Identification of potential miRNA-mRNA regulatory network contributing to pathogenesis of HBV-related HCC.

BACKGROUND: Hepatitis B virus (HBV) is one of the major risk factors of hepatocellular carcinoma (HCC). Increasing evidence indicates that microRNA (miRNA)-mRNA axis is involved in HCC. However, a comprehensive miRNA-mRNA regulatory network in HBV-related HCC is still absent. This study aims to identify potential miRNA-mRNA regulatory pathways contributing to pathogenesis of HBV-related HCC. METHODS: Microarray GSE69580 was downloaded from Gene Expression Omnibus (GEO) database. GEO2R and 'R-limma' were used to conduct differential expression analysis. The common miRNAs appeared in the two analytic sets were screened as potential differentially expressed miRNAs (DE-miRNAs). The prognostic roles of screened DE-miRNAs in HCC were further evaluated using Kaplan-Meier plotter database. Target genes of DE-miRNAs were predicted by miRNet. Then, protein-protein interaction (PPI) networks were established for these targets via the STRING database, after which hub genes in the networks were identified by Cytoscape. Functional annotation and pathway enrichment analyses for the target genes were performed through DAVID database. Three enriched pathways related to HBV-related HCC were selected for further analysis and potential target genes commonly appeared in all three pathways were screened. Cytoscape was employed to construct miRNA-hub gene network. The expression and correlation of potential miRNAs and targets were further detected in clinical HBV-related HCC samples by qRT-PCR. RESULTS: 7 upregulated and 9 downregulated DE-miRNAs were accessed. 5 of 7 upregulated DE-miRNAs and 5 of 7 downregulated DE-miRNAs indicated significant prognostic roles in HCC. 2312 and 1175 target genes were predicted for the upregulated and downregulated DE-miRNAs, respectively. TP53 was identified as the hub gene in the PPI networks. Pathway enrichment analysis suggested that these predicted targets were linked to hepatitis B, pathways in cancer, microRNAs in cancer and viral carcinogenesis. Further analysis of these pathways screened 20 and 16 target genes for upregulated and downregulated DE-miRNAs, respectively. By detecting the expression of 36 target genes, six candidate target genes were identified. Finally, a potential miRNA-mRNA regulatory network was established based on the results of qRT-PCR and expression correlation analysis. CONCLUSIONS: In the study, potential miRNA-mRNA regulatory pathways were identified, exploring the underlying pathogenesis and effective therapy strategy of HBV-related HCC.

In silico analysis excavates potential biomarkers by constructing miRNA-mRNA networks between non-cirrhotic HCC and cirrhotic HCC.

BACKGROUND: Mounting evidences have demonstrated that HCC patients with or without cirrhosis possess different clinical characteristics, tumor development and prognosis. However, few studies directly investigated the underlying molecular mechanisms between non-cirrhotic HCC and cirrhotic HCC. METHODS: The clinical information and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) of HCC with or without cirrhosis were obtained by R software. Functional annotation and pathway enrichment analysis were performed by Enrichr. Protein-protein interaction (PPI) network was established through STRING and mapped to Cytoscape to identify hub genes. MicroRNAs were predicted through miRDB database. Furthermore, correlation analysis between selected genes and miRNAs were conducted via starBase database. MiRNAs expression levels between HCC with or without cirrhosis and corresponding normal liver tissues were further validated through GEO datasets. Finally, expression levels of key miRNAs and target genes were validated through qRT-PCR. RESULTS: Between 132 non-cirrhotic HCC and 79 cirrhotic HCC in TCGA, 768 DEGs were acquired, mainly involved in neuroactive ligand-receptor interaction pathway. According to the result from gene expression analysis in TCGA, CCL19, CCL25, CNR1, PF4 and PPBP were renamed as key genes and selected for further investigation. Survival analysis indicated that upregulated CNR1 correlated with worse OS in cirrhotic HCC. Furthermore, ROC analysis revealed the significant diagnostic values of PF4 and PPBP in cirrhotic HCC, and CCL19, CCL25 in non-cirrhotic HCC. Next, 517 miRNAs were predicted to target the 5 key genes. Correlation analysis confirmed that 16 of 517 miRNAs were negatively regulated the key genes. By detecting the expression levels of these key miRNAs from GEO database, we found 4 miRNAs have high research values. Finally, potential miRNA-mRNA networks were constructed based on the results of qRT-PCR. CONCLUSION: In silico analysis, we first constructed the miRNA-mRNA regulatory networks in non-cirrhotic HCC and cirrhotic HCC.

Non-Negative Low-Rank Representation With Similarity Correction for Cell Type Identification in scRNA-Seq Data.

Single-cell RNA sequencing (scRNA-Seq) technology has emerged as a powerful tool to investigate cellular heterogeneity within tissues, organs, and organisms. One fundamental question pertaining to single-cell gene expression data analysis revolves around the identification of cell types, which constitutes a critical step within the data processing workflow. However, existing methods for cell type identification through learning low-dimensional latent embeddings often overlook the intercellular structural relationships. In this paper, we present a novel non-negative low-rank similarity correction model (NLRSIM) that leverages subspace clustering to preserve the global structure among cells. This model introduces a novel manifold learning process to address the issue of imbalanced neighbourhood spatial density in cells, thereby effectively preserving local geometric structures. This procedure utilizes a position-sensitive hashing algorithm to construct the graph structure of the data. The experimental results demonstrate that the NLRSIM surpasses other advanced models in terms of clustering effects and visualization experiments. The validated effectiveness of gene expression information after calibration by the NLRSIM model has been duly ascertained in the realm of relevant biological studies. The NLRSIM model offers unprecedented insights into gene expression, states, and structures at the individual cellular level, thereby contributing novel perspectives to the field.

In situ embedding of glucose oxidase in amorphous ZIF-7 with high catalytic activity and stability and mechanism investigation.

Glucose oxidase (GOx) has a great application potential in the determination of glucose concentration. However, its sensitivity to the environment and poor recyclability limited its broader application. Herein, with the assistance of DA-PEG-DA, a novel immobilized GOx based on amorphous Zn-MOFs (DA-PEG-DA/GOx@aZIF-7/PDA) was developed to impart excellent properties to the enzyme. SEM, TEM, XRD, and BET analyses confirmed that GOx was embedded in amorphous ZIF-7 with ∼5 wt% loading. Compared with free GOx, DA-PEG-DA/GOx@aZIF-7/PDA exhibited enhanced stability, excellent reusability, and promising potential for glucose detection. After 10 repetitions, the catalytic activity of DA-PEG-DA/GOx@aZIF-7/PDA can maintain 95.53 % ± 3.16 %. In understanding the in situ embedding of GOx in ZIF-7, the interaction of zinc ion and benzimidazole with GOx was studied by using molecular docking and multi-spectral methods. Results showed that zinc ions and benzimidazole had multiple binding sites on the enzyme, which induced the accelerated synthesis of ZIF-7 around the enzyme. During binding, the structure of the enzyme changes, but such changes hardly affect the activity of the enzyme. This study provides not only a preparation strategy of immobilized enzyme with high activity, high stability, and low enzyme leakage rate for glucose detection, but also a more comprehensive understanding of the formation of immobilized enzymes using the in situ embedding strategy.

Host plant-mediated effects on Buchnera symbiont: implications for biological characteristics and nutritional metabolism of pea aphids (Acyrthosiphon pisum).

Introduction: The pea aphid, Acyrthosiphon pisum, is a typical sap-feeding insect and an important worldwide pest. There is a primary symbiont-Buchnera aphidicola, which can synthesize and provide some essential nutrients for its host. At the same time, the hosts also can actively adjust the density of bacterial symbiosis to cope with the changes in environmental and physiological factors. However, it is still unclear how symbionts mediate the interaction between herbivorous insects' nutrient metabolism and host plants. Methods: The current study has studied the effects of different host plants on the biological characteristics, Buchnera titer, and nutritional metabolism of pea aphids. This study investigated the influence of different host plants on biological characteristics, Buchnera titer, and nutritional metabolism of pea aphids. Results and discussion: The titer of Buchnera was significantly higher on T. Pretense and M. officinalis, and the relative expression levels were 1.966±0.104 and 1.621±0.167, respectively. The content of soluble sugar (53.46±1.97µg/mg), glycogen (1.12±0.07µg/mg) and total energy (1341.51±39.37µg/mg) of the pea aphid on V. faba were significantly higher and showed high fecundity (143.86±11.31) and weight (10.46±0.77µg/mg). The content of total lipids was higher on P. sativum and T. pretense, which were 2.82±0.03µg/mg and 2.92±0.07µg/mg, respectively. Correlation analysis found that the difference in Buchnera titer was positively correlated with the protein content in M. officinalis and the content of total energy in T. pratense (P < 0.05). This study confirmed that host plants not only affected the biological characteristics and nutritional metabolism of pea aphids but also regulated the symbiotic density, thus interfering with the nutritional function of Buchnera. The results can provide a theoretical basis for further studies on the influence of different host plants on the development of pea aphids and other insects.

Impact of insulin therapy on outcomes of diabetic patients with heart failure: A systematic review and meta-analysis.

OBJECTIVE: To compare clinical outcomes in diabetic patients with heart failure managed by insulin with those managed by non-insulin (oral hypoglycemic agents and/or lifestyle modification) based therapy. METHODS: PubMed and Scopus databases were searched for studies conducted on diabetic patients with heart failure. Studies were to compare outcomes of patients managed by insulin versus non-insulin therapies. RESULTS: 15 studies were included. Compared to those who were managed using non-insulin therapy, insulin-treated patients had increased risk of all-cause mortality (RR 1.46, 95% CI: 1.14, 1.88) and cardiovascular specific mortality (RR 1.62, 95% CI: 1.33, 1.96). Those managed using insulin also had increased risk of hospitalization (RR 1.45, 95% CI: 1.09, 1.93) and readmission (RR 1.49, 95% CI: 1.32, 1.67). There was no additional risk for stroke (RR 1.07, 95% CI: 0.91, 1.27) or myocardial infarction (MI) (RR 1.10, 95% CI: 0.96, 1.27) between the two groups of patients. CONCLUSIONS: Receipt of insulin among diabetic patients with heart failure was associated with an increased risk of mortality, hospitalization and readmission compared to management using oral hypoglycemic agents and/or lifestyle modification. Such patients should be closely monitored for any adverse events.

Development of an Inflammation-Related lncRNA-miRNA-mRNA Network Based on Competing Endogenous RNA in Breast Cancer at Single-Cell Resolution.

The role and mechanism of inflammation in breast cancer is unclear. This study aims to probe the relationship between inflammation and long non-coding RNAs (lncRNAs) and to stablish an inflammation-related competing endogenous RNA (ceRNA) network in breast cancer. Inflammation-related lncRNAs and target genes were screened based on the data from four single-cell RNA sequencing (scRNA-seq) studies and miRNAs were bioinformatically predicted according to ceRNA hypothesis. A series of in silico analyses were performed to construct an inflammation-related ceRNA network in breast cancer. Consequently, a total of seven inflammation-related lncRNAs were selected, after which LRRC75A-AS1 was identified as the most potential lncRNA in view of its expression and prognostic predictive value in breast cancer. Finally, an inflammation-related ceRNA network in breast cancer at the single cell level was established based on lncRNA LRRC75A-AS1, miR-3127-5p, miR-2114-3p, RPL36 and RPL27A mRNAs. Collectively, the lncRNA LRRC75A-AS1 and the LRRC75A-AS1-based on ceRNA network may exert crucial roles in modulating inflammation response during the initiation and progression of breast cancer.

A Comprehensive Analysis for Expression, Diagnosis, and Prognosis of m5C Regulator in Breast Cancer and Its ncRNA-mRNA Regulatory Mechanism.

Recent studies have well demonstrated that 5-methylcytosine (m5C) regulators play pivotal roles in pathological conditions, including cancer. This study first tried to identify potential 5-methylcytosine (m5C) regulators in breast cancer by combination of expression, diagnosis, and survival analyses, and then established an ncRNA-mRNA network accounting for m5C regulators' roles in breast cancer. Among 13 m5C regulators, DNMT3B and ALYREF were significantly upregulated in breast cancer and their high expression indicated unfavorable prognosis. Both DNMT3B and ALYREF possessed the statistical abilities to distinguish breast cancer from normal breast samples. Moreover, five potential upstream miRNAs (let-7b-5p, miR-195-5p, miR-29a-3p, miR-26a-5p, and miR-26b-5p) of m5C regulators could not only serve as independent prognostic predictors but also together made up a promising miRNA prognostic signature in breast cancer. Next, upstream potential lncRNAs of the five miRNAs were predicted and analyzed. Pathway enrichment analysis revealed that the target genes of these miRNAs were markedly enriched in some cancer-related pathways, and further investigation indicated VEGFA and EZH2 were found to be the most potential target genes in the m5C regulators-related ncRNA-mRNA network in breast cancer. These findings comprehensively provided key clues for developing m5C regulators-related effective therapeutic targets and promising diagnostic biomarkers in breast cancer.

Inclisiran inhibits oxidized low-density lipoprotein-induced foam cell formation in Raw264.7 macrophages via activating the PPARγ pathway.

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a well-known proprotein convertase that influences foam cell formation and modulates atherosclerosis. Inclisiran is a novel chemosynthetic small interfering RNA that inhibits PCSK9 synthesis. This study aimed to explore the effect of inclisiran on oxidized low-density lipoprotein (ox-LDL)-induced foam cell formation in Raw264.7 macrophages and to investigate the underlying mechanisms. Raw264.7 cells were treated with ox-LDL to induce the formation of macrophage-derived foam cells. Oil Red O staining and high-performance liquid chromatography were performed to detect lipid accumulation and cholesterol levels. Dil-ox-LDL uptake assay, CCK-8, RT-qPCR, and Western blotting analysis were performed to examine ox-LDL uptake, cell viability, and expression of scavenger receptor-related factors. Inclisiran reduced lipid accumulation in ox-LDL-treated macrophages in a dose-dependent manner. Inclisiran significantly inhibited the levels of total cholesterol, free cholesterol, and cholesterol ester in the supernatant of Raw264.7 cells. Inclisiran reduced ox-LDL uptake and increased Raw264.7 cell viability. Meanwhile, inclisiran downregulated the expression of SR-A, LOX-1, and CD36 and upregulated SR-BI, ApoE, and ABCA1. Furthermore, inclisiran increased PPARγ activity and decreased NF-κB activity. An inhibitor of PPARγ (T0070907) reversed the beneficial effects of inclisiran on ox-LDL uptake, NF-κB inactivation, and cytokine expression. In conclusion, these data suggested that inclisiran inhibited the formation of macrophage-derived foam cells by activating the PPARγ pathway.HighlightsInclisiran reduces lipid accumulation in Raw264.7 cells;Inclisiran reduces ox-LDL uptake and increases Raw264.7 cell viability;Inclisiran inhibits foam cell formation by activating the PPARγ pathway.

Theoretical Study of O-CH3 Bond Dissociation Enthalpy in Anisole Systems.

Understanding ubiquitous methyl transfer reactions requires a systematic study of thermodynamical parameters that could reveal valuable information about the nature of the chemical bond and the feasibility of those processes. In the present study, the O-CH3 bond dissociation enthalpies (BDEs) of 67 compounds belonging to phenol/anisole systems were calculated employing the Gaussian-4 (G4) method. Those compounds contain different substituents including alkyl groups, electron-donating groups (EDGs), and electron-withdrawing groups (EWGs). The results show that the bigger branched alkyl groups and EDGs will destabilize the O-CH3 bond, while EWGs have the opposite effect. A combination of different effects including steric effects, hydrogen bonds, and substituents and their position can achieve around 20 kcal/mol difference compared to the basic phenyl frame. Also, the linear correlation between σp + and O-CH3 BDE can provide a reference for the O-CH3 BDE prediction. The present study represents a step forward to establish a comprehensive O-CH3 BDE database to understand the substituent effect and make its contribution to the rational design of inhibitors and drugs.

A novel antimicrobial polymer efficiently treats multidrug-resistant MRSA-induced bloodstream infection.

The present study aimed to ascertain if polymer 2a, a novel synthesized antimicrobial polyionene, could treat methicillin-resistant Staphylococcus aureus (MRSA)-induced bloodstream infection. The minimum inhibitory concentration (MIC) of polymer 2a against MRSA was detected. A time-kill assay was employed to determine the killing kinetic of polymer 2a. Potential antimicrobial mechanisms of polymer 2a, including membrane disruption and programmed cell death (PCD), were explored. A resistance development assay was introduced to determine the propensity of polymer 2a toward resistance against MRSA. A mouse model of MRSA bacteremia was established to assess in vivo efficacy of polymer 2a. Furthermore, in vivo toxicity of polymer 2a was also evaluated through injection by tail vein. Polymer 2a exhibited more superior antimicrobial activity and faster killing kinetic than the control antibiotics against clinically isolated MRSA strains. Polymer 2a resulted in an obvious leakage of cellular components (concentration more than 1× MIC). mRNA expression of PCD pathway-related gene (recA) was significantly up-regulated in the presence of polymer 2a with low concentration (concentration less than 1× MIC). Repeated use of polymer 2a did not lead to drug resistance. In a MRSA-induced bloodstream infection mouse model, polymer 2a displayed superior therapeutic efficacy with negligible systemic toxicity. Moreover, polymer 2a treatment by tail vein could evidently reduce MRSA counts in blood and major organs and markedly improve living conditions. In conclusion, all these findings presented in this work convincingly suggested that polymer 2a may be a promising therapeutic alternative for treating MRSA-induced infections, especially bloodstream infection.

Distinct expression pattern and prognostic values of pituitary tumor transforming gene family genes in non-small cell lung cancer.

Members of the pituitary tumor transforming gene (PTTG) family, including PTTG1, PTTG2 and PTTG3P, exhibit pivotal roles in the onset and progression of certain types of human cancer. However, to the best of our knowledge, a systematic study regarding the expression pattern and the prognostic values of PTTG family genes in non-small cell lung cancer (NSCLC) remains to be performed. The expression levels of PTTG family genes in NSCLC were successively determined using the Gene Expression Profiling Interactive Analysis, UALCAN and Oncomine databases. Subsequently, the Kaplan-Meier plotter database was used to assess the prognostic value of the PTTG family genes in patients with NSCLC, and to determine the associations between PTTG expression levels and the prognosis of patients based on different clinicopathological features, including cancer stage, grade, chemotherapy, radiotherapy, lymph node status, smoking history, and sex. PTTG1 was identified to be significantly upregulated in NSCLC in all three databases, whereas PTTG2 and PTTG3P were significantly upregulated in NSCLC in only the UALCAN database. Patients with NSCLC with higher expression levels of the three PTTG genes demonstrated shorter overall survival times. In summary, the results of the present study suggested that increased expression of PTTG family genes may serve as promising prognostic biomarkers for patients with NSCLC.

Five miRNAs-mediated PIEZO2 downregulation, accompanied with activation of Hedgehog signaling pathway, predicts poor prognosis of breast cancer.

Roles of Piezo-type mechanosensitive ion channel component 2 (PIEZO2) in cancer remain largely unknown. Herein, we explored PIEZO2 expression, prognosis and underlying mechanisms in cancer. Breast was selected as the candidate as its relatively higher expression level of PIEZO2 than other human tissues. Next, we identified a decreased expression of PIEZO2 in breast cancer compared with normal controls, and found that PIEZO2 expression positively correlated with estrogen receptor (ER) and progesterone receptor (PR) status but negatively correlated with human epidermal growth factor receptor 2 (HER2) status, Nottingham Prognostic Index (NPI) score, Scarff-Bloom-Richardson (SBR) grade, basal-like and triple-negative status. Subsequent analysis revealed that high expression of PIEZO2 had a favorable prognosis in breast cancer. 182 miRNAs were predicted to target PIEZO2. Among these miRNAs, five miRNAs (miR-130b-3p, miR-196a-5p, miR-301a-3p, miR-421 and miR-454-3p) possess the greatest potential in targeting PIEZO2. 109 co-expressed genes of PIEZO2 were identified. Pathway enrichment analysis showed that these genes were enriched in Hedgehog signaling pathway, including Cell adhesion molecule-related/downregulated by oncogenes (CDON). CDON expression was decreased in breast cancer and downregulation of CDON indicated a poor prognosis. Altogether, these findings suggest that decreased expression of PIEZO2 may be utilized as a prognostic biomarker of breast cancer.

Identification of chemoresistance-associated miRNAs in breast cancer.

BACKGROUND: Neoadjuvant chemotherapy (NAC) is an effective therapeutic regimen for patients with breast cancer. However, some individuals cannot benefit from NAC because of drug resistance. To date, valid strategies about enhancing sensitivity of breast cancer to NAC are still scarce. miRNAs have been reported to proverbially be involved in the onset and development of malignancies including drug resistance. METHODS: GSE73736 was downloaded from the GEO database. Student's t-test was conducted to acquire differentially expressed-miRNAs (DE-miRNAs). Potential target genes of DE-miRNAs were predicted by miRTarBase. Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses for these target genes were performed by database for annotation, visualization, and integrated discovery. Protein-protein interaction network was constructed by STRING database and visualized through Cytoscape software. The hub target gene-miRNA network was also established by Cytoscape software. Next, the expression of potential functional miRNAs in breast cancer cell lines and tissues was determined. Finally, the roles of miR-3617-3p, miR-3136-3p, and miR-520b in modulating breast cancer chemoresistance were further examined. RESULTS: A total of 123 DE-miRNAs were identified, including 60 upregulated miRNAs and 63 downregulated miRNAs in the chemoresistant breast cancer group when compared with the chemosensitive group. Six hundred and seventeen and 1,146 potential target genes for the top 10 most upregulated and downregulated miRNAs were predicted, respectively. Enrichment analyses revealed that these target genes were enriched in some cancer-associated or chemo-resistance-associated pathways, such as MAPK signaling pathway, wnt signaling pathway, and p53 signaling pathway. MAPK1 and PRDM10 were identified as hub genes in the protein-protein interaction network. The top 25 hub genes were potentially regulated by 16 DE-miRNAs, among which miR-3617-3p and miR-3136-3p were commonly upregulated, whereas miR-520b was downregulated in two chemoresistant breast cancer cells compared with chemosensitive cell. By analyzing TCGA data, we found that expression of miR-3136-3p and miR-520b was increased and decreased in breast cancer tissues, respectively. Moreover, functional experiments demonstrated that miR-3136-3p and miR-3617-3p could reduce chemosensitivity of breast cancer, whereas miR-520b could reverse chemoresistance. CONCLUSION: The present study, based on bioinformatics analysis and experimental validation, brings to light novel mechanisms of breast cancer NAC resistance.

MicroRNA regulation of liver cancer stem cells.

MicroRNAs (miRNAs), a class of emerging small non-coding RNAs, serve as vital players in modulating multiple biological processes via the post-transcriptional regulation of gene expression. Dysregulated expression of miRNAs in liver cancer is well documented, and the involvement of miRNAs in liver cancer initiation and progression has also been described. Cancer stem cells (CSCs) are a subset of cells known to be at the root of cancer recurrence and resistance to therapy. In this review, we highlight recent reports indicating that miRNAs participate in the regulation of liver cancer stem cells (LCSCs). The Wnt signaling pathway, TGF-beta signaling pathway, JAK/STAT signaling pathway and epithelial-mesenchymal transition (EMT) are all closely correlated with the miRNA modulation of LCSCs. In addition, several miRNAs have been demonstrated to be involved in the regulation of LCSCs in response to therapy sensitivity. Targeting LCSCs by regulating the expression of these miRNAs represents a potential therapeutic strategy for treating cancer drug resistance, metastasis and recurrence in the near future.

MicroRNAs in cancer metastasis and angiogenesis.

Cancer metastasis is a malignant process by which tumor cells migrate from their primary site of origin to other organs. It is the main cause of poor prognosis in cancer patients. Angiogenesis is the process of generating new blood capillaries from pre-existing vasculature. It plays a vital role in primary tumor growth and distant metastasis. MicroRNAs are small non-coding RNAs involved in regulating normal physiological processes as well as cancer pathogenesis. They suppress gene expression by specifically binding to the 3'-untranslated region (3'-UTR) of their target genes. They can thus act as oncogenes or tumor suppressors depending on the function of their target genes. MicroRNAs have shown great promise for use in anti-metastatic cancer therapy. In this article, we review the roles of various miRNAs in cancer angiogenesis and metastasis and highlight their potential for use in future therapies against metastatic cancer.

Improvement of genome assembly completeness and identification of novel full-length protein-coding genes by RNA-seq in the giant panda genome.

High-quality and complete gene models are the basis of whole genome analyses. The giant panda (Ailuropoda melanoleuca) genome was the first genome sequenced on the basis of solely short reads, but the genome annotation had lacked the support of transcriptomic evidence. In this study, we applied RNA-seq to globally improve the genome assembly completeness and to detect novel expressed transcripts in 12 tissues from giant pandas, by using a transcriptome reconstruction strategy that combined reference-based and de novo methods. Several aspects of genome assembly completeness in the transcribed regions were effectively improved by the de novo assembled transcripts, including genome scaffolding, the detection of small-size assembly errors, the extension of scaffold/contig boundaries, and gap closure. Through expression and homology validation, we detected three groups of novel full-length protein-coding genes. A total of 12.62% of the novel protein-coding genes were validated by proteomic data. GO annotation analysis showed that some of the novel protein-coding genes were involved in pigmentation, anatomical structure formation and reproduction, which might be related to the development and evolution of the black-white pelage, pseudo-thumb and delayed embryonic implantation of giant pandas. The updated genome annotation will help further giant panda studies from both structural and functional perspectives.

In vitro anti-coxsackievirus B(3) effect of ethyl acetate extract of Tian-hua-fen.

AIM: To investigation the anti-coxsackievirus B(3) (CVB(3m)) effect of the ethyl acetate extract of Tian-hua-fen on HeLa cells infected with CVB(3m). METHODS: HeLa cells were infected with CVB(3m) and the cytopathic effects (CPE) were observed through light microscope and crystal violet staining on 96-well plate and A(600) was detected using spectrophotometer. The protective effect of the extract to HeLa cells and the mechanism of the effect were also evaluated through the change of CPE and value of A(600). RESULTS: The extract had some toxicity to HeLa cells at a higher concentration while had a marked inhibitory effect on cell pathological changes at a lower concentration. Consistent results were got through these two methods. We also investigated the mechanism of its anti-CVB(3m) effect and the results indicated that the extract represented an inhibitory effect through all the processes of CVB(3m) attachment, entry, biosynthesis and assemble in cells. CONCLUSION: The results demonstrate that the ethyl acetate extract of Tian-hua-fen has a significant protective effect on HeLa cells infected with CVB(3m) in a dose-dependent manner and this effect exists through the process of CVB(3m) attachment, entry, biosynthesis and assemble in cells, suggesting that the ethyl acetate extract of Tian-hua-fen can be developed as an anti-virus agent.

Cassava genome from a wild ancestor to cultivated varieties.

Cassava is a major tropical food crop in the Euphorbiaceae family that has high carbohydrate production potential and adaptability to diverse environments. Here we present the draft genome sequences of a wild ancestor and a domesticated variety of cassava and comparative analyses with a partial inbred line. We identify 1,584 and 1,678 gene models specific to the wild and domesticated varieties, respectively, and discover high heterozygosity and millions of single-nucleotide variations. Our analyses reveal that genes involved in photosynthesis, starch accumulation and abiotic stresses have been positively selected, whereas those involved in cell wall biosynthesis and secondary metabolism, including cyanogenic glucoside formation, have been negatively selected in the cultivated varieties, reflecting the result of natural selection and domestication. Differences in microRNA genes and retrotransposon regulation could partly explain an increased carbon flux towards starch accumulation and reduced cyanogenic glucoside accumulation in domesticated cassava. These results may contribute to genetic improvement of cassava through better understanding of its biology.