factors for PID: age <20 versus ≥20, black ethnicity versus other ethnicity, chlamydia positive versus negative at baseline and ≥2 sexual partners in the previous year versus 0-1 partners. RESULTS: Samples from 106 women (median age 19 years, 40% black ethnicity, 22% chlamydia positive, 54% reporting multiple partners) were suitable for analysis. Three main taxonomic clusters were identified dominated by L. iners, L. crispatus and Gardnerella vaginalis. There was no association between a more diverse, G. vaginalis dominated microbiome and subsequent PID, although increased Shannon diversity was associated with black ethnicity (p=0.002) and bacterial vaginosis (diagnosed by Gram stain p<0.0001). Women who developed PID had similar relative abundance of protective D(-) isomer lactic acid producing lactobacilli to women without PID, but numbers of PID cases were small. CONCLUSIONS: In the first-ever community-based prospective study of PID, there was no clear association between the vaginal microbiome and subsequent development of PID. Future studies using serial samples may identify vaginal microbial communities that may predispose to PID.

Cell type diversity in scallop adductor muscles revealed by single-cell RNA-Seq.

Studies on cell atlas in marine invertebrates provide a better understanding of cell types, stem cell maintenance, and lineages of cell differentiation. To investigate the molecular features of various cell types in molluscan muscles, we performed single-cell RNA sequencing (scRNA-seq) to map cell types in scallop adductor muscles. We uncovered the cell type-specific features of 20 cell clusters defined by the expression of multiple specific molecular markers. These cell clusters are mainly classified into four broad classes, including mesenchymal stem cells, muscle cells, neurons, and haemolymph cells. In particular, we identified a diverse repertoire of neurons in the striated adductor muscle, but not in the smooth muscle. We further reconstructed the cell differentiation events using all the cell clusters by single-cell pseudotemporal trajectories. By integrating dual BrdU-PCNA immunodetection, neuron-specific staining and electron microscopy observation, we showed the spatial distribution of mesenchymal stem cells and neurons in striated adductor muscle of scallops. The present findings will not only be useful to address the cell type-specific gene expression profiles in scallop muscles, but also provide valuable resources for cross-species comparison of marine organisms.

LncRNA H19 modulated by miR-146b-3p/miR-1539-mediated allelic regulation in transarterial chemoembolization of hepatocellular carcinoma.

Transarterial chemoembolization (TACE) is an effective treatment for unresectable hepatocellular carcinoma (HCC) patients. Although overall survival (OS) of TACE-treated patients has been evidently prolonged, not all unresectable HCC patients can benefit from TACE. Genome-wide association studies identified multiple HCC susceptibility single nucleotide polymorphisms (SNPs). However, it is still unclear how lncRNAs and their functional SNPs impact therapeutic responses of TACE. In the study, we hypothesized that the functional lncRNA H19 SNP(s) might impact H19 expression and, thus, prognosis of TACE-treated HCC patients. We found that the H19 rs3741219 SNP was significantly associated with OS of HCC patients received TACE. Cox proportional hazards model demonstrated that the rs3741219 CC genotype was associated with longer OS and a 37% decreased death risk compared with the TT carriers after TACE therapy (P = 0.001). Interestingly, the rs3741219 T-to-C change led to allelic down-regulation of lncRNA H19 expression via creating the binding sites of miR-146b-3p and miR-1539. Luciferase reporter gene assays indicated that miR-146b-3p and miR-1539 could markedly silence the rs3741219 C-allelic H19 expression but not lncRNA H19 with the T allele. Consistently, there was significantly reduced expression of lncRNA H19 in HCC and normal tissues of the C allele carriers compared with the H19 levels in patients with the T allele. Knock-down of lncRNA H19 significantly promoted the anti-viability efficiency of oxaliplatin (the main chemotherapy drug used in TACE) to HCC cells. In view of these results, we assume that lncRNA H19 might be a potential therapeutic target for unresectable HCC patients.

N6-methyladenosine reader YTHDC2 and eraser FTO may determine hepatocellular carcinoma prognoses after transarterial chemoembolization.

Transarterial chemoembolization (TACE) has significantly improved overall survival (OS) of unresectable hepatocellular carcinoma (HCC) patients. Unfortunately, a portion of patients show no therapeutic responses to TACE. N6-methyladenosine (m6A) as well as its epigenetic writers, erasers, and readers play a crucial role in HCC development. However, it is still largely unclear how functional small nucleotide polymorphisms (SNPs) in m6A-regulating genes contribute to prognosis of TACE-treated HCC patients. In this study, potential functional SNPs were systematically evaluated to identify their roles in the prognosis of HCC patients after TACE in a Chinese Han population. Employing multiple databases, we successfully annotated 55 candidate SNPs. After genotyping these SNPs in our TACE cohort, we identified three genetic variants in YTHDC2 (rs6594732, rs10071816, and rs2303718) and one SNP in FTO (rs7202116) having statistically significant associations with the OS of HCC patients treated with TACE. For example, multivariate Cox proportional hazards model indicated that the rs7202116 GG genotype carriers had markedly shorter OS and an 87% increased death risk compared with the AA carriers after TACE therapy (P = 0.002). When investigating functional relevance of these SNPs, we observed an allelic regulation of rs7202116 on FTO expression in HCC tissue samples, with higher tumor suppressor FTO expression among the A allele carriers. Our findings reported the first evidence supporting the prognostic value of m6A reader YTHDC2 and m6A eraser FTO SNPs in TACE-treated HCC patients. Importantly, our data implicated that m6A-regulating genes may be targets to improve therapeutic strategy for unresectable HCC patients.

Peripheral blood cell count ratios are predictive biomarkers of clinical response and prognosis for non-surgical esophageal squamous cell carcinoma patients treated with radiotherapy.

BACKGROUND: Peripheral blood cell count ratios, including the neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and lymphocyte-to-monocyte ratio (LMR), have been reported to be prognostic factors in many malignancies as markers of inflammation and immune status. The aim of this study was to determine whether NLR, PLR, or LMR can be clinical response and prognostic biomarkers of non-surgical esophageal squamous cell carcinoma (ESCC) patients treated with radiotherapy. METHODS: 193 non-surgical ESCC patients who underwent radiotherapy were retrospectively analyzed. The peripheral blood cell count ratios were obtained before, during (weekly) and at the end of the treatment. Then, we compared the subsequent results with the corresponding pretreatment values and computed the rates of change, which were defined as cNLR, cPLR, and cLMR. Univariate and multivariate Cox regression analyses were used for overall survival (OS). Ordinal logistic regression was used to analyze the clinical response. RESULTS: In multivariate analysis, cNLR at week 4(P = .026) and week 5(P = .025) during radiotherapy were significantly associated with OS, along with BMI, tumor stage, tumor length, tumor location, and grade of adverse events. Besides, BMI, tumor stage, tumor length, adverse event grade, cNLR at week 4(P = .044) and week 5(P = .013), and cPLR at week 4(P = .034) and week 5(P = .015) were significantly associated with the clinical response in the multivariate logistic regression analysis. CONCLUSIONS: The cNLR at weeks 4 and 5 was negatively correlated with the OS and clinical response of non-surgical ESCC patients treated with radiotherapy. The elevated cPLR at weeks 4 and 5 was only related to poor clinical response.

The long noncoding RNA GAS8-AS1 suppresses hepatocarcinogenesis by epigenetically activating the tumor suppressor GAS8.

Long noncoding RNAs (lncRNAs) are vital players in cancers, including hepatocellular carcinoma (HCC). We previously identified an lncRNA, GAS8-AS1, that is located in intron 2 of GAS8 However, its involvement in HCC is still largely unknown. In this study, we report that both GAS8-AS1 and its host gene GAS8 act as HCC tumor suppressors. We found that expression of GAS8-AS1 or GAS8 is significantly decreased in HCC tissues and is associated with a poor prognosis among HCC patients. Interestingly, lncRNA GAS8-AS1 could promote GAS8 transcription. We detected a CpG island in the GAS8 promoter, but lncRNA GAS8-AS1 did not affect DNA methylation at this GAS8 promoter site. Moreover, we identified two GAS8-AS1-interacting proteins, mixed-lineage leukemia 1 (MLL1), a histone 3 Lys-4 (H3K4) methyltransferase, and its partner WD-40 repeat protein 5 (WDR5). RNA pulldown, ChIP, and RNA immunoprecipitation assays revealed that GAS8-AS1 is required for maintaining the GAS8 promoter in an open chromatin state by recruiting the MLL1/WDR5 complex and for enhancing RNA polymerase II activity and GAS8 transcription. Of note, GAS8-AS1-dependent GAS8 hyperactivation inhibited malignant transformation of hepatocytes. Our results provide important insights into how lncRNA GAS8-AS1 suppresses HCC development and suggest potential strategies for treating patients with liver cancer.

Transcriptomics analysis revealing candidate genes and networks for sex differentiation of yesso scallop (Patinopecten yessoensis).

BACKGROUND: The Yesso scallop, Patinopecten (Mizuhopecten) yessoensis, is a commercially important bivalve in the coastal countries of Northeast Asia. It has complex modes of sex differentiation, but knowledge of the mechanisms underlying this sex determination and differentiation is limited. RESULTS: In this study, the gonad tissues from females and males at three developmental stages were used to investigate candidate genes and networks for sex differentiation via RNA-Req. A total of 901,980,606 high quality clean reads were obtained from 18 libraries, of which 417 expressed male-specific genes and 754 expressed female-specific genes. Totally, 10,074 genes differentially expressed in females and males were identified. Weighted gene co-expression network analysis (WGCNA) revealed that turquoise and green gene modules were significantly positively correlated with male gonads, while coral1 and black modules were significantly associated with female gonads. The most important gene for sex determination and differentiation was Pydmrt 1, which was the only gene discovered that determined the male sex phenotype during early gonadal differentiation. Enrichment analyses of GO terms and KEGG pathways revealed that genes involved in metabolism, genetic and environmental information processes or pathways are sex-biased. Forty-nine genes in the five modules involved in sex differentiation or determination were identified and selected to construct a gene co-expression network and a hypothesized sex differentiation pathway. CONCLUSIONS: The current study focused on screening genes of sex differentiation in Yesso scallop, highlighting the potential regulatory mechanisms of gonadal development in P. yessoensis. Our data suggested that WCGNA can facilitate identification of key genes for sex differentiation and determination. Using this method, a hypothesized P. yessoensis sex determination and differentiation pathway was constructed. In this pathway, Pydmrt 1 may have a leading function.

Integrative Functional Genomics Implicates EPB41 Dysregulation in Hepatocellular Carcinoma Risk.

Genome-wide association studies (GWASs) have provided many insights into cancer genetics. However, the molecular mechanisms of many susceptibility SNPs defined by GWASs in cancer heritability and in promoting cancer risk remain elusive. New research strategies, including functional evaluations, are warranted to systematically explore truly causal genetic variants. In this study, we developed an integrative functional genomics methodology to identify cancer susceptibility SNPs in transcription factor-binding sites across the whole genome. Employing integration of functional genomic data from c-Myc cistromics, 1000 Genomes, and the TRANSFAC matrix, we successfully annotated 12 SNPs present in the c-Myc cistrome with properties consistent with modulating c-Myc binding affinity in hepatocellular carcinoma (HCC). After genotyping these 12 SNPs in 1,806 HBV-related HCC case subjects and 1,708 control subjects, we identified a HCC susceptibility SNP, rs157224G>T, in Chinese populations (T allele: odds ratio = 1.64, 95% confidence interval = 1.32-2.02; p = 5.2 × 10(-6)). This polymorphism leads to HCC predisposition through modifying c-Myc-mediated transcriptional regulation of EPB41, with the risk rs157224T allele showing significantly decreased gene expression. Based on cell proliferation, wound healing, and transwell assays as well as the mouse xenograft model, we identify EPB41 as a HCC susceptibility gene in vitro and in vivo. Consistent with this notion, we note that EPB41 expression is significantly decreased in HCC tissue specimens, especially in portal vein metastasis or intrahepatic metastasis, compared to normal tissues. Our results highlight the involvement of regulatory genetic variants in HCC and provide pathogenic insights of this malignancy via a genome-wide approach.

Impact of mass drug administration of azithromycin for trachoma elimination on prevalence and azithromycin resistance of genital Mycoplasma genitalium infection.

BACKGROUND: Mass drug administration (MDA) of 20 mg/kg (maximum 1 g in adults) azithromycin for ocular Chlamydia trachomatis (CT) infection is a key component of the WHO trachoma elimination strategy. However, this dose may be suboptimal in Mycoplasma genitalium infection and may encourage emergence of antimicrobial resistance (AMR) to azithromycin. OBJECTIVES: To determine the effect of MDA for trachoma elimination on M. genitalium prevalence, strain type and azithromycin resistance. METHODS: A secondary analysis of CT-negative vulvovaginal swabs from three outpatient antenatal clinics (Honiara, Solomon Islands) from patients recruited either pre-MDA, or 10 months post-MDA in two cross-sectional surveys was carried out. Swabs were tested for M. genitalium infection using Fast Track Diagnostics Urethritis Plus nucleic acid amplification assay. M. genitalium-positive samples were subsequently tested for azithromycin resistance by sequencing domain V of the 23S rRNA DNA region of M. genitalium and underwent phylogenetic analysis by dual locus sequence typing. RESULTS: M. genitalium prevalence was 11.9% (28/236) in women pre-MDA and 10.9% (28/256) 10 months post-MDA (p=0.7467). Self-reported receipt of azithromycin as part of MDA was 49.2% in women recruited post-MDA and 17.9% (5/28) in those who tested M. genitalium positive. Of samples sequenced (21/28 pre-MDA, 22/28 post-MDA), all showed a macrolide susceptible genotype. Strain typing showed that sequence types diverged into two lineages, with a suggestion of strain replacement post-MDA. CONCLUSION: A single round of azithromycin MDA in an island population with high baseline M. genitalium prevalence did not appear to impact on either prevalence or azithromycin resistance, in contrast to reported decreased genital CT prevalence in the same population. This may be due to limitations such as sample size, including CT-negative samples only, and low MDA coverage. Further investigation of the impact of multiple rounds of MDA on M. genitalium azithromycin AMR in antibiotic experienced and naïve populations is warranted.

Ark shell Scapharca broughtonii hemocyte response against Vibrio anguillarum challenge.

Scapharca broughtonii is one of the most important Arcidae aquaculture species in the Asia-Pacific region. We aimed to investigate the immune responses of hemocytes from ark shell S. broughtonii hemolymph against pathogens. Hemocyte ultrastructure and immunological activity in response to Vibrio anguillarum challenge were observed by scanning and transmission electron microscopy. Before ultrastructure observation, we used the API ZYM semi-quantitative kit to evaluate the levels of hydrolytic enzymes in the plasma and hemocytes following V. anguillarum infection. An enzyme-linked immunosorbent assay kit was used to investigate the variation in the lysozyme activity and hemocytes following bacterial infection. The results showed that hemocytes were the main defense cells against bacterial infection, whereas plasma played a role in the transport and support of hemocytes. It was presumed that an important function of lysozymes and hydrolytic enzymes in lysosomes was for bacterial digestion. Three major types of hemocytes were observed, namely, red blood cells (RBCs), white blood cells (WBCs), and thrombocytes (TCs). Scanning electron microscopy showed that the normal RBCs appeared pie-shaped with 10 µm diameter and 4 µm central thickness, whereas WBCs were spherical in shape with varying sizes, 4-8 µm diameter, and included small lymphocytes. TCs were long, spindle-shaped, and 12-20 µm in length. The cell membrane surface was smooth and even for all cells before pathogen challenge. Under transmission electron microscopy, RBCs displayed a limited ability to devour and digest bacteria adherent to the cell surface following infection. Many hemoglobin particles were observed in the RBC cytoplasm. WBCs were very active against bacterial invasion and showed a strong ability to digest and decompose infected and wrapped V. anguillarum through phagocytosis and lysosome fusion. Digestive vacuoles rapidly became transparent and were thought to contain increasing quantities of pathogen-induced lysozymes. WBCs that devoured pathogenic bacteria were prone to deformation as well as adhesion to each other. TCs were rich in endoplasmic reticulum (ER) content in their cytoplasm and were widely connected in a net-shaped structure. Mitochondria in TCs formed clusters upon invasion of V. anguillarum in the hemolymph. TCs disintegrated to release the ER into the plasma to form a mesh that facilitated clotting. The ability of circulating hemocytes to quickly modify their morphologies and stainability suggests that S. broughtonii is endowed with highly dynamic hemocyte populations capable of coping with environmental changes and rapidly growing pathogens.

Differences between fast and slow muscles in scallops revealed through proteomics and transcriptomics.

BACKGROUND: Scallops possess striated and catch adductor muscles, which have different structure and contractile properties. The striated muscle contracts very quickly for swimming, whereas the smooth catch muscle can keep the shells closed for long periods with little expenditure of energy. In this study, we performed proteomic and transcriptomic analyses of differences between the striated (fast) and catch (slow) adductor muscles in Yesso scallop Patinopecten yessoensis. RESULTS: Transcriptomic analysis reveals 1316 upregulated and 8239 downregulated genes in slow compared to fast adductor muscle. For the same comparison, iTRAQ-based proteomics reveals 474 differentially expressed proteins (DEPs), 198 up- and 276 downregulated. These DEPs mainly comprise muscle-specific proteins of the sarcoplasmic reticulum, extracellular matrix, and metabolic pathways. A group of conventional muscle proteins-myosin heavy chain, myosin regulatory light chain, myosin essential light chain, and troponin-are enriched in fast muscle. In contrast, paramyosin, twitchin, and catchin are preferentially expressed in slow muscle. The association analysis of proteomic and transcriptomic data provides the evidences of regulatory events at the transcriptional and posttranscriptional levels in fast and slow muscles. Among 1236 differentially expressed unigenes, 22.7% show a similar regulation of mRNA levels and protein abundances. In contrast, more unigenes (53.2%) exhibit striking differences between gene expression and protein abundances in the two muscles, which indicates the existence of fiber-type specific, posttranscriptional regulatory events in most of myofibrillar proteins, such as myosin heavy chain, titin, troponin, and twitchin. CONCLUSIONS: This first, global view of protein and mRNA expression levels in scallop fast and slow muscles reveal that regulatory mechanisms at the transcriptional and posttranscriptional levels are essential in the maintenance of muscle structure and function. The existence of fiber-type specific, posttranscriptional regulatory mechanisms in myofibrillar proteins will greatly improve our understanding of the molecular basis of muscle contraction and its regulation in non-model invertebrates.

Whole exome sequencing identifies lncRNA GAS8-AS1 and LPAR4 as novel papillary thyroid carcinoma driver alternations.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer. However, we know little of mutational spectrum in the Chinese population. Thus, here we report the identification of somatic mutations for Chinese PTC using 402 tumor-normal pairs (Discovery: 91 pairs via exome sequencing; validation: 311 pairs via Sanger sequencing). We observed three distinct mutational signatures, evidently different from the two mutational signatures among Caucasian PTCs. Ten significantly mutated genes were identified, most previously uncharacterized. Notably, we found that long non-coding RNA (lncRNA) GAS8-AS1 is the secondary most frequently altered gene and acts as a novel tumor suppressor in PTC. As a mutation hotspot, the c.713A>G/714T>C dinucleotide substitution was found among 89.1% patients with GAS8-AS1 mutations and associated with advanced PTC disease (P = 0.009). Interestingly, the wild-type lncRNA GAS8-AS1 (A713T714) showed consistently higher capability to inhibit cancer cell growth compared to the mutated lncRNA (G713C714). Further studies also elucidated the oncogene nature of the G protein-coupled receptor LPAR4 and its c.872T>G (p.Ile291Ser) mutation in PTC malignant transformation. The BRAF c.1799T>A (p.Val600Glu) substitution was present in 59.0% Chinese PTCs, more frequently observed in patients with lymph node metastasis (P = 1.6 × 10(-4)). Together our study defines a exome mutational spectrum of PTC in the Chinese population and highlights lncRNA GAS8-AS1 and LPAR4 as potential diagnostics and therapeutic targets.

Molecular cloning, expression and biochemical characterization of hemoglobin gene from ark shell Scapharca broughtonii.

Hemoglobin, the main component of haemolymph, is widely distributed in animals. Although its important oxygen transport functions has been extensively reported, studies on the immune function of hemoglobin in mollusc are few. Research on immune of hemoglobin of ark shell Scapharca broughtonii attracted more and more attention due to its ownership of erythrocyte comparing with many other shellfish. In this study, the hemoglobin cDNA of S. broughtonii was cloned by EST and RACE methods (named as SbHb). Sequence analysis revealed that the cDNA was 946 bp in length, including an open reading frame (ORF) of 459 bp which encoded a polypeptide of 152 amino acid residues, and a 5'-untranslated region (UTR) of 313 bp, a 3'-UTR of 174 bp. Sequence and homology analysis showed that the SbHb shared similarity with that of other related species. The mRNA expression profiles of SbHb in tested tissues analyzed by quantitative real-time PCR (qRT-PCR) revealed that the mRNA of SbHb could be all detected in foot, gill, mantle, adductor muscle, haemocytes and hepatopancreas, and the highest level was found in the haemocytes, which is 163.2 times higher than that in adductor muscle. Vibrio anguillarum stimulation and hypoxia treatment both had significant impact on the expression of SbHb, which showed the same trends as increasing first to the highest at 16 h after treatment and then followed by declining. Recombinant protein of SbHb (rSbHb) was successfully obtained by prokaryotic expression, and further function analysis indicated obviously that the rSbHb had very strong phenoloxidase-like activity (PO-like activity) and it could remarkably inhibit growth of gram-negative bacteria V. anguillarum. All the data suggested that the SbHb plays a significant role in the process of antibacterial and anoxia tolerance reaction in S. broughtonii, providing the evidence that SbHb is a key immune factor.

MiRNA-192 [corrected] and miRNA-204 Directly Suppress lncRNA HOTTIP and Interrupt GLS1-Mediated Glutaminolysis in Hepatocellular Carcinoma.

Accumulated evidence demonstrated that long non-coding RNAs (lncRNAs) play a pivotal role in tumorigenesis. However, it is still largely unknown how these lncRNAs were regulated by small ncRNAs, such as microRNAs (miRNAs), at the post-transcriptional level. We here use lncRNA HOTTIP as an example to study how miRNAs impact lncRNAs expression and its biological significance in hepatocellular carcinoma (HCC). LncRNA HOTTIP is a vital oncogene in HCC, one of the deadliest cancers worldwide. In the current study, we identified miR-192 and miR-204 as two microRNAs (miRNAs) suppressing HOTTIP expression via the Argonaute 2 (AGO2)-mediated RNA interference (RNAi) pathway in HCC. Interaction between miR-192 or miR-204 and HOTTIP were further confirmed using dual luciferase reporter gene assays. Consistent with this notion, a significant negative correlation between these miRNAs and HOTTIP exists in HCC tissue specimens. Interestingly, the dysregulation of the three ncRNAs was associated with overall survival of HCC patients. In addition, the posttranscriptional silencing of HOTTIP by miR-192, miR-204 or HOTTIP siRNAs could significantly suppress viability of HCC cells. On the contrary, antagonizing endogenous miR-192 or miR-204 led to increased HOTTIP expression and stimulated cell proliferation. In vivo mouse xenograft model also support the tumor suppressor role of both miRNAs. Besides the known targets (multiple 5' end HOX A genes, i.e. HOXA13), glutaminase (GLS1) was identified as a potential downstream target of the miR-192/-204-HOTTIP axis in HCC. Considering glutaminolysis as a crucial hallmark of cancer cells and significantly inhibited cell viability after silencingGLS1, we speculate that the miR-192/-204-HOTTIP axis may interrupt HCC glutaminolysis through GLS1 inhibition. These results elucidate that the miR-192/-204-HOTTIP axis might be an important molecular pathway during hepatic cell tumorigenesis. Our data in clinical HCC samples highlight miR-192, miR-204 and HOTTIP with prognostic and potentially therapeutic implications.

Evaluation of the Clinical and Microbiological Response to Salmonella Paratyphi A Infection in the First Paratyphoid Human Challenge Model.

Background: To expedite the evaluation of vaccines against paratyphoid fever, we aimed to develop the first human challenge model of Salmonella enterica serovar Paratyphi A infection. Methods: Two groups of 20 participants underwent oral challenge with S. Paratyphi A following sodium bicarbonate pretreatment at 1 of 2 dose levels (group 1: 1-5 × 103 colony-forming units [CFU] and group 2: 0.5-1 × 103 CFU). Participants were monitored in an outpatient setting with daily clinical review and collection of blood and stool cultures. Antibiotic treatment was started when prespecified diagnostic criteria were met (temperature ≥38°C for ≥12 hours and/or bacteremia) or at day 14 postchallenge. Results: The primary study objective was achieved following challenge with 1-5 × 103 CFU (group 1), which resulted in an attack rate of 12 of 20 (60%). Compared with typhoid challenge, paratyphoid was notable for high rates of subclinical bacteremia (at this dose, 11/20 [55%]). Despite limited symptoms, bacteremia persisted for up to 96 hours after antibiotic treatment (median duration of bacteremia, 53 hours [interquartile range, 24-85 hours]). Shedding of S. Paratyphi A in stool typically preceded onset of bacteremia. Conclusions: Challenge with S. Paratyphi A at a dose of 1-5 × 103 CFU was well tolerated and associated with an acceptable safety profile. The frequency and persistence of bacteremia in the absence of clinical symptoms was notable, and markedly different from that seen in previous typhoid challenge studies. We conclude that the paratyphoid challenge model is suitable for the assessment of vaccine efficacy using endpoints that include bacteremia and/or symptomatology. Clinical Trials Registration: NCT02100397.

Changes in hemolymph characteristics of ark shell Scapharaca broughtonii dealt with Vibrio anguillarum challenge in vivo and various of anticoagulants in vitro.

The ark shell Scapharca broughtonii is a commercially important shellfish in China. Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS) are common anticoagulants used for shellfish blood. To observe the immune response mediated by its hemocytes, we challenged in vivo S. broughtonii hemolymph with Vibrio anguillarum and dealt with the following three anticoagulants in vitro: Alserver's solution (AS), modified Alserver's solution (MAS) and Heparin sodium solution (HSS). The methodologies we used were immunostimulation with V. anguillarum, Wright-Giemsa staining, micro-examination, and flow cytometric and hydrolyzing enzyme activity analysis. The results showed that all three types of anticoagulants effectively prevented blood clotting in ark shellfish. The morphology of hemocytes did not significantly change 30 h after anticoagulant treatment, except for the shrinking of hemocytes after administering HSS. The size and permeability of hemocytes changed when treated with the anticoagulants and when stimulated with V. anguillarum. Both alkaline phosphatase (AKP) and acid phosphatase (ACP) in hemocytes and Plasma were measured at different times after they were stimulated with V. anguillarum in HSS and MAS. The AKP enzymatic activity in HSS was somewhat higher than in the MAS anticoagulant, but changes in response to V. anguillarum challenge of enzymatic activity were almost the same in HSS and MAS groups. In conclusion, all three types of anticoagulants may be used for ark shell blood preservation. They all changed the cell-surface characteristics of hemocytes to inhibit clot formation. The AS anticoagulant was appropriate for maintaining white and red cell shapes, while MAS was ideal for retaining throbus cell function. Lastly, HSS was appropriate for maintaining enzymatic activity in hemolymph and function of hemocytes. Following this investigation, we gained insight into the changes in hemolymph characteristic during immune response.