Prognostic significance and value of further classification of lymphovascular invasion in invasive breast cancer: a retrospective observational study.

PURPOSE: To investigate the prognostic significance of lymphovascular invasion in invasive breast cancer and the value of using specific vascular endothelial markers to further classify lymphovascular invasion. METHODS: We collected 2124 patients with invasive breast cancer who were hospitalized at the First Hospital of Dalian Medical University from 2012 to 2020. Statistical methods were used to investigate the relationship between lymphovascular invasion and clinicopathological characteristics of breast cancer, and the correlation between lymphovascular invasion on overall survival (OS) and disease-free survival (DFS) of various categories of breast cancers. Immunohistochemical staining of breast cancer samples containing lymphovascular invasion using specific vascular endothelial markers D2-40 and CD34 was used to classify lymphovascular invasion and to investigate the relationship between lymphovascular invasion and breast cancer progression. RESULTS: There was a high correlation between lymphovascular invasion and T stage, N stage and nerve invasion. Survival analyses showed that patients with lymphovascular invasion, especially luminal B, triple-negative, and Her-2 overexpression breast cancer patients, had poorer OS and DFS prognosis, and that lymphovascular invasion was an independent prognostic factor affecting OS and DFS in breast cancer. The immunohistochemical staining results showed that positive D2-40 staining of lymphovascular invasion was linked to the N stage and localized recurrence of breast cancer. CONCLUSION: Lymphovascular invasion is associated with aggressive clinicopathological features and is an independent poor prognostic factor in invasive breast cancer. Breast cancer localized recurrence rate and lymph node metastases are influenced by lymphatic vessel invasion. Immunohistochemical techniques should be added to the routine diagnosis of lymphovascular invasion.

Specialized cis-Acting RNA Elements Balance Genome Cyclization to Ensure Efficient Replication of Yellow Fever Virus.

Genome cyclization is essential for viral RNA (vRNA) replication of the vertebrate-infecting flaviviruses, and yet its regulatory mechanisms are not fully understood. Yellow fever virus (YFV) is a notorious pathogenic flavivirus. Here, we demonstrated that a group of cis-acting RNA elements in YFV balance genome cyclization to govern efficient vRNA replication. It was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) is conserved in the YFV clade and is important for efficient YFV propagation. By using two different replicon systems, we found that the function of the DCS-HP is determined primarily by its secondary structure and, to a lesser extent, by its base-pair composition. By combining in vitro RNA binding and chemical probing assays, we found that the DCS-HP orchestrates the balance of genome cyclization through two different mechanisms, as follows: the DCS-HP assists the correct folding of the 5' end in a linear vRNA to promote genome cyclization, and it also limits the overstabilization of the circular form through a potential crowding effect, which is influenced by the size and shape of the DCS-HP structure. We also provided evidence that an A-rich sequence downstream of the DCS-HP enhances vRNA replication and contributes to the regulation of genome cyclization. Interestingly, diversified regulatory mechanisms of genome cyclization, involving both the downstream of the 5'-cyclization sequence (CS) and the upstream of the 3'-CS elements, were identified among different subgroups of the mosquito-borne flaviviruses. In summary, our work highlighted how YFV precisely controls the balance of genome cyclization to ensure viral replication. IMPORTANCE Yellow fever virus (YFV), the prototype of the Flavivirus genus, can cause devastating yellow fever disease. Although it is preventable by vaccination, there are still tens of thousands of yellow fever cases per year, and no approved antiviral medicine is available. However, the understandings about the regulatory mechanisms of YFV replication are obscure. In this study, by a combination of bioinformatics, reverse genetics, and biochemical approaches, it was shown that the downstream of the 5'-cyclization sequence hairpin (DCS-HP) promotes efficient YFV replication by modulating the conformational balance of viral RNA. Interestingly, we found specialized combinations for the downstream of the 5'-cyclization sequence (CS) and upstream of the 3'-CS elements in different groups of the mosquito-borne flaviviruses. Moreover, possible evolutionary relationships among the various downstream of the 5'-CS elements were implied. This work highlighted the complexity of RNA-based regulatory mechanisms in the flaviviruses and will facilitate the design of RNA structure-targeted antiviral therapies.

Key Residue in the Precursor Region of M Protein Contributes to the Neurovirulence and Neuroinvasiveness of the African Lineage of Zika Virus.

The Zika virus (ZIKV) represents an important global health threat due to its unusual association with congenital Zika syndrome. ZIKV strains are phylogenetically grouped into the African and Asian lineages. However, the viral determinants underlying the phenotypic differences between the lineages remain unknown. Here, multiple sequence alignment revealed a highly conserved residue at position 21 of the premembrane (prM) protein, which is glutamic acid and lysine in the Asian and African lineages, respectively. Using reverse genetics, we generated a recombinant virus carrying an E21K mutation based on the genomic backbone of the Asian lineage strain FSS13025 (termed E21K). The E21K mutation significantly increased viral replication in multiple neural cell lines with a higher ratio of M to prM production. Animal studies showed E21K exhibited increased neurovirulence in suckling mice, leading to more severe defects in mouse brains by causing more neural cell death and destruction of hippocampus integrity. Moreover, the E21K substitution enhanced neuroinvasiveness in interferon alpha/beta (IFN-α/ß) receptor knockout mice, as indicated by the increased mortality, and enhanced replication in mouse brains. The global transcriptional analysis showed E21K infection profoundly altered neuron development networks and induced stronger antiviral immune response than wild type (WT) in both neural cells and mouse brains. More importantly, the reverse K21E mutation based on the genomic backbone of the African strain MR766 caused less mouse neurovirulence. Overall, our findings support the 21st residue of prM functions as a determinant for neurovirulence and neuroinvasiveness of the African lineage of ZIKV. IMPORTANCE The suspected link of Zika virus (ZIKV) to birth defects led the World Health Organization to declare ZIKV a Public Health Emergency of International Concern. ZIKV has been identified to have two dominant phylogenetic lineages, African and Asian. Significant differences exist between the two lineages in terms of neurovirulence and neuroinvasiveness in mice. However, the viral determinants underlying the phenotypic differences are still unknown. Here, combining reverse genetics, animal studies, and global transcriptional analysis, we provide evidence that a single E21K mutation of prM confers to the Asian lineage strain FSS130125 significantly enhanced replication in neural cell lines and more neurovirulent and neuroinvasiveness phenotypes in mice. Our findings support that the highly conserved residue at position 21 of prM functions as a determinant of neurovirulence and neuroinvasiveness of the African lineage of ZIKV in mice.

Effect of flocculant dosage on the settling properties and underflow concentration of thickener for flocculated tailing suspensions.

Flocculation is important in the thickening process to improve the underflow concentration in thickeners for tailing suspensions. Traditional zone settling velocity (ZSV) functions ignore the effect of flocculant dosage on the ZSV and the thickening behavior of thickeners. To investigate the effect of flocculant dosage on the settling flux function, a series of batch settling tests were conducted at various flocculant dosages for unclassified and fine tailings. The correlation among flocculant dosage, solid fraction, and parameters in the ZSV function was revealed. Moreover, a simulation of continuous thickening based on the ZSV function was performed. Results indicated that flocculation influenced settling velocity and floc density. With an increased flocculant dosage, the settling velocity of floc increased, resulting in increased underflow concentration. Conversely, floc density decreased due to a stronger particle-particle interaction, leading to a decreased underflow concentration.

Short Direct Repeats in the 3' Untranslated Region Are Involved in Subgenomic Flaviviral RNA Production.

Mosquito-borne flaviviruses consist of a positive-sense genome RNA flanked by the untranslated regions (UTRs). There is a panel of highly complex RNA structures in the UTRs with critical functions. For instance, Xrn1-resistant RNAs (xrRNAs) halt Xrn1 digestion, leading to the production of subgenomic flaviviral RNA (sfRNA). Conserved short direct repeats (DRs), also known as conserved sequences (CS) and repeated conserved sequences (RCS), have been identified as being among the RNA elements locating downstream of xrRNAs, but their biological function remains unknown. In this study, we revealed that the specific DRs are involved in the production of specific sfRNAs in both mammalian and mosquito cells. Biochemical assays and structural remodeling demonstrate that the base pairings in the stem of these DRs control sfRNA formation by maintaining the binding affinity of the corresponding xrRNAs to Xrn1. On the basis of these findings, we propose that DRs functions like a bracket holding the Xrn1-xrRNA complex for sfRNA formation.IMPORTANCE Flaviviruses include many important human pathogens. The production of subgenomic flaviviral RNAs (sfRNAs) is important for viral pathogenicity as a common feature of flaviviruses. sfRNAs are formed through the incomplete degradation of viral genomic RNA by the cytoplasmic 5'-3' exoribonuclease Xrn1 halted at the Xrn1-resistant RNA (xrRNA) structures within the 3'-UTR. The 3'-UTRs of the flavivirus genome also contain distinct short direct repeats (DRs), such as RCS3, CS3, RCS2, and CS2. However, the biological functions of these ancient primary DR sequences remain largely unknown. Here, we found that DR sequences are involved in sfRNA formation and viral virulence and provide novel targets for the rational design of live attenuated flavivirus vaccine.

Predictive value of CHADS2 and CHA2DS2-VASc scores for coronary artery lesions and in-hospital prognosis of patients with acute ST-segment elevation myocardial infarction.

OBJECTIVE: To evaluate the predictive value of CHADS2 and CHA2DS2-VASc scores for coronary artery lesions and in-hospital prognosis of patients with acute ST-segment elevation myocardial infarction (STEMI). METHODS: A total of 524 patients who were diagnosed with STEMI from January 2016 to August 2017 were retrospectively reviewed. The correlation between CHADS2 and CHA2DS2-VASc scores with the patients' clinical data, number of coronary lesions, Gensini scores, the target vessel and hospitalization time and in-hospital adverse events (AEs) was analyzed. RESULTS: The number of coronary lesions in STEMI patients was mainly single and double lesions. The CHADS2 and CHA2DS2-VASc scores were not meaningful for predicting the number of coronary lesions. However, for left main coronary artery lesion, CHADS2 score was significantly increased when the number increased (P < 0.05), but CHA2DS2-VASc score showed no statistical difference (P > 0.05). The incidence of target lesions in STMEI patients was mainly left anterior descending coronary artery (LAD) and right coronary artery (RCA). The two scores were not meaningful for predicting target lesions (P > 0.05). For the severity of coronary lesions, there was positive correlation between CHADS2 score with Gensini score (P < 0.05), but no exact correlation between CHA2DS2-VASc score and Gensini score (P > 0.05). The stratifications of CHADS2 score and CHA2DS2-VASc score were significantly associated with hospitalization time and adverse events during hospitalization (P < 0.05). The high score group had longer hospitalization time and more AEs during hospitalization than the low score group and the middle group statistically (P < 0.05). CONCLUSION: CHADS2 score had a certain value to predict the severity of coronary lesion and the presence of left main coronary artery in STEMI. The CHA2DS2-VASc score had no predictive ability to do it. There was no significant value in predicting the number of coronary lesions and the location of the target lesions in STEMI patients. However, both scores had the predictive ability for patient hospitalization and AEs during hospitalization.

The long-term impact of a chronic total occlusion in a non-infarct-related artery on acute ST-segment elevation myocardial infarction after primary coronary intervention.

OBJECTIVES: To investigate the long-term outcome of patients with acute ST-segment elevation myocardial infarction (STEMI) and a chronic total occlusion (CTO) in a non-infarct-related artery (IRA) and the risk factors for mortality. METHODS: The enrolled cohort comprised 323 patients with STEMI and multivessel diseases (MVD) that received a primary percutaneous coronary intervention between January 2008 and November 2013. The patients were divided into two groups: the CTO group (n = 97) and the non-CTO group (n = 236). The long-term major adverse cardiovascular and cerebrovascular events (MACCE) experienced by each group were compared. RESULTS: The rates of all-cause mortality and MACCE were significantly higher in the CTO group than they were in the non-CTO group. Cox regression analysis showed that an age ≥ 65 years (OR = 3.94, 95% CI: 1.47-10.56, P = 0.01), a CTO in a non-IRA(OR = 5.09, 95% CI: 1.79 ~ 14.54, P < 0.01), an in-hospital Killip class ≥ 3 (OR = 4.32, 95% CI: 1.71 ~ 10.95, P < 0.01), and the presence of renal insufficiency (OR = 5.32, 95% CI: 1.49 ~ 19.01, P = 0.01), stress ulcer with gastraintestinal bleeding (SUB) (OR = 6.36, 95% CI: (1.45 ~ 28.01, P = 0.01) were significantly related the 10-year mortality of patients with STEMI and MVD; an in-hospital Killip class ≥ 3 (OR = 2.97,95% CI:1.46 ~ 6.03, P < 0.01) and the presence of renal insufficiency (OR = 5.61, 95% CI: 1.19 ~ 26.39, P = 0.03) were significantly related to the 10-year mortality of patients with STEMI and a CTO. CONCLUSIONS: The presence of a CTO in a non-IRA, an age ≥ 65 years, an in-hospital Killip class ≥ 3, and the presence of renal insufficiency, and SUB were independent risk predictors for the long-term mortality of patients with STEMI and MVD; an in-hospital Killip class ≥ 3 and renal insufficiency were independent risk predictors for the long-term mortality of patients with STEMI and a CTO.

Effects of antidiabetic drugs on left ventricular function/dysfunction: a systematic review and network meta-analysis.

BACKGROUND: Although a variety of antidiabetic drugs have significant protective action on the cardiovascular system, it is still unclear which antidiabetic drugs can improve ventricular remodeling and fundamentally delay the process of heart failure. The purpose of this network meta-analysis is to compare the efficacy of sodium glucose cotransporter type 2 (SGLT-2) inhibitors, dipeptidyl peptidase-4 (DPP-4) inhibitors, glucagon-like peptide-1 (GLP-1) agonists, metformin (MET), sulfonylurea (SU) and thiazolidinediones (TZDs) in improving left ventricular (LV) remodeling in patients with type 2 diabetes (T2DM) and/or cardiovascular disease (CVD). METHODS: We searched articles published before October 18, 2019, regardless of language or data, in 4 electronic databases: PubMed, EMBASE, Cochrane Library and Web of Science. We included randomized controlled trials in this network meta-analysis, as well as a small number of cohort studies. The differences in the mean changes in left ventricular echocardiographic parameters between the treatment group and control group were evaluated. RESULTS: The difference in the mean change in LV ejection fraction (LVEF) between GLP-1 agonists and placebo in treatment effect was greater than zero (MD = 2.04% [0.64%, 3.43%]); similar results were observed for the difference in the mean change in LV end-diastolic diameter (LVEDD) between SGLT-2 inhibitors and placebo (MD = - 3.3 mm [5.31, - 5.29]), the difference in the mean change in LV end-systolic volume (LVESV) between GLP-1 agonists and placebo (MD = - 4.39 ml [- 8.09, - 0.7]); the difference in the mean change in E/e' between GLP-1 agonists and placebo (MD = - 1.05[- 1.78, - 0.32]); and the difference in the mean change in E/e' between SGLT-2 inhibitors and placebo (MD = - 1.91[- 3.39, - 0.43]). CONCLUSIONS: GLP-1 agonists are more significantly associated with improved LVEF, LVESV and E/e', SGLT-2 inhibitors are more significantly associated with improved LVEDD and E/e', and DPP-4 inhibitors are more strongly associated with a negative impact on LV end-diastolic volume (LVEDV) than are placebos. SGLT-2 inhibitors are superior to other drugs in pairwise comparisons.

IL-17A/IL-17RA promotes invasion and activates MMP-2 and MMP-9 expression via p38 MAPK signaling pathway in non-small cell lung cancer.

The present study is to investigate the effect and mechanism of action of interleukin (IL)-17A and its receptor IL-17RA on non-small cell lung cancer (NSCLC). A total of 139 NSCLC patients were included in the study. NSCLC tissues and tumor-adjacent tissues were collected from the patients. Human NSCLC cell lines H157, H1975, and A549 were used for in vitro studies. MTT assay was performed to determine cell proliferation. Wound healing assay was used to determine cell motility. Transwell assay was carried out to detect migration and invasion. Quantitative real-time polymerase chain reaction was conducted to measure mRNA expression, while Western blotting was used for determine protein expression. Immunohistochemistry was employed to evaluate IL-17RA expression in 139 primary human NSCLC tissues. Levels of IL-17RA in NSCLC tissues were higher than tumor-adjacent normal tissues, and associated with clinical outcomes. Kaplan-Meier survival analysis indicated that NSCLC patients with positive IL-17RA expression had a poor survival. In addition, IL-17A/IL-17RA affected NSCLC cell migration and invasion in vitro. Treatment with IL-17A/IL-17RA increased the expression of MMP-2 and MMP-9 in NSCLC cells. Furthermore, phosphorylation of p38 was enhanced in IL-17RA-overexpressing NSCLC cells. P38 MAPK-specific inhibitor SB203580 suppressed the migration and invasion of NSCLC cells. MMP-2 and MMP-9 were downstream effectors of IL-17RA and p38 signaling pathways. The present study demonstrates that P38 MAPK activity is crucial for IL-17A/IL-17RA to promote NSCLC metastasis. In addition, IL-17A/IL-17RA signaling may be a novel and promising cancer therapeutic target for the treatment of NSCLC.

Evaluation of short-term strength development of cemented backfill with varying sulphide contents and the use of additives.

The disposal of sulphide tailings and control of strength deterioration of sulphide cemented backfills are of both environmental and financial interest. The uniaxial compressive strength (UCS) of samples with varying sulphide contents (6-25 wt%) was evaluated over a short-term period. A severe strength loss occurred after 14 days on those with low sulphide and the sulphide-rich samples keep increasing over 28 days. With SEM and XRD, it was inferred that the expansive phases were presumably responsible for the strength failure and the mechanical performance is closely in relation to the microstructure. They are deemed to present a positive to negative turn of effects on the strength. Initially, the precipitation in voids and micropores contributes to strength development until the already hardened structure cannot bear the inner pressure, microcracks begin to occur. For sulphide-rich cemented backfills, two kinds of additives, set retarder and accelerator, were adopted to measure their effects on the mechanical strength and setting time as the hardening process of cemented backfills was varied, and the results show that the accelerator gave rise to a rapid gain of strength while followed by a sharp deterioration after 14 days. In contrast, the retarder contributes to the continuous strength growth. From the economic perspective, the use of set retarder or accelerator involves 6.25% and 9.5% of binder cost respectively.

The role of microRNAs in heart failure.

MicroRNAs are small non-coding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. In the past decade, many in vitro and in vivo studies have explored the involvement of microRNAs in various cardiovascular diseases. In this paper, studies focused upon the target genes and functionality of miRNAs in the pathophysiological processes of heart failure are reviewed. The selected miRNAs are categorized according to the biological relevance of their target genes in relation to four cardiovascular pathologies, namely angiogenesis, cardiac hypertrophy, fibrosis and apoptosis. This review illustrates the involvement of miRNAs in different biological signaling pathways and provides an overview of current understanding of the roles of miRNAs in cardiovascular health and diseases. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

The Emerging Duck Flavivirus Is Not Pathogenic for Primates and Is Highly Sensitive to Mammalian Interferon Antiviral Signaling.

UNLABELLED: Flaviviruses pose a significant threat to both animals and humans. Recently, a novel flavivirus, duck Tembusu virus (DTMUV), was identified to be the causative agent of a serious duck viral disease in Asia. Its rapid spread, expanding host range, and uncertain transmission routes have raised substantial concerns regarding its potential threats to nonavian hosts, including humans. Here, we demonstrate that DTMUV is not pathogenic for nonhuman primates and is highly sensitive to mammal type I interferon (IFN) signaling. In vitro assays demonstrated that DTMUV infected and replicated efficiently in various mammalian cell lines. Further tests in mice demonstrated high neurovirulence and the age-dependent neuroinvasiveness of the virus. In particular, the inoculation of DTMUV into rhesus monkeys did not result in either viremia or apparent clinical symptoms, although DTMUV-specific humoral immune responses were detected. Furthermore, we revealed that although avian IFN failed to inhibit DTMUV in avian cells, DTMUV was more sensitive to the antiviral effects of type I interferon than other known human-pathogenic flaviviruses. Knockout of the type I IFN receptor in mice caused apparent viremia, viscerotropic disease, and mortality, indicating a vital role of IFN signaling in protection against DTMUV infection. Collectively, we provide direct experimental evidence that this novel avian-origin DTMUV possesses a limited capability to establish infection in immunocompetent primates due to its decreased antagonistic activity in the mammal IFN system. Furthermore, our findings highlight the potential risk of DTMUV infection in immunocompromised individuals and warrant studies on the cross-species transmission and pathogenesis of this novel flavivirus. IMPORTANCE: Mosquito-borne flaviviruses comprise a large group of pathogenic and nonpathogenic members. The pathogenic flaviviruses include dengue, West Nile, and Japanese encephalitis viruses, and the nonpathogenic flaviviruses normally persist in a natural cycle and rarely cause disease in humans. A novel flavivirus, DTMUV (also known as duck egg drop syndrome flavivirus [DEDSV]) was identified in 2012 in ducks and then rapidly spread to several Asian countries. This new flavivirus was then shown to infect multiple avian species, resulting in neurological symptoms with unknown routes of transmission. There is public concern regarding its potential transmission from birds to humans and other nonavian hosts. Our present study shows that the mammalian IFN system can efficiently eliminate DTMUV infection and that the emergence of severe DTMUV-associated disease in mammals, especially humans, is unlikely. Currently, DTMUV infection mostly affects avian species.

In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2.

Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate.

Rapid detection of genetic mutations in individual breast cancer patients by next-generation DNA sequencing.

Breast cancer is the most common malignancy in women and the leading cause of cancer deaths in women worldwide. Breast cancers are heterogenous and exist in many different subtypes (luminal A, luminal B, triple negative, and human epidermal growth factor receptor 2 (HER2) overexpressing), and each subtype displays distinct characteristics, responses to treatment, and patient outcomes. In addition to varying immunohistochemical properties, each subtype contains a distinct gene mutation profile which has yet to be fully defined. Patient treatment is currently guided by hormone receptor status and HER2 expression, but accumulating evidence suggests that genetic mutations also influence drug responses and patient survival. Thus, identifying the unique gene mutation pattern in each breast cancer subtype will further improve personalized treatment and outcomes for breast cancer patients. In this study, we used the Ion Personal Genome Machine (PGM) and Ion Torrent AmpliSeq Cancer Panel to sequence 737 mutational hotspot regions from 45 cancer-related genes to identify genetic mutations in 80 breast cancer samples of various subtypes from Chinese patients. Analysis revealed frequent missense and combination mutations in PIK3CA and TP53, infrequent mutations in PTEN, and uncommon combination mutations in luminal-type cancers in other genes including BRAF, GNAS, IDH1, and KRAS. This study demonstrates the feasibility of using Ion Torrent sequencing technology to reliably detect gene mutations in a clinical setting in order to guide personalized drug treatments or combination therapies to ultimately target individual, breast cancer-specific mutations.

MicroRNA-181c targets Bcl-2 and regulates mitochondrial morphology in myocardial cells.

Apoptosis is an important mechanism for the development of heart failure. Mitochondria are central to the execution of apoptosis in the intrinsic pathway. The main regulator of mitochondrial pathway of apoptosis is Bcl-2 family which includes pro- and anti-apoptotic proteins. MicroRNAs are small noncoding RNA molecules that regulate gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. It has been proposed that microRNAs play critical roles in the cardiovascular physiology and pathogenesis of cardiovascular diseases. Our previous study has found that microRNA-181c, a miRNA expressed in the myocardial cells, plays an important role in the development of heart failure. With bioinformatics analysis, we predicted that miR-181c could target the 3' untranslated region of Bcl-2, one of the anti-apoptotic members of the Bcl-2 family. Thus, we have suggested that miR-181c was involved in regulation of Bcl-2. In this study, we investigated this hypothesis using the Dual-Luciferase Reporter Assay System. Cultured myocardial cells were transfected with the mimic or inhibitor of miR-181c. We found that the level of miR-181c was inversely correlated with the Bcl-2 protein level and that transfection of myocardial cells with the mimic or inhibitor of miR-181c resulted in significant changes in the levels of caspases, Bcl-2 and cytochrome C in these cells. The increased level of Bcl-2 caused by the decrease in miR-181c protected mitochondrial morphology from the tumour necrosis factor alpha-induced apoptosis.

Genotype-specific neutralization determinants in envelope protein: implications for the improvement of Japanese encephalitis vaccine.

Japanese encephalitis remains the leading cause of viral encephalitis in children in Asia and is expanding its geographical range to larger areas in Asia and Australasia. Five genotypes of Japanese encephalitis virus (JEV) co-circulate in the geographically affected areas. In particular, the emergence of genotype I (GI) JEV has displaced genotype III (GIII) as the dominant circulating genotype in many Asian regions. However, all approved vaccine products are derived from GIII strains. In the present study, bioinformatic analysis revealed that GI and GIII JEV strains shared two distinct amino acid residues within the envelope (E) protein (E222 and E327). By using reverse genetics approaches, A222S and S327T mutations were demonstrated to decrease live-attenuated vaccine (LAV) SA14-14-2-induced neutralizing antibodies in humans, without altering viral replication. A222S or S327T mutations were then rationally engineered into the infectious clone of SA14-14-2, and the resulting mutant strains retained the same genetic stability and attenuation characteristics as the parent strain. More importantly, immunization of mice with LAV-A222S or LAV-S327T elicited increased neutralizing antibodies against GI strains. Together, these results demonstrated that E222 and E327 are potential genotype-related neutralization determinants and are critical in determining the protective efficacy of live Japanese encephalitis vaccine SA14-14-2 against circulating GI strains. Our findings will aid in the rational design of the next generation of Japanese encephalitis LAVs capable of providing broad protection against all JEV strains belonging to different genotypes.

Lysine-specific demethylase 5C promotes hepatocellular carcinoma cell invasion through inhibition BMP7 expression.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of tumor and is associated with high morbidity and mortality rates. Patients with HCC routinely undergo surgery followed by adjuvant radiation therapy and chemotherapy. Despite such aggressive treatment approaches, median survival times remain under 1 year in most cases. KDM5C is a member of the family of JmjC domain-containing proteins that removes methyl residues from methylated lysine 4 on histone H3 lysine 4 (H3K4). KDM5C has been proposed as an oncogene in many types of tumors; however, its role and underlying mechanisms in HCC remain unclear. METHODS: Expression level of KDM5C was examined by RT-PCR, and IHC. Forced expression of KDM5C was mediated by retroviruses, and KDM5C was downregulated by shRNAs expressing lentiviruses. Migration and invasion of HCC cells was measured by wound healing, Transwell and Matrigel assays respectively. RESULTS: In this study, we report that KDM5C is abundantly expressed in invasive human HCC cells. Cellular depletion of KDM5C by shRNA inhibited HCC cell migration, invasion and epithelial-mesenchymal transition in vitro, and markedly decreased the metastasis capacity of invasive HCC cells in the liver and lung. Furthermore, ectopic expression of KDM5C in HCC cells promoted cell migration, invasion and epithelial-mesenchymal transition via the inactivation of BMP7. Knockdown of BMP7 significantly promotes shKDM5C-induced cell migration inhibition. CONCLUSIONS: Taken together, these data suggest that KDM5C-mediated BMP7 inactivation is essential for HCC cell invasion.

Fumaric acid production by Torulopsis glabrata: engineering the urea cycle and the purine nucleotide cycle.

A multi-vitamin auxotrophic Torulopsis glabrata strain, a pyruvate producer, was further engineered to produce fumaric acid. Using the genome-scale metabolic model iNX804 of T. glabrata, four fumaric acid biosynthetic pathways, involving the four cytosolic enzymes, argininosuccinate lyase (ASL), adenylosuccinate lyase (ADSL), fumarylacetoacetase (FAA), and fumarase (FUM1), were found. Athough single overexpression of each of the four enzymes in the cytosol improved fumaric acid production, the highest fumaric acid titer (5.62 g L(-1) ) was obtained with strain T.G-ASL(H) -ADSL(L) by controlling the strength of ASL at a high level and ADSL at a low level. In order to further improve the production of fumaric acid, the SpMAE1 gene encoding the C4 -dicarboxylic acids transporter was overexpressed in strain T.G-ASL(H) -ADSL(L) -SpMAE1 and the final fumaric acid titer increased to 8.83 g L(-1) . This study provides a novel strategy for fumaric acid biosynthesis by utilizing the urea cycle and the purine nucleotide cycle to enhance the bridge between carbon metabolism and nitrogen metabolism.

Novel cis-acting element within the capsid-coding region enhances flavivirus viral-RNA replication by regulating genome cyclization.

cis-Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis-acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The downstream of 5' cyclization sequence (5'CS) pseudoknot (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis-acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis-acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.

Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.