Search

The presumed DNA helicase encoded by ORF44 of Kaposi's sarcoma-associated herpesvirus (KSHV) plays a crucial role in unwinding viral double-stranded DNA and initiating DNA replication during lytic reactivation. However, the regulatory mechanism of KSHV ORF44 has not been fully elucidated. In a previous study, we identified that N-Myc downstream regulated gene 1 (NDRG1), a host scaffold protein, facilitates viral genome replication by interacting with proliferating cell nuclear antigen (PCNA) and the latent viral protein latency-associated nuclear antigen (LANA) during viral latency. In the present study, we further demonstrated that NDRG1 can interact with KSHV ORF44 during viral lytic replication. We also found that the mRNA and protein levels of NDRG1 were significantly increased by KSHV ORF50-encoded replication and transcription activator (RTA). Remarkably, knockdown of NDRG1 greatly decreased the protein level of ORF44 and impaired viral lytic replication. Interestingly, NDRG1 enhanced the stability of ORF44 and inhibited its ubiquitin-proteasome-mediated degradation by reducing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44. In summary, NDRG1 is a novel binding partner of ORF44 and facilitates viral lytic replication by maintaining the stability of ORF44. This study provides new insight into the mechanisms underlying KSHV lytic replication. Author summary: During lytic replication, KSHV ORF44 unwinds viral DNA and initiates DNA replication. Here, we report that the host protein NDRG1, a novel ORF44 binding partner, is significantly upregulated during viral lytic replication and facilitates this process. Mechanistically, NDRG1 can increase the stability of ORF44, impairing the polyubiquitination of the lysine residues at positions 79 and 368 in ORF44, thus inhibiting ubiquitin-proteasome-mediated degradation of ORF44. Our study demonstrates that NDRG1 plays an important role in KSHV lytic replication and may thus constitute a promising therapeutic target for KSHV infection. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the associations of pre-endoscopy anxiety with discomfort and tolerance in patients undergoing unsedated esophagogastroduodenoscopy (EGD). Methods: This is a hospital-based cohort study of 348 patients undergoing routine, non-advanced EGD without sedation. The primary outcomes were discomfort and tolerance. The anxiety before endoscopy was evaluated with a 10-point visual analogue scale (VAS). The associations of pre-endoscopy anxiety with the outcomes were evaluated with logistic regression adjusting for potential confounders like age, sex, and body mass index. Results: Seventy patients reported severe discomfort and 56 patients reported poor tolerance after endoscopy. The risk of severe discomfort increased with pre-endoscopy anxiety and reached a platform around 7–10 points. Compared with the participants with low pre-endoscopy anxiety, those with moderate (adjusted odds ratio [OR] 2.70, 95% confidence interval [CI] 1.17 to 6.22) and high level of anxiety (adjusted OR 6.87, 95% CI 2.16 to 21.79) were associated with a gradual increase in the risk of severe discomfort (P-trend < 0.001). The association between pre-endoscopy anxiety and tolerance was linear, with an adjusted OR of 1.67(95% CI 1.33 to 2.08) for a 1-score increase in pre-endoscopy anxiety VAS. The associations were not modified by age, sex, pharyngitis, duration of endoscopy, and diameter of the endoscope. Conclusions: Pre-endoscopy anxiety was an independent predictor of severe discomfort and poor tolerance in Chinese patients undergoing unsedated EGD. Our findings suggested the importance of the management of anxiety to reduce adverse endoscopic experience and taking high level of anxiety as an indication for sedation. [ABSTRACT FROM AUTHOR]

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases. [ABSTRACT FROM AUTHOR]

Bats are important reservoirs of many viruses, which are capable of infecting the host without inducing obvious clinical diseases. Interferon and the downstream interferon regulated genes (IRGs) are known to act as the first line of defense against viral infections. Little is known about the transcriptional profile of genes being induced by interferon in bats and their role in controlling virus infection. In this study, we constructed IFNAR2 knockout bat cell lines using CRISPR technology and further characterized gene expression profiles induced by the most abundant IFN-α (IFN-α3). Firstly, we demonstrated that the CRISPR/Cas9 system is for bat cells as this represents the first CRIPSR knockout cell line for bats. Our results showed the pleiotropic effect of IFN-α3 on the bat kidney cell line, PaKiT03. As expected, we confirmed that IFNAR2 is indispensable for IFN-a signaling pathway and plays an important role in antiviral immunity. Unexpectedly, we also identified novel IFNAR2-dependent IRGs which are enriched in pathways related to cancer. To our knowledge, this seems to be bat-specific as no such observation has been reported for other mammalian species. This study expands our knowledge about bat immunology and the cell line established can provide a powerful tool for future study into virus-bat interaction and cancer biology. [ABSTRACT FROM AUTHOR]

The endocytosis of transferrin receptor (TfR) has served as a model to study the receptor-targeted cargo delivery system for cancer therapy for many years. To accurately evaluate and optically measure this TfR targeting delivery in vitro, a CHO cell line with enhanced green fluorescent protein (EGFP)-tagged human TfR was established. A chimera of the hTfR and EGFP was engineered by fusing EGFP to the amino terminus of hTfR. Data were provided to demonstrate that hTfR-EGFP chimera was predominantly localized on the plasma membrane with some intracellular fluorescent structures on CHO cells and the EGFP moiety did not affect the endocytosis property of hTfR. Receptor internalization occurred similarly to that of HepG2 cells expressing wild-type hTfR. The internalization percentage of this chimeric receptor was about 81±3% of wild type. Time-dependent co-localization of hTfR-EGFP and PE-conjugated anti-hTfR mAb in living cells demonstrated the trafficking of mAb-receptor complexes through the endosomes followed by segregation of part of the mAb and receptor at the late stages of endocytosis. The CHO-hTfR cells preferentially took up anti-hTfR mAb conjugated nanoparticles. This CHO-hTfR cell line makes it feasible for accurate evaluation and visualization of intracellular trafficking of therapeutic agents conjugated with transferrin or Abs targeting the hTfRs. [ABSTRACT FROM AUTHOR]

In related research on queuing systems, in order to determine the system state, there is a widespread practice to assume that the system is stable and that distributions of the customer arrival ratio and service ratio are known information. In this study, the queuing system is looked at as a black box without any assumptions on the distribution of the arrival and service ratios and only keeping the assumption on the stability of the queuing system. By applying the principle of maximum entropy, the performance distribution of queuing systems is derived from some easily accessible indexes, such as the capacity of the system, the mean number of customers in the system, and the mean utilization of the servers. Some special cases are modeled and their performance distributions are derived. Using the chi-square goodness of fit test, the accuracy and generality for practical purposes of the principle of maximum entropy approach is demonstrated. [ABSTRACT FROM AUTHOR]

In this paper, a parametric method is introduced to solve fuzzy transportation problem. Considering that parameters of transportation problem have uncertainties, this paper develops a generalized fuzzy transportation problem with fuzzy supply, demand and cost. For simplicity, these parameters are assumed to be fuzzy trapezoidal numbers. Based on possibility theory and consistent with decision-makers' subjectiveness and practical requirements, the fuzzy transportation problem is transformed to a crisp linear transportation problem by defuzzifying fuzzy constraints and objectives with application of fractile and modality approach. Finally, a numerical example is provided to exemplify the application of fuzzy transportation programming and to verify the validity of the proposed methods. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is a worldwide liver disease and nearly 25% of chronic HBV infections terminate in hepatocellular carcinoma (HCC). Currently, there is no effective therapy to inhibit HBV replication and to eliminate hepatoma cells, making it highly desired to develop novel therapies for these two stages of the HBV-caused detrimental disease. Recently, short hairpin RNA (shRNA) has emerged as a potential therapy for virus-infected disease and cancer. Here, we have generated a shRNA, pGenesil-siHBV4, which effectively inhibits HBV replication in the human hepatoma cell line HepG2.2.15. The inhibitory effects of pGenesil-siHBV4 are manifested by the decrease of both the HBV mRNA level and the protein levels of the secreted HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), and by the reduction of secreted HBV DNA. Using mouse hydrodynamic tail vein injection, we demonstrate that pGenesil-siHBV4 is effective in inhibiting HBV replication in vivo. Because survivin plays a key role in cancer cell escape from apoptosis, we further generated pGenesil-siSurvivin, a survivin-silencing shRNA, and showed its effect of triggering apoptosis of HBV-containing hepatoma cells. To develop targeted shRNA therapy, we have identified that as a specific binder of the asialoglycoprotein receptor (ASGPR), jetPEI-Hepatocyte delivers pGenesil-siHBV4 and pGenesil-siSurvivin specifically to hepatocytes, not other types of cells. Finally, co-transfection of pGenesil-siHBV4 and pGenesil-siSurvivin exerts synergistic effects in inducing hepatoma cell apoptosis, a novel approach to eliminate hepatoma by downregulating survivin via multiple mechanisms. The application of these novel shRNAs with the jetPEI-Hepatocyte targeting strategy demonstrates the proof-of-principle for a promising approach to inhibit HBV replication and eliminate hepatoma cells with high specificity.

This study evaluated the performance of the Maxwell 16 System (Promega) for extraction of influenza virus (flu-v) RNA from diverse samples compared to a classical manual method (QIAamp Kit, QIAGEN). Following extraction by the two methods, all samples were analyzed by Real-time RT-PCR. Results revealed that the use of the standard Maxwell 16 protocol (Maxwell 16-S) resulted in good linearity and precision across a wide concentration range and higher sensitivity of detection from flu-v stock suspensions than the manual method. Compared with the latter method, Maxwell 16-S extracted RNA more efficiently (higher RNA yield and/or fewer PCR inhibitors) from throat swabs and bronchoalveolar lavage fluids, while both methods performed comparably on fecal samples from human and poultry in terms of overall threshold cycle values and detection rates although the Maxwell 16-S co-purified more inhibitors from fecal samples. The capacity of this system to remove inhibitors from fecal matrix was improved by using a modified Maxwell 16 protocol with a reduced sample input, which eliminated all false-negatives produced by the Maxwell 16-S. These findings suggest that the Maxwell 16 System is suitable for RNA extraction from multiple-source samples for diagnosis of influenza and viral load determination and that a proper reduction in starting sample volume may improve the detection of flu-v from complex matrices such as feces. Additionally, this system allows flexible sample throughput and labor-saving sample processing with little or no risk of cross-contamination.