Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Theranostic Robot-Assisted Radical Prostatectomy: Things Understood and Not Understood.

OBJECTIVE: This study aimed to explore the benefits of theranostic robot-assisted radical prostatectomy (T-RARP) for clinically highly suspicious prostate cancer (PCa) without proven biopsies. MATERIAL AND METHODS: Between February 2016 and December 2020, we included men with clinically highly suspicious PCa in this study. They were assessed to have possible localized PCa without any initial treatments, and were categorized into previous benign biopsies or without biopsies. Furthermore, another group of malignant biopsies with RARP in the same time frame was adopted as the control group. The endpoints were to compare the oncological outcome and functional outcome between malignant biopsies with RARP and T-RARP. p < 0.05 was considered to be significant. RESULTS: We included 164 men with proven malignant biopsies treated with RARP as the control group. For T-RARP, we included 192 men. Among them, 129 were preoperatively benign biopsies, and 63 had no biopsies before T-RARP. Approximately 75% of men in the T-RARP group had malignant pathology in their final reports, and the other 25% had benign pathology. T-RARP provides several oncological advantages, such as a higher initial pathological T stage, lower Gleason grade, and lower odds of positive surgical margins. However, the biochemical recurrence rates were not significantly decreased. From our cohort, T-RARP (odds ratio with 95% confidence interval; erectile recovery: 3.19 (1.84-5.52), p < 0.001; continence recovery: 2.25 (1.46-3.48), p < 0.001) could result in better recovery of functional outcomes than malignant biopsies with RARP. CONCLUSIONS: For clinically highly suspicious PCa, T-RARP was able to detect around 75% of PCa cases and preserved their functional outcomes maximally. However, in 25% of men with benign pathology, approximately 6% would have incontinence and 10% would have erectile impairment. This part should be sufficiently informed of the potential groups considering T-RARP.

Oncolytic viruses-modulated immunogenic cell death, apoptosis and autophagy linking to virotherapy and cancer immune response.

Recent reports have revealed that oncolytic viruses (OVs) play a significant role in cancer therapy. The infection of OVs such as oncolytic vaccinia virus (OVV), vesicular stomatitis virus (VSV), parvovirus, mammalian reovirus (MRV), human adenovirus, Newcastle disease virus (NDV), herpes simplex virus (HSV), avian reovirus (ARV), Orf virus (ORFV), inactivated Sendai virus (ISV), enterovirus, and coxsackievirus offer unique opportunities in immunotherapy through diverse and dynamic pathways. This mini-review focuses on the mechanisms of OVs-mediated virotherapy and their effects on immunogenic cell death (ICD), apoptosis, autophagy and regulation of the immune system.

Oncolytic Avian Reovirus σA-Modulated Upregulation of the HIF-1α/C-myc/glut1 Pathway to Produce More Energy in Different Cancer Cell Lines Benefiting Virus Replication.

Our previous reports proved that the structural protein σA of avian reovirus (ARV) is an energy activator which can regulate cellular metabolism that is essential for virus replication. This study has further demonstrated that the ARV protein σA is able to upregulate the HIF-1α/myc/glut1 pathway in three cancer cell lines (A549, B16-F10, and HeLa) to alter the metabolic pathway of host cells. Quantitative real-time RT-PCR and Western blotting results have revealed that σA protein could enhance both mRNA and the protein levels of HIF-1α, c-myc, and glut1 in these cancer cell lines. In this work, ATeam immunofluorescence staining was used to reveal that knockdown of HIF-1α, c-myc, and glut1 by shRNAs decreased cellular ATP levels. Our data reveal that the ARV σA protein can downregulate lactate fermentation and upregulate glutaminolysis. The σA protein upregulates glutaminase, which converts glutamate into the TCA cycle intermediate α-ketoglutarate, activating the TCA cycle. In the lactate fermentation pathway, ARV σA protein suppresses lactate dehydrogenase A (LDHA), implying the Warburg effect does not occur in these cancer cell lines. This study provides a novel finding revealing that ARV σA protein upregulates glycolysis and glutaminolysis to produce energy using the HIF-1α/c-myc/glut1 pathway to benefit virus replication in these cancer cell lines.

TWEAK-Fn14 Axis Induces Calcium-Associated Autophagy and Cell Death To Control Mycobacterial Survival in Macrophages.

Autophagy is a natural defense mechanism that protects the host against pathogens. We previously demonstrated that mycobacterial infection upregulated tumor necrosis factor-like weak inducer of apoptosis (TWEAK) to promote autophagy and mycobacterial autophagosome maturation through activation of AMP-activated protein kinase (AMPK). Fibroblast growth factor-inducible 14 (Fn14) is the receptor of TWEAK. But the role of Fn14 in mycobacterial infection remains elusive. Herein, we observed increased expression of Fn14 in peripheral blood mononuclear cells of active tuberculosis (TB) patients. Downregulation of cellular Fn14 enhanced mycobacterial survival in macrophages. Conversely, Fn14 overexpression inhibited mycobacterial growth, suggesting that Fn14 can inhibit mycobacterial infection. The in vitro results revealed that TWEAK-promoted mycobacterial phagosome maturation is Fn14-dependent. We demonstrated that TWEAK-Fn14 signaling promotes oxidative stress to enhance the expression of stromal interaction molecule 1 (STIM1) and its activation of the Ca2+ channel ORAI1. Elevated calcium influx stimulated the activation of CaMCCK2 (calcium/calmodulin-dependent protein kinase kinase 2) and its downstream effector AMPK, thus inducing autophagy in early infection. Persistently TWEAK-Fn14 signaling caused cell death in late infection by reducing mitochondrial membrane potential, leading to mitochondrial ROS accumulation, and activating cell death-associated proteins. Genetic Fn14 deficiency or TWEAK blockers decreased oxidative stress-induced calcium influx, thus suppressing autophagy and cell death in mycobacteria-infected macrophages, and resulting in elevated mycobacterial survival. We propose that the TWEAK-Fn14 axis and calcium influx could be manipulated for anti-TB therapeutic purposes. Our results offer a new molecular machinery to understand the association between the TWEAK-Fn14 axis, calcium influx, and mycobacterial infection. IMPORTANCE Tuberculosis remains a major cause of morbidity and mortality worldwide. We previously demonstrated a relationship between TWEAK and activation of the autophagic machinery, which promotes anti-mycobacterial immunity. The TWEAK-Fn14 axis is multi-functional and involved in the pathogenesis of many diseases, thus blockade of TWEAK-Fn14 axis has been considered as a potential therapeutic target. Here, we demonstrated that the TWEAK-Fn14 axis plays a novel role in anti-mycobacterial infection by regulating calcium-associated autophagy. Persistently, TWEAK-Fn14 signaling caused cell death in late infection by reducing mitochondrial membrane potential, leading to mitochondrial ROS accumulation, and activating cell death-associated proteins. TWEAK blocker or Fn14 deficiency could suppress oxidative stress and calcium-associated autophagy, resulting in elevated mycobacterial survival. We propose that the TWEAK-Fn14 axis and calcium influx could be manipulated for anti-TB therapeutic purposes. This study offers a new molecular machinery to understand the association between the TWEAK-Fn14 axis, calcium influx, and mycobacterial infection.

Oncolytic Avian Reovirus p17-Modulated Inhibition of mTORC1 by Enhancement of Endogenous mTORC1 Inhibitors Binding to mTORC1 To Disrupt Its Assembly and Accumulation on Lysosomes.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Oncolytic avian reovirus σA-modulated fatty acid metabolism through the PSMB6/Akt/SREBP1/acetyl-CoA carboxylase pathway to increase energy production for virus replication.

We have demonstrated previously that the σA protein of avian reovirus (ARV) functions as an activator of cellular energy, which upregulates glycolysis and the TCA cycle for virus replication. To date, there is no report with respect to σA-modulated regulation of cellular fatty acid metabolism. This study reveals that the σA protein of ARV inhibits fatty acids synthesis and enhance fatty acid oxidation by upregulating PSMB6, which suppresses Akt, sterol regulatory element-binding protein 1 (SREBP1), acetyl-coA carboxylase α (ACC1), and acetyl-coA carboxylase ß (ACC2). SREBP1 is a transcription factor involved in fatty acid and cholesterol biosynthesis. Overexpression of SREBP1 reversed σA-modulated suppression of ACC1 and ACC2. In this work, a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams, was used to study σA-modulated inhibition of fatty acids synthesis which enhances cellular ATP levels in Vero cells and human cancer cell lines (A549 and HeLa). By using Ateams, we demonstrated that σA-modulated inhibition of Akt, SREBP1, ACC1, and ACC2 leads to increased levels of ATP in mammalian and human cancer cells. Furthermore, knockdown of PSMB6 or overexpression of SREBP1 reversed σA-modulated increased levels of ATP in cells, indicating that PSMB6 and SREBP1 play important roles in ARV σA-modulated cellular fatty acid metabolism. Furthermore, we found that σA R155/273A mutant protein loses its ability to enter the nucleolus, which impairs its ability to regulate fatty acid metabolism and does not increase ATP formation, suggesting that nucleolus entry of σA is critical for regulating cellular fatty acid metabolism to generate more energy for virus replication. Collectively, this study provides novel insights into σA-modulated inhibition of fatty acid synthesis and enhancement of fatty acid oxidation to produce more energy for virus replication through the PSMB6/Akt/SREBP1/ACC pathway.

p17-Modulated Hsp90/Cdc37 Complex Governs Oncolytic Avian Reovirus Replication by Chaperoning p17, Which Promotes Viral Protein Synthesis and Accumulation of Viral Proteins σC and σA in Viral Factories.

In this work we have determined that heat shock protein 90 (Hsp90) is essential for avian reovirus (ARV) replication by chaperoning the ARV p17 protein. p17 modulates the formation of the Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation. Inhibition of the Hsp90/Cdc37 complex by inhibitors (17-N-allylamino-17-demethoxygeldanamycin 17-AGG, and celastrol) or short hairpin RNAs (shRNAs) significantly reduced expression levels of viral proteins and virus yield, suggesting that the Hsp90/Cdc37 chaperone complex functions in virus replication. The expression levels of p17 were decreased at the examined time points (2 to 7 h and 7 to 16 h) in 17-AAG-treated cells in a dose-dependent manner while the expression levels of viral proteins σA, σC, and σNS were decreased at the examined time point (7 to 16 h). Interestingly, the expression levels of σC, σA, and σNS proteins increased along with coexpression of p17 protein. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability, maturation, and virus production. Virus factories of ARV are composed of nonstructural proteins σNS and µNS. We found that the Hsp90/Cdc37 chaperone complex plays an important role in accumulation of the outer-capsid protein σC, inner core protein σA, and nonstructural protein σNS of ARV in viral factories. Depletion of Hsp90 inhibited σA, σC, and p17 proteins colocalized with σNS in viral factories. This study provides novel insights into p17-modulated formation of the Hsp90/Cdc37 chaperone complex governing virus replication via stabilization and maturation of viral proteins and accumulation of viral proteins in viral factories for virus assembly. IMPORTANCE Molecular mechanisms that control stabilization of ARV proteins and the intermolecular interactions among inclusion components remain largely unknown. Here, we show that the ARV p17 is an Hsp90 client protein. The Hsp90/Cdc37 chaperone complex is essential for ARV replication by protecting p17 chaperone from ubiquitin-proteasome degradation. p17 modulates the formation of Hsp90/Cdc37 complex by phosphorylation of Cdc37, and this chaperone machinery protects p17 from ubiquitin-proteasome degradation, suggesting a feedback loop between p17 and the Hsp90/Cdc37 chaperone complex. p17 together with the Hsp90/Cdc37 complex does not increase viral genome replication but enhances viral protein stability and virus production. Depletion of Hsp90 prevented viral proteins σA, σC, and p17 from colocalizing with σNS in viral factories. Our findings elucidate that the Hsp90/Cdc37 complex chaperones p17, which, in turn, promotes the synthesis of viral proteins σA, σC, and σNS and facilitates accumulation of the outer-capsid protein σC and inner core protein σA in viral factories for virus assembly.

Molecular chaperone TRiC governs avian reovirus replication by protecting outer-capsid protein σC and inner core protein σA and non-structural protein σNS from ubiquitin- proteasome degradation.

Avian reoviruses (ARVs) are important pathogens that cause considerable economic losses in poultry farming. To date, host factors that control stabilization of ARV proteins remain largely unknown. In this work we determined that the eukaryotic chaperonin T-complex protein-1 (TCP-1) ring complex (TRiC) is essential for avian reovirus (ARV) replication by stabilizing outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV. TriC serves as a chaperone of viral proteins and prevent their degradation via the ubiquitin-proteasome pathway. Furthermore, reciprocal co-immunoprecipitation assays confirmed the association of viral proteins (σA, σC, and σNS) with TRiC. Immunofluorescence staining indicated that the TRiC chaperonins (CCT2 and CCT5) are colocalized with viral proteins σC, σA, and σNS of ARV. In this study, inhibition of TRiC chaperonins (CCT2 and CCT5) by the inhibitor HSF1A or shRNAs significantly reduced expression levels of the σC, σA, and σNS proteins of ARV as well as virus yield, suggesting that the TRiC complex functions in stabilization of viral proteins and virus replication. This study provides novel insights into TRiC chaperonin governing virus replication via stabilization of outer-capsid protein σC, inner core protein σA, and the non-structural protein σNS of ARV.

Genomic and phylogenetic analysis of avian polyomaviruses isolated from parrots in Taiwan.

Avian polyomavirus (APV) is a non-enveloped virus with a circular double-stranded DNA genome approximately 5000 bp in length. APV was first reported in fledgling budgerigars (Melopsittacus undulatus) as the causative agent of budgerigar fledgling disease, resulting in high parrot mortality rates in the 1980s. This disease has been observed worldwide, and APV has a wide host range including budgerigars, cockatoos, lorikeets, lovebirds, and macaws. Twenty APV isolates have been collected from healthy and symptomatic parrots in Taiwan from 2015 to 2019. These isolates were then amplified via polymerase chain reaction, after which the whole genomes of these isolates were sequenced. The overall APV-positive rate was 14.2%, and the full lengths of the APV Taiwan isolates varied from 4971 to 4982 bps. The APV genome contains an early region that encodes two regulatory proteins (the large tumor antigen (Large T-Ag) and the small tumor antigen (Small t-Ag)) and a late region which encodes the capsid proteins VP1, VP2, VP3, and VP4. The nucleotide identities of the VP1 and VP4 genes ranged from 98.7 to 100%, whereas the nucleotide sequence of the Large T-Ag gene had the highest identity (99.2-100%) relative to other APV isolates from the GenBank database. A phylogenetic tree based on the whole genome demonstrated that the APV Taiwan isolates were closely related to Japanese and Portuguese isolates. Recombination events were analyzed using the Recombination Detection Program version 4 and APV Taiwan isolate TW-3 was identified as a minor parent of the APV recombinants. In this study, we first reported the characterization of the whole genome sequences of APV Taiwan isolates and their phylogenetic relationships with all APV isolates available in the GenBank database.

Hepatitis C Virus-Induced Exosomal MicroRNAs and Toll-Like Receptor 7 Polymorphism Regulate B-Cell Activating Factor.

There are large gaps in understanding the molecular machinery accounting for the association of hepatitis C virus (HCV) infection with autoimmunity. Mixed cryoglobulinemia (MC) is the most common HCV-associated extrahepatic manifestation, which is characterized by B-cell lymphoproliferation and autoantibody production. B-cell activating factor (BAFF) is a member of the tumor necrosis factor family and plays an important role in B-cell proliferation. We explored the roles of hepatocyte-derived exosomal microRNAs (exo-miRNAs) and BAFF in the extrahepatic diseases of HCV infection. The exo-miRNA profiles were explored using a next-generation sequencing approach, followed by quantitative reverse transcription-PCR validation. The Toll-like receptor 7 (TLR7) polymorphism were analyzed using quantitative PCR. The biological function of exo-miRNAs and TLR7 polymorphism in BAFF expression was evaluated by using immunoblotting and enzyme-linked immunosorbent assay. Significantly increased levels of BAFF, exosomes, and TLR7 were found in HCV patients, particularly in those with MC (P < 0.005). HCV-infected hepatocyte-derived miR-122/let-7b/miR-206 upregulated BAFF expression in human macrophages through exosome transmission and TLR7 activation. Analysis of a TLR7 single-nucleotide polymorphism (rs3853839) revealed that G-allele carriers had increased TLR7 transcripts, resulting in more BAFF expression induced by hepatocyte-derived exo-miR-122, compared to those in C-allele carriers (P < 0.005). We identified HCV-infected hepatocyte-derived GU-enriched miRNAs (e.g., miR-122/let-7b/miR-206) as a TLR7 ligand that could induce BAFF production in macrophages through exosome transmission. The polymorphism in TLR7 is associated with the BAFF levels induced by exo-miR-122. It may be a potential predisposing factor of MC syndrome development. IMPORTANCE HCV remains an important cause of liver disease worldwide. Accumulating evidence has demonstrated that HCV infection is associated with B cell lymphoproliferative disorders such as MC. Approximately half of the patients infected with HCV develop MC, but the real reason and regulatory mechanism is still uncertain. Here, we demonstrate a novel relationship between HCV-infected hepatocyte-derived exo-miRNAs, host genetic background in TLR7, and BAFF expression. We validate that HCV-induced GU-enriched miRNAs (e.g., miR-122, let-7b, and miR-206) upregulated BAFF expression through exosome transmission and TLR7 activation. This mechanism of miRNAs action is implicated in HCV-infected hepatocyte-immune system communication and is important in extrahepatic manifestation development, thus representing a possible target for HCV infection and extrahepatic diseases treatment. In addition, we show that a functional polymorphism in TLR7 is a potential predisposing factor of MC development. Our results elucidate the molecular machinery in order to better understand the association of HCV infection with autoimmunity.

MicroRNA-223 inhibits neutrophil extracellular traps formation through regulating calcium influx and small extracellular vesicles transmission.

Modulation of miRNAs and neutrophil extracellular traps (NETs) formation are both implicated in inflammatory disorders. Adult-onset Still's disease (AOSD) is a systemic autoinflammatory disease with neutrophilic leukocytosis and unknown etiology. Although the NETs formation is elevated in AOSD patients, the regulatory roles of miRNAs in NETs formation in AOSD remains unclear. We revealed that the circulating levels of IL-18, NETs, and miR-223 were significantly higher in active AOSD patients, compared with inactive AOSD patients or healthy controls (P < 0.005). Moreover, IL-18 increased calcium influx into neutrophils, which led to mitochondrial ROS (mROS) production and NETs formation. Elevated levels of NETs-DNA could induce miR-223 expression in neutrophils through activating Toll-like receptor 9. The upregulated miR-223 expression in neutrophils suppressed mROS production by blocking calcium influx, and subsequently inhibited IL-18-mediated NETs formation. Besides, the increased neutrophil-derived exosomal miR-223 levels were observed in active AOSD patients compared with healthy controls (P < 0.005). Our in vitro assays demonstrated that the neutrophil-derived small extracellular vesicles carried miR-223, which could repress IL-18 production in macrophages. Together, these results suggest a fine-tuned mechanism between inflammatory (IL-18 induced NETs) and anti-inflammatory (miR-223) factors in AOSD. MiR-223, mROS inhibitors, and calcium channel blockers are the potential therapeutics for autoinflammatory diseases such as AOSD.

Aspirin and 5-Aminoimidazole-4-carboxamide Riboside Attenuate Bovine Ephemeral Fever Virus Replication by Inhibiting BEFV-Induced Autophagy.

Recent study in our laboratory has demonstrated that BEFV-induced autophagy via activation of the PI3K/Akt/NF-κB and Src/JNK pathways and suppression of the PI3K-AKt-mTORC1 pathway is beneficial for virus replication. In the current study, we found that both aspirin and 5-aminoimidazole-4-carboxamide-1-ß-riboside (AICAR) siginificantly attenuated virus replication by inhibiting BEFV-induced autophagy via suppressing the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways as well as inducing reversion of the BEFV-suppressed PI3K-Akt-mTORC1 pathway. AICAR reversed the BEFV-activated PI3K/Akt/NF-κB and Src/JNK pathways at the early to late stages of infection and induced reversion of the BEFV-suppressed PI3K-AKt-mTORC1 pathway at the late stage of infection. Our findings reveal that inhibition of BEFV-induced autophagy by AICAR is independent of AMPK. Furthermore, we found that AICAR transcriptionally downregulates the ATG related genes ULK1, Beclin 1, and LC3 and enhances Atg7 degradation by the proteasome pathway. Aspirin suppresses virus replication by inhibiting BEFV-induced autophagy. It directly suppressed the NF-κB pathway and reversed the BEFV-activated Src/JNK pathway at the early stage of infection and reversed the BEFV-suppressed PI3K/Akt/mTOR pathway at the late stage of infection. The current study provides mechanistic insights into the effects of aspirin and AICAR on BEFV replication through suppression of BEFV-induced autophagy.

CUX2, BRAP and ALDH2 are associated with metabolic traits in people with excessive alcohol consumption.

Molecular mechanisms that prompt or mitigate excessive alcohol consumption could be partly explained by metabolic shifts. This genome-wide association study aims to identify the susceptibility gene loci for excessive alcohol consumption by jointly measuring weekly alcohol consumption and γ-GT levels. We analysed the Taiwan Biobank data of 18,363 Taiwanese people, including 1945 with excessive alcohol use. We found that one or two copies of the G allele in rs671 (ALDH2) increased the risk of excessive alcohol consumption, while one or two copies of the C allele in rs3782886 (BRAP) reduced the risk of excessive alcohol consumption. To minimize the influence of extensive regional linkage disequilibrium, we used the ridge regression. The ridge coefficients of rs7398833, rs671 and rs3782886 were unchanged across different values of the shrinkage parameter. The three variants corresponded to posttranscriptional activity, including cut-like homeobox 2 (a protein coded by CUX2), Glu504Lys of acetaldehyde dehydrogenase 2 (a protein encoded by ALDH2) and Glu4Gly of BRCA1-associated protein (a protein encoded by BRAP). We found that Glu504Lys of ALDH2 and Glu4Gly of BRAP are involved in the negative regulation of excessive alcohol consumption. The mechanism underlying the γ-GT-catalytic metabolic reaction in excessive alcohol consumption is associated with ALDH2, BRAP and CUX2. Further study is needed to clarify the roles of ALDH2, BRAP and CUX2 in the liver-brain endocrine axis connecting metabolic shifts with excessive alcohol consumption.

MicroRNA-889 Inhibits Autophagy To Maintain Mycobacterial Survival in Patients with Latent Tuberculosis Infection by Targeting TWEAK.

Autophagy plays an important role in protecting the host against pathogens. Mycobacterium tuberculosis can suppress autophagy and then remain dormant and survive within the host for an extended period, which is responsible for latent tuberculosis infection (LTBI). Here, we explored the role of microRNAs (miRNAs) in LTBI. The miRNA profiles were explored using the next-generation sequencing approach, followed by quantitative reverse transcription-PCR validation. The biological function of candidate miRNA was evaluated using immunoblotting, immunofluorescence techniques, and enzyme-linked immunosorbent assay in an in vitro human TB granuloma model. An increased miR-889 expression was observed in patients with LTBI compared with that in patients without infection. The reporter assay identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as the target of miR-889. Mycobacterial infection induced TWEAK upregulation in the early phase. TWEAK induced autophagy and promoted mycobacterial autophagosome maturation through activation of AMP-activated protein kinase (AMPK). Upon entry to LTBI status, elevated miR-889 levels were associated with TNF alpha (TNF-α) and granuloma formation/maintenance. MiR-889 inhibited autophagy via posttranscriptional suppression of TWEAK expression to maintain mycobacterial survival in granulomas. Adalimumab (anti-TNF-α monoclonal antibody) treatment reduced levels of both TNF-α and miR-889 and caused granuloma destruction and LTBI reactivation. The circulating miR-889 and TWEAK levels were correlated with LTBI and subsequently associated with anti-TNF-α-related LTBI reactivation in patients. We propose that miR-889 and TWEAK can act as targets that can be manipulated for antimycobacterial therapeutic purposes and act as candidate biomarkers for LTBI and LTBI reactivation, respectively.IMPORTANCE TB remains a leading cause of morbidity and mortality worldwide. Approximately one-quarter of the world's population has latent TB infection. TWEAK is a multiple-function cytokine and may be used as a target for the treatment of rheumatic diseases, cardiovascular diseases, and renal diseases. Here, we demonstrated a novel relationship between TWEAK and activation of the autophagic machinery which promotes antimycobacterial immunity. Additionally, TB infection is highly dynamic and determined by the interaction between the host and mycobacterium. We demonstrated a mechanism of fine-tuned balance between the mycobacterium and host for granuloma formation and/or maintenance in LTBI status. Once patients entered LTBI status, the upregulation of miR-889 was associated with TNF-α levels and granuloma formation to maintain mycobacterial survival. Adalimumab (a TNF-α inhibitor) reduced both TNF-α and miR-889 levels and caused LTBI reactivation and, thus, TWEAK enhancement. MiR-889 and TWEAK may become potential diagnostic biomarkers or therapeutic targets for LTBI and LTBI reactivation, respectively.

Cdc20 and molecular chaperone CCT2 and CCT5 are required for the Muscovy duck reovirus p10.8-induced cell cycle arrest and apoptosis.

This study demonstrates that the Muscovy duck reovirus (MDRV) p10.8 protein is one of many viral non-structural proteins that induces both cell cycle arrest and apoptosis. The p10.8 but not σC is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm. Our results reveal that p10.8-induced apoptosis in cultured cells occurs by the nucleoporin Tpr/p53-dependent and Fas/caspase 8-mediated pathways. Furthermore, a compelling finding from this study is that the p10.8 and σC proteins of MDRV facilitate CDK2 and CDK4 degradation via the ubiquitin-proteasome pathway. We found that depletion of Cdc20 reversed the p10.8- and σC- mediated CDK4 degradation and p10.8-induced apoptosis, suggesting that Cdc20 plays a critical role in modulating p10.8-mediated cell cycle and apoptosis. Furthermore, we found that depletion of chaperonin-containing tailless complex polypeptide 1 (CCT) 2 and CCT5 reduced the level of Cdc20 and reversed the p10.8- and σC-mediated CDK4 degradation and p10.8-induced apoptosis, indicating that molecular chaperone CCT2 and CCT5 are required for stabilization of Ccd20 for mediating both cell cycle arrest and apoptosis. This study provides mechanistic insights into how p10.8 induces both cell cycle arrest and apoptosis.

Avian reovirus σA-modulated suppression of lactate dehydrogenase and upregulation of glutaminolysis and the mTOC1/eIF4E/HIF-1α pathway to enhance glycolysis and the TCA cycle for virus replication.

Adenosine triphosphate (ATP) is an energy source for many types of viruses for facilitating virus replication. This is the first report to demonstrate that the structural protein σA of avian reovirus (ARV) functions as an activator of cellular energy. Three cellular factors, isocitrate dehydrogenase 3 subunit beta (IDH3B), lactate dehydrogenase A (LDHA), and vacuolar-type H+-ATPase (vATPase) co-immunoprecipitated with ARV σA and were identified by 2D-LC/MS/MS. ARV enhances glycolytic flux through upregulation of glycolytic enzymes. Increased ATP levels in both ARV-infected and σA-transfected cells were observed by a fluorescence resonance energy transfer-based genetically encoded indicator, Ateams. Furthermore, σA upregulates IDH3B and glutamate dehydrogenase (GDH) to promote glutaminolysis, activating HIF-1α. Both HIF-1α level and viral yield in IDH3B-depleted and glutamine-deprived cells, and inhibition of glutaminolysis was significantly reduced. The σAR155/273A mutant loses its ability to enter the nucleolus, impairing its ability to regulate glycolysis. In addition, we have identified the conserved untranslated regions (UTR) of the 5'- and 3'-termini of the ARV genome segments that are required for viral protein synthesis in an ATP-dependent manner. Deletion of either the 5'- or 3'-UTR impaired viral protein synthesis. Knockdown of σA reduced the ATP level and significantly decreased virus yield, suggesting that σA enhances ATP formation to promote virus replication. Collectively, this study provides novel insights into σA-modulated suppression of LDHA and activation of IDH3B and GDH to activate the mTORC1/eIF4E/HIF-1α pathways to upregulate glycolysis and the TCA cycle for virus replication.

Mechanistic insights into avian reovirus p17-modulated suppression of cell cycle CDK-cyclin complexes and enhancement of p53 and cyclin H interaction.

The avian reovirus p17 protein is a nucleocytoplasmic shuttling protein. Although we have demonstrated that p17 causes cell growth retardation via activation of p53, the precise mechanisms remain unclear. This is the first report that avian reovirus p17 possesses broad inhibitory effects on cell cycle CDKs, cyclins, CDK-cyclin complexes, and CDK-activating kinase activity in various mammalian, avian, and cancer cell lines. Suppression of CDK activity by p17 occurs by direct binding to CDKs, cyclins, and CDK-cyclin complexes; transcriptional down-regulation of CDKs; cytoplasmic retention of CDKs and cyclins; and inhibition of CDK-activating kinase activity by promoting p53-cyclin H interaction. p17 binds to CDK-cyclin except for CDK1-cyclin B1 and CDK7-cyclin H complexes. We have determined that the negatively charged 151LAVXDVDA(E/D)DGADPN165 motif in cyclin B1 interacts with a positively charged region of CDK1. p17 mimics the cyclin B1 sequence to compete for CDK1 binding. The PSTAIRE motif is not required for interaction of CDK1-cyclin B1, but it is required for other CDK-cyclin complexes. p17 interacts with cyclins by its cyclin-binding motif, 125RXL127 Sequence and mutagenic analyses of p17 indicated that a 140WXFD143 motif and residues Asp-113 and Lys-122 in p17 are critical for CDK2 and CDK6 binding, leading to their sequestration in the cytoplasm. Exogenous expression of p17 significantly enhanced virus replication, whereas p17 mutants with low binding ability to cell cycle CDKs had no effect on virus yield, suggesting that p17 inhibits cell growth and the cell cycle, benefiting virus replication. An in vivo tumorigenesis assay also showed a significant reduction in tumor size.

A set of cancer stem cell homing peptides associating with the glycan moieties of glycosphingolipids.

Cancer stem cells (CSCs) are currently believed to be involved in tumor metastasis and relapse. And treatments against CSCs are well concerned issues. Peptides targeting to mouse and human CSCs were screened from an M13 phage display library. The first subset of cancer stem cell homing peptides (CSC HPs), CSC HP-1 to -12, were screened with mouse EMT6 breast cancer stem cells. Among them, CSC HP-1, CSC HP-3, CSC HP-8, CSC HP-9, and CSC HP-10 can bind to mouse CT26 colon CSCs; CSC HP-1, CSC HP-2, CSC HP-3, and CSC HP-8 can bind to mouse Hepa1-6 liver CSCs; as well as CSC HP-1, CSC HP-2, CSC HP-3, CSC HP-8, CSC HP-9, CSC HP-10, and CSC HP-11 can bind to human PANC-1 pancreatic CSCs. The second subset of cancer stem cell homing peptides, CSC HP-hP1 to -hP3, were screened with human PANC-1 pancreatic CSCs. Both CSC HP-hP1 and CSC HP-hP2 were demonstrated able to bind mouse EMT6, CT26 and Hepa1-6 CSCs as well as human colorectal HT29 and lung H1650 CSCs. CSC HP-1 and CSC HP-hP1 could strongly associate with the Globo 4 and Lewis Y glycan epitopes coupled on a microarray chip or Globo 4 and Globo H conjugated on bovine serum albumin. CSC HP-10, CSC HP-11 and CSC HP-hP2 could associate with the disialylated saccharide Neu5Ac-α-2,6-Gal-ß-1,3-(Neu5Ac-α-2,6)-GalNAc coupled on a microarray chip. These results indicate that the CSC HPs may target to the known stem cell glycan markers GbH and Lewis Y as well as the disialylated saccharide.