Selection and evaluation of quality markers (Q-markers) of vladimiriae radix extract for cholestatic liver injury based on spectrum-effect relationship, pharmacokinetics, and molecular docking.

ETHNOPHARMACOLOGICAL RELEVANCE: As a representative local medicinal herb produced in China, Vladimiriae Radix (VR) has been proven to exert hepatoprotective and choleretic effects, with particular therapeutic efficacy in cholestatic liver injury (CLI), as demonstrated by the VR extract (VRE). However, the quality markers (Q-markers) of VRE for the treatment of CLI remain unclear. AIM OF THE STUDY: A new strategy based on the core element of "efficacy" was proposed, using a combination of spectrum-effect relationship, pharmacokinetics, and molecular docking methods to select and confirm Q-markers of VRE. MATERIAL AND METHODS: First, the HPLC fingerprinting of 10 batches of VRE was studied, and the in vivo pharmacological index of anti-CLI in rats was determined. The spectrum-effect relationship was utilized as a screening method to identify the Q-markers of VRE. Secondly, Q-markers were used as VRE pharmacokinetic markers to measure their concentrations in normal and CLI rat plasma, and to analyze their disposition. Finally, molecular docking was utilized to predict the potential interaction between the identified Q-markers and crucial targets of CLI. RESULTS: The fingerprints of 10 batches of VRE was established. The in vivo pharmacological evaluation of rats showed that VRE had a significant therapeutic effect on CLI. The spectrum-effect correlation analysis showed that costunolide (COS) and dehydrocostus lactone (DEH) were the Q-markers of VRE anti-CLI. The pharmacokinetic results showed that AUC(0-t), Cmax, CLZ/F, and VZ/F of COS and DEH in CLI rats had significant differences (P < 0.01). They were effectively absorbed into the blood plasma of CLI rats, ensuring ideal bioavailability, and confirming their role as Q-markers. Molecular docking results showed that COS, DEH had good affinity with key targets (FXR, CAR, PXR, MAPK, TGR5, NRF2) for CLI treatment (Binding energy < -4.52 kcal mol-1), further verifying the correctness of Q-marker selection. CONCLUSIONS: In this study, through the combination of experimental and theoretical approaches from the aspects of pharmacodynamic expression, in vivo process rules, and interaction force prediction, the therapeutic effect of VRE and Q-markers (COS、DEH) were elucidated. Furthermore, a new idea based on the principle of "efficacy" was successfully proposed for screening and evaluating Q-markers.

Emergence of mcr-8.2-harboring hypervirulent ST412 Klebsiella pneumoniae strain from pediatric sepsis: A comparative genomic survey.

Emerging mobile colistin resistance (mcr) genes pose a significant threat to public health for colistin was used as the last resort to treat multidrug-resistant (MDR) pathogenic bacterial infections. Hypervirulent Klebsiella pneumoniae (hvKP) is a clinically significant pathogen resulting in highly invasive infections, often complicated by devastating dissemination. Worryingly, the untreatable and severe infections caused by mcr-harbouring hvKP leave the selection of antibiotics for clinical anti-infective treatment in a dilemma. Herein, we screened 3,461 isolates from a tertiary teaching hospital from November 2018 to March 2021, and an mcr-8.2-harbouring hvKP FAHZZU2591 with a conjugative plasmid was identified from paediatric sepsis. This is the first report of MCR-8-producing hvKP from paediatric sepsis to our best knowledge. The susceptibility, genetic features, and plasmid profiles of the isolate were investigated. Further, we assessed the virulence potential of FAHZZU2591 and verified its pathogenicity and invasive capacity using a mouse model. The phylogenetic analysis of mcr-8-bearing K. pneumoniae revealed that China is the predominant reservoir of the mcr-8 gene, and the clinic is the primary source. Our work highlights the risk for the spread of mcr-positive hvKP in clinical, especially in paediatric sepsis, and the persistent surveillance of colistin-resistance hvKP is urgent.

Establishment of three heterogeneous subtypes and a risk model of low-grade gliomas based on cell senescence-related genes.

Background: Cellular senescence is a key element in the occurrence and progression of a variety of tumors. As a result, cellular senescence-related markers can be categorized based on the prognosis status of patients. Due to the heterogeneity and the complexity of the tumor microenvironment (TME), the long-term effectiveness of low-grade glioma (LGG) treatment remains a clinical challenge. Consequently, developing and refining effective treatment approaches to aid with LGG management is critical. Methods: Based on the expressions of cell senescence-related genes (CSRGs) acquired from the cellAge database, consensus clustering was utilized to identify stable molecular subtypes. Clinical features, immune infiltration, route modifications, and genetic changes of various subtypes were also assessed. Following that, the least absolute shrinkage and selection operator (LASSO) regression and univariate Cox regression analysis were used for developing the cell senescence-related risk score (CSRS) model. Finally, a correlation study of the CSRS model with molecular, immunological, and immunotherapy parameters was performed. Results: C1, C2, and C3, are the three senescence-related subtypes that were identified. Patients belonging to the C1 subtype had poor prognoses and a substantial proportion of them was in the grade G3. The differentially expressed genes (DEGs) among the three subtypes were used to develop the CSRS model. In both the training and independent validation cohort, the model had a high area under the receiver operating characteristic (ROC) curve in predicting the overall survival (OS) of patients. As a result, this model can predict clinical features and responses to immunotherapy in a variety of patients and it is a potential independent prognostic factor for LGG. Conclusion: This research discovered three LGG subtypes related to cell senescence and created a CSRS model for six genes. Cell senescence was highly associated with unfavorable prognosis in LGG. The CSRS model can be used to predict the prognosis of patients and identify patients who would benefit from immunotherapy.

Emergence of Neonatal Sepsis Caused by MCR-9- and NDM-1-Co-Producing Enterobacter hormaechei in China.

Mobile colistin resistance (mcr) genes represent an emerging threat to public health. Reports on the prevalence, antimicrobial profiles, and clonality of MCR-9-producing Enterobacter cloacae complex (ECC) isolates on a national scale in China are limited. We screened 3,373 samples from humans, animals, and the environment and identified eleven MCR-9-positive ECC isolates. We further investigated their susceptibility, epidemiology, plasmid profiles, genetic features, and virulence potential. Ten strains were isolated from severe bloodstream infection cases, especially three of them were recovered from neonatal sepsis. Enterobacter hormaechei was the most predominant species among the MCR-9-producing ECC population. Moreover, the co-existence of MCR-9, CTX-M, and SHV-12 encoding genes in MCR-9-positive isolates was globally observed. Notably, mcr-9 was mainly carried by IncHI2 plasmids, and we found a novel ~187 kb IncFII plasmid harboring mcr-9, with low similarity with known plasmids. In summary, our study presented genomic insights into genetic characteristics of MCR-9-producing ECC isolates retrieved from human, animal, and environment samples with one health perspective. This study is the first to reveal NDM-1- and MCR-9-co-producing ECC from neonatal sepsis in China. Our data highlights the risk for the hidden spread of the mcr-9 colistin resistance gene.

Early age at menarche and risk of postpartum hemorrhage: a retrospective study in Chinese women.

OBJECTIVE: The objective of this study is to investigate whether early age at menarche is associated with increased risk of postpartum hemorrhage among Chinese women. MATERIALS AND METHODS: Clinical data from 6,383 Chinese women who gave birth to live singleton infants at The First Affiliated Hospital of Chengdu Medical College between October 2016 and October 2019 were extracted from the electronic medical records system. Patients were categorized into four groups according to their age at menarche (≤12, 13, 14 and ≥15 years). Logistic regression analysis was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for postpartum hemorrhage for the different menarche age groups. RESULTS: After controlling for potential confounders, women with menarche at an early age (≤12 years) had a significantly higher risk of developing postpartum hemorrhage than women with an age at menarche of 13 years, and the ORs (95% CIs) for postpartum hemorrhage across the menarche age categories (≤12, 13, 14 and ≥15 years) were 1.27 (1.02-1.81), 1.00 (reference), 0.95 (0.61-1.57), and 0.91 (0.51-1.58), respectively. Moreover, age at menarche was inversely associated with the risk of postpartum hemorrhage after adjustment for all relevant confounding factors, and the OR (95% CI) for postpartum hemorrhage per year of increasing in the age at menarche was 0.93 (0.74-0.99). CONCLUSION: Early age at menarche was associated with a significantly increased risk of postpartum hemorrhage after adjustment for known confounding factors. This finding could help obstetricians and midwives to identify pregnant women at higher risk of developing postpartum hemorrhage, and allow early preventative strategies to be implemented.

Comparative genomic characterization of multidrug-resistant Citrobacter spp. strains in Fennec fox imported to China.

BACKGROUND: To investigate the antimicrobial profiles and genomic characteristics of MDR-Citrobacter spp. strains isolated from Fennec fox imported from Sudan to China. METHODS: Four Citrobacter spp. strains were isolated from stool samples. Individual fresh stool samples were collected and subsequently diluted in phosphate buffered saline as described previously. The diluted fecal samples were plated on MacConkey agar supplemented with 1 mg/l cefotaxime and incubated for 20 h at 37 °C. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was used for identification. Antimicrobial susceptibility testing was performed using the broth microdilution method. Whole-genome sequencing was performed on an Illumina Novaseq-6000 platform. Acquired antimicrobial resistance genes and plasmid replicons were detected using ResFinder 4.1 and PlasmidFinder 1.3, respectively. Comparative genomic analysis of 277 Citrobacter genomes was also performed. RESULTS: Isolate FF141 was identified as Citrobacter cronae while isolate FF371, isolate FF414, and isolate FF423 were identified as Citrobacter braakii. Of these, three C. braakii isolates were further confirmed to be extended-spectrum ß-lactamases (ESBL)-producer. All isolates are all multidrug resistance (MDR) with resistance to multiple antimicrobials. Plasmid of pKPC-CAV1321 belong to incompatibility (Inc) group. Comparative genomics analysis of Citrobacter isolates generated a large core-genome. Genetic diversity was observed in our bacterial collection, which clustered into five main clades. Human, environmental and animal Citrobacter isolates were distributed into five clusters. CONCLUSIONS: To our knowledge, this is the first investigation of MDR-Citrobacter from Fennec Fox. Our phenotypic and genomic data further underscore the threat of increased ESBL prevalence in wildlife and emphasize that increased effort should be committed to monitoring the potentially rapid dissemination of ESBL-producers with one health perspective.

Comparative transcriptome among Euscaphis konishii Hayata tissues and analysis of genes involved in flavonoid biosynthesis and accumulation.

BACHGROUND: Euscaphis konishii Hayata, a member of the Staphyleaceae Family, is a plant that has been widely used in Traditional Chinese Medicine and it has been the source for several types of flavonoids. To identify candidate genes involved in flavonoid biosynthesis and accumulation, we analyzed transcriptome data from three E. konishii tissues (leaf, branch and capsule) using Illumina Hiseq 2000 platform. RESULTS: A total of 91.7, 100.3 and 100.1million clean reads were acquired for the leaf, branch and capsule, respectively; and 85,342 unigenes with a mean length of 893.60 bp and N50 length of 1307 nt were assembled using Trinity program. BLASTx analysis allowed to annotate 40,218 unigenes using public protein databases, including NR, KOG/COG/eggNOG, Swiss-Prot, KEGG and GO. A total of 14,291 (16.75%) unigenes were assigned to 128 KEGG pathways, and 900 unigenes were annotated into 22 KEGG secondary metabolites, including flavonoid biosynthesis. The structure enzymes involved in flavonoid biosynthesis, such as phenylalanine ammonia lyase, cinnamate 4-hydroxylase, 4-coumarate CoA ligase, shikimate O-hydroxycinnamoyltransferase, coumaroylquinate 3'-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, chalcone isomerase, flavanone 3-hydroxylase, flavonoid 3'-hydroxylase, flavonoid 3',5'-hydroxylase, flavonolsynthese, dihydroflavonol 4-reductase, anthocyanidinreductase, leucoanthocyanidin dioxygenase, leucoanthocyanidin reductase, were identified in the transcriptome data, 40 UDP-glycosyltransferase (UGT), 122 Cytochrome P450 (CYP) and 25 O-methyltransferase (OMT) unigenes were also found. A total of 295 unigenes involved in flavonoid transport and 220 transcription factors (97 MYB, 84 bHLH and 39 WD40) were identified. Furthermore, their expression patterns among different tissues were analyzed by DESeq, the differentially expressed genes may play important roles in tissues-specific synthesis, accumulation and modification of flavonoids. CONCLUSION: We present here the de novo transcriptome analysis of E. konishii and the identification of candidate genes involved in biosynthesis and accumulation of flavonoid. In general, these results are an important resource for further research on gene expression, genomic and functional genomics in E. konishii and other related species.

Selection and evaluation of reference genes for qRT-PCR analysis in Euscaphis konishii Hayata based on transcriptome data.

BACKGROUND: Quantitative real-time reverse transcription-polymerase chain reaction has been widely used in gene expression analysis, however, to have reliable and accurate results, reference genes are necessary to normalize gene expression under different experimental conditions. Several reliable reference genes have been reported in plants of Traditional Chinese Medicine, but none have been identified for Euscaphis konishii Hayata. RESULTS: In this study, 12 candidate reference genes, including 3 common housekeeping genes and 9 novel genes based on E. konishii Hayata transcriptome data were selected and analyzed in different tissues (root, branch, leaf, capsule and seed), capsule and seed development stages. Expression stability was calculated using geNorm and NormFinder, the minimal number of reference genes required for accurate normalization was calculated by Vn/Vn + 1 using geNorm. EkEEF-5A-1 and EkADF2 were the two most stable reference genes for all samples, while EkGSTU1 and EkGAPDH were the most stable reference genes for tissue samples. For seed development stages, EkGAPDH and EkEEF-5A-1 were the most stable genes, whereas EkGSTU1 and EkGAPDH were identified as the two most stable genes in the capsule development stages. Two reference genes were sufficient to normalize gene expression across all sample sets. CONCLUSION: Results of this study revealed that suitable reference genes should be selected for different experimental samples, and not all the common reference genes are suitable for different tissue samples and/or experimental conditions. In this study, we present the first data of reference genes selection for E. konishii Hayata based on transcriptome data, our data will facilitate further studies in molecular biology and gene function on E. konishii Hayata and other closely related species.

Metallothionein 1 family profiling identifies MT1X as a tumor suppressor involved in the progression and metastastatic capacity of hepatocellular carcinoma.

Metallothionein 1 (MT1s) is a family of cysteine-rich proteins with diverse functions such as metal homeostasis, oxidative stress, and carcinogenesis. However, its involvement in hepatocellular carcinoma (HCC) remains not fully understood. We aimed to explore the contribution of the individual member of MT1s to HCC. Its member mRNA levels were determined in cohort 1 of normal (n = 30), cirrhotic (n = 30), peritumoral (n = 135), and HCC (n = 135). In cohort 1, seven of eight members were down-regulated during the transition from normal liver to HCC, and only MT1G and MT1X were correlated with tumor features and outcomes. The MT1X was selected to be further stained in cohort 2 consisting of a series of liver nodules (15 normal livers, 33 cirrhotic livers, 12 dysplastic nodules, 31 HCC, and 9 HCC metastasis), and in cohort 3 (HCC, n = 85). In cohort 2, MT1X immunoreactivity was reduced in HCC and lost in metastatic HCC and showed good diagnostic performance for HCC (AUC = 0.754, 95%IC = 0.659-0.849). In cohort 3, MT1X expression in peritumoral tissues was independent predictor for HCC (recurrence free survival: HR = 0.34, 95%CI = 0.17-0.66; overall survival: HR = 0.32, 95%CI = 0.16-0.60). Moreover, we found that ectopic overexpression of MT1X delayed G1/S progression of cell cycle and promoted apoptosis in HCC cells in vitro, and suppressed tumor growth and lung metastasis in nude mice in vivo. We further demonstrated that MT1X induces cell cycle arrest and apoptosis by inactivating NF-κB signaling in HCC. In conclusion, MT1X may serve as a candidate of prognostic indicator and inhibits the progression and metastasis of HCC.

Whole genome sequence of an MCR-1-carrying, extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli ST746 isolate recovered from a community-acquired urinary tract infection.

OBJECTIVES: Colistin is regarded as one of the last-resort antimicrobials for severe infections. Isolates carrying the plasmid-borne mobile colistin resistance gene mcr-1 were rarely reported in community-acquired infections. Here we report the draft genome sequence of an MCR-1-carrying, extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli isolate from community-acquired urinary tract infection. METHODS: Antimicrobial susceptibility testing (AST) was performed by the broth microdilution method. Transferability of the mcr-bearing plasmid was determined by filter mating using E. coli EC600 as recipient strain. Multilocus sequence typing (MLST) was undertaken using the E. coli MLST database. The draft genome sequence of isolate LX13 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SOAPdenovo. Acquired antimicrobial resistance genes were identified using ResFinder 2.1. RESULTS: AST showed that LX13 was resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefazolin, cefepime and polymyxins. MLST showed that isolate LX13 belongs to ST746. The MCR-1-producing plasmid was conjugative and conferred increased resistance to colistin the transconjugant. The draft genome of E. coli LX13 was 4914035bp in size. In silico analysis revealed the presence of eight putative acquired resistance genes, including blaCTX-M-14, blaTEM-1B, aadA5, mcr-1, dfrA17, sul2, tet34 and tetA. plasmidSPAdes revealed that the mcr-1 gene was harboured by a plasmid of replicon type IncI2. CONCLUSIONS: This study highlights the potential risk of spread of MCR-1-carrying, ESBL-producing E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant E. coli.

Profiling, clinicopathological correlation and functional validation of specific long non-coding RNAs for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent and aggressive malignancies worldwide. Studies seeking to advance the overall understanding of lncRNA profiling in HCC remain rare. METHODS: The transcriptomic profiling of 12 HCC tissues and paired adjacent normal tissues was determined using high-throughput RNA sequencing. Fifty differentially expressed mRNAs (DEGs) and lncRNAs (DELs) were validated in 21 paired HCC tissues via quantitative real-time PCR. The correlation between the expression of DELs and various clinicopathological characteristics was analyzed using Student's t-test or linear regression. Co-expression networks between DEGs and DELs were constructed through Pearson correlation co-efficient and enrichment analysis. Validation of DELs' functions including proliferation and migration was performed via loss-of-function RNAi assays. RESULTS: In this study, we identified 439 DEGs and 214 DELs, respectively, in HCC. Furthermore, we revealed that multiple DELs, including NONHSAT003823, NONHSAT056213, NONHSAT015386 and especially NONHSAT122051, were remarkably correlated with tumor cell differentiation, portal vein tumor thrombosis, and serum or tissue alpha fetoprotein levels. In addition, the co-expression network analysis between DEGs and DELs showed that DELs were involved with metabolic, cell cycle, chemical carcinogenesis, and complement and coagulation cascade-related pathways. The silencing of the endogenous level of NONHSAT122051 or NONHSAT003826 could significantly attenuate the mobility of both SK-HEP-1 and SMMC-7721 HCC cells. CONCLUSION: These findings not only add knowledge to the understanding of genome-wide transcriptional evaluation of HCC but also provide promising targets for the future diagnosis and treatment of HCC.

Identification of Specific Long Non-Coding RNA Expression: Profile and Analysis of Association with Clinicopathologic Characteristics and BRAF Mutation in Papillary Thyroid Cancer.

BACKGROUND: In recent years, long non-coding RNAs (lncRNAs) have been shown to play a critical regulatory role in cancer biology. However, the contribution of lncRNAs to papillary thyroid cancer (PTC) remains largely unknown. METHODS: RNA sequencing and quantitative reverse transcription polymerase chain reaction were used to detect and verify changes to the transcriptome profile in 12 PTC tissues compared to paired normal adjacent tissues. The statistical correlation between differentially expressed lncRNAs and clinicopathologic characteristics was analyzed, and potential lncRNA functions were predicted by examining annotations for the co-expressed mRNAs. Furthermore, the specific subgroup patterns of the PTC transcriptome remodeled by BRAF mutations were also analyzed. RESULTS: A total of 188 lncRNAs and 505 mRNAs were differentially expressed in 50% or more of the PTC tissues (fold change >2; p < 0.05) as assessed by RNA-sequencing compared with paired normal adjacent tissues. Forty-seven lncRNAs and 39 mRNAs were verified in 31 pairs of PTC specimens using quantitative reverse transcription polymerase chain reaction, and the results were consistent with the RNA sequencing data. The lncRNAs NONHSAT076747 and NONHSAT122730 were associated with lymph node metastasis, and NONHSAG051968 expression was negatively correlated with tumor size. A co-expression network between differentially expressed lncRNAs and protein-coding RNAs was constructed and analyzed, and functional analysis suggested that the differentially expressed genes mainly participate in ECM-receptor interactions and the focal adhesion pathway. Furthermore, a specific PTC transcriptome subtype pattern stratified by BRAF mutation was also uncovered. The p53 signaling pathway was the most highly enriched pathway among the BRAF mutation-related genes. CONCLUSIONS: This study reveals specific changes to the lncRNA profile associated with PTC, and provides new insight into its pathogenesis. The PTC-associated lncRNAs NONHSAG051968, NONHSAT076747, and NONHSAT122730 might be potential diagnostic and therapeutic targets for PTC patients, and lncRNAs with subtype-specific expression stratified by BRAF mutation might be significant in individual molecular subtypes.

Influence of H7N9 virus infection and associated treatment on human gut microbiota.

Between March and June, 2013, forty H7N9 patients were hospitalized in our hospital. Next-generation sequencing technologies have been used to sequence the fecal DNA samples of the patient, the within sample diversity analysis, enterotyping, functional gene and metagenomic species analysis have been carried on both the patients and healthy controls. The influence of associated treatment in H7N9 infected patients is dramatic and was firstly revealed in species level due to deep sequencing technology. We found that most of the MetaGenomic Species (MGS) enriched in the control samples were Roseburia inulinivorans DSM 16841, butyrate producing bacterium SS3/4 and most of MGS enriched in the H7N9 patients were Clostridium sp. 7 2 43FAA and Enterococcus faecium. It was concluded that H7N9 viral infection and antibiotic administration have a significant effect on the microbiota community with decreased diversity and overgrowth of the bacteria such as Escherichia coli and Enterococcus faecium. Enterotype analysis showed that the communities were unstable. Treatment including antivirals, probiotics and antibiotics helps to improve the microbiota diversity and the abundance of beneficial bacteria in the gut.

MicroRNA let-7c Inhibits Cell Proliferation and Induces Cell Cycle Arrest by Targeting CDC25A in Human Hepatocellular Carcinoma.

Down-regulation of the microRNA let-7c plays an important role in the pathogenesis of human hepatocellular carcinoma (HCC). The aim of the present study was to determine whether the cell cycle regulator CDC25A is involved in the antitumor effect of let-7c in HCC. The expression levels of let-7c in HCC cell lines were examined by quantitative real-time PCR, and a let-7c agomir was transfected into HCC cells to overexpress let-7c. The effects of let-7c on HCC proliferation, apoptosis and cell cycle were analyzed. The in vivo tumor-inhibitory efficacy of let-7c was evaluated in a xenograft mouse model of HCC. Luciferase reporter assays and western blotting were conducted to identify the targets of let-7c and to determine the effects of let-7c on CDC25A, CyclinD1, CDK6, pRb and E2F2 expression. The results showed that the expression levels of let-7c were significantly decreased in HCC cell lines. Overexpression of let-7c repressed cell growth, induced cell apoptosis, led to G1 cell cycle arrest in vitro, and suppressed tumor growth in a HepG2 xenograft model in vivo. The luciferase reporter assay showed that CDC25A was a direct target of let-7c, and that let-7c inhibited the expression of CDC25A protein by directly targeting its 3' UTR. Restoration of CDC25A induced a let-7c-mediated G1-to-S phase transition. Western blot analysis demonstrated that overexpression of let-7c decreased CyclinD1, CDK6, pRb and E2F2 protein levels. In conclusion, this study indicates that let-7c suppresses HCC progression, possibly by directly targeting the cell cycle regulator CDC25A and indirectly affecting its downstream target molecules. Let-7c may therefore be an effective therapeutic target for HCC.

MicroRNA let-7g and let-7i inhibit hepatoma cell growth concurrently via downregulation of the anti-apoptotic protein B-cell lymphoma-extra large.

Let-7 family members have been identified as tumor-suppressing microRNAs, which are important in human hepatocellular carcinoma (HCC). These family members may function differently as a result of different base sequences at the 3'end. The aim of this study was to determine the antitumor effects of miR-let-7g/i (let-7g/i) on HCC cells and to investigate whether let-7g and let-7i have a combinatorial effect on HCC. The expression levels of let-7g/i in hepatoma cells were determined by quantitative reverse transcription polymerase chain reaction. In addition, a 5-ethynyl-2'-deoxyuridine retention assay and flow cytometry analysis were used to detect the effect of let-7g/i on the proliferation and apoptosis of BEL-7402 cells, respectively. The expression of anti-apoptotic protein B-cell lymphoma-extra large (Bcl-xL) was analyzed using western blot analysis. The results revealed that the expression levels of let-7g/i were significantly decreased in HCC cell lines when compared with L-02 cells. Furthermore, the overexpression of let-7g/i significantly suppressed DNA replication, inhibited cell proliferation and promoted apoptosis of BEL-7402 hepatoma cells. The expression of the anti-apoptotic protein, Bcl-xL, was inhibited by the combined role of let-7g and let-7i. We hypothesize that let-7g and let-7i exhibit a concurrent effect to regulate cell proliferation and the apoptosis of hepatoma cells, and this function is mediated by the Bcl-xL protein.

Alterations of the human gut microbiome in liver cirrhosis.

Liver cirrhosis occurs as a consequence of many chronic liver diseases that are prevalent worldwide. Here we characterize the gut microbiome in liver cirrhosis by comparing 98 patients and 83 healthy control individuals. We build a reference gene set for the cohort containing 2.69 million genes, 36.1% of which are novel. Quantitative metagenomics reveals 75,245 genes that differ in abundance between the patients and healthy individuals (false discovery rate < 0.0001) and can be grouped into 66 clusters representing cognate bacterial species; 28 are enriched in patients and 38 in control individuals. Most (54%) of the patient-enriched, taxonomically assigned species are of buccal origin, suggesting an invasion of the gut from the mouth in liver cirrhosis. Biomarkers specific to liver cirrhosis at gene and function levels are revealed by a comparison with those for type 2 diabetes and inflammatory bowel disease. On the basis of only 15 biomarkers, a highly accurate patient discrimination index is created and validated on an independent cohort. Thus microbiota-targeted biomarkers may be a powerful tool for diagnosis of different diseases.

MiR-29a modulates the angiogenic properties of human endothelial cells.

Although extensive investigation has been made on miR-29a in relation to malignancies, only a little information has been provided about the angiogenic property of this miRNA so far. Herein, we sought to investigate the role of miR-29a in regulating cell cycle and angiogenic phenotype of endothelial cells. The results showed that miR-29a is highly expressed and upregulated by hypoxia-mimicking reagents in human umbilical vein endothelial cells (HUVEC). Consistent with this preliminary finding, introduction of exogenous agomiR-29a, or Antagomir-29a altered cell cycle progression and promoted, or repressed the proliferation and tube formation of HUVEC, respectively. Furthermore, by using luciferase reporter assay, the expression of HBP1, a suppressor transcription factor was directly regulated by miR-29a through 3'-UTR. Increased or decreased HBP1 protein level was associated with the inhibition or overexpression of miR-29a, respectively. We conclude that miR-29a has a significant role in regulating cell cycle, proliferation and angiogenic properties of HUVEC, and this function is likely mediated through HBP1 protein at the post-transcriptional level. As a novel molecular target, miR-29a may have a potential value for the treatment of angiogenesis-associated diseases such as cardiovascular diseases and cancers.

Peripheral blood mononuclear cells inhibit proliferation and promote apoptosis of HeLa cells following stimulation with Bacillus Calmette-Guerin.

Bacillus Calmette-Guerin (BCG) immunotherapy is established as an effective adjuvant intravesical treatment for non-muscle invasive bladder cancer. BCG is also effective in the treatment of Condylomata acuminata caused by low-risk human papilloma virus (HPV). The aim of this study was to determine the efficacy of BCG for the treatment of cervical cancer or HPV high-risk infections. BCG-activated killer (BAK) cells were incubated with a high-risk HPV18-infected cervical cancer cell line, HeLa. The cell cycle distribution and apoptotic index of the HeLa cells were analyzed by flow cytometry. The alterations of HPV-E7, retinoblastoma (RB) and E2F1 levels were detected at the transcriptional and translational levels. The BAK cell cytotoxicity to HeLa cells was 24.08, 14.74 and 6.8% and the natural killer (NK) cell cytotoxicity was 17.62, 10.78 and 5.8% at the E/T ratios of 40:1, 20:1 and 10:1, respectively. The BAK cells significantly induced the apoptosis of HeLa cells to result in an apoptosis level of 24.2% compared with 13.45% by the NK cell treatment at the ratio of 20:1. BAK cells inhibit the proliferation of HeLa cells by G(1)/S cell cycle arrest and this may be associated with the RB/E2F1 pathway. However, G(1)/S arrest and the alteration of RB protein (pRB) and E2F1 levels in the HeLa cells did not show significant differences between the BAK cell- and NK cell-treated groups. HPV-E7 appeared not to be associated with the alteration in cell cycle progression. This study showed that immunotherapy may be a potential treatment for cervical cancer and that BCG immunotherapy may be an alternative and effective method, but further experiments and clinical trials are required to verify this effect.

Genome sequence of Staphylococcus capitis QN1, which causes infective endocarditis.

Staphylococcus capitis is a subtype of coagulase-negative staphylococci (CoNS) which could emerge as a significant pathogen causing infective endocarditis, prosthetic valve endocarditis, and late-onset sepsis. We isolated S. capitis strain QN1 from the skin swab sample of a female. Here we prepared a genome sequence for this strain consisting of 30 contigs totaling 2,430,101 bases and a GC content of 32.76%.

MicroRNA-122 suppresses cell proliferation and induces cell apoptosis in hepatocellular carcinoma by directly targeting Wnt/ß-catenin pathway.

AIMS: To validate whether the anti-cancer effect of microRNA-122 (miR-122) on hepatocellular carcinoma (HCC) is mediated through regulating Wnt/ß-catenin signalling pathways. METHODS: The expression levels of miR-122 in HCC tissues and varied hepatoma cells were quantified by real-time PCR. MiR-122 agomir was transfected into HepG2, Hep3B cells to over-express miR-122. The effect of over-expression miR-122 on proliferation and apoptosis of HepG2 and Hep3B cells was evaluated using CCK-8 kit and flow cytometer respectively. The 3'-UTR segments of Wnt1 containing the miR-122 binding sites were amplified by PCR and the luciferase activity in the transfected cells was assayed. Wnt1 mRNA level was quantified using RT-PCR. Protein levels of Wnt1, ß-catenin and TCF-4 were detected using Western blotting. RESULTS: In comparison with the expression level of miR-122 in para-cancerous tissues or Chang liver cell, the expression level in HCC tissues or varied hepatoma cells was significantly decreased (P < 0.05). Over-expression of miR-122 significantly inhibited the proliferation (P < 0.05), and promoted the apoptosis of HepG2 and Hep3B cells. Over-expressed miR-122 down-regulated the protein levels of Wnt1, ß-catenin and TCF-4 (P < 0.05). MiR-122 suppressed the luciferase activity of the pmiR-Wnt1-wt by approximately 50% compared with the negative control, while mutation or removal of the miR-122 binding site using siRNA or mir-122 inhibitor blocked the suppressive effect (P < 0.05). CONCLUSIONS: MiR-122 expression is down-regulated in human HCC. Over-expression of miR-122 inhibits HCC cell growth and promotes the cell apoptosis by affecting Wnt/ß-catenin-TCF signalling pathway.