Tristetraprolin down-regulates IL-23 expression in colon cancer cells.

mRNA 3'UTR demonstrated that the ARE cluster between the third and fifth AREs was responsible for TTP-mediated destabilization of IL-23 mRNA. A RNA electrophoretic mobility shift assay confirmed that TTP binds to this ARE cluster. Taken together, these results demonstrate that TTP acts as a negative regulator of IL-23 gene expression in mouse colon cancer cells and suggest its potential application as a novel therapeutic target to control IL-23-mediated tumor promotion

Ectopic over-expression of tristetraprolin in human cancer cells promotes biogenesis of let-7 by down-regulation of Lin28

Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3′-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3′-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells

Viral Etiology of Encephalitis in Children in Southern Vietnam: Results of a One-Year Prospective Descriptive Study

Viral encephalitis is associated with high morbidity and mortality in Vietnam. However little is known about the causes of the disease due to a lack of diagnostic facilities in this relatively resource-poor setting. Knowledge about the etiologies and clinical outcome of viral encephalitis is necessary for future design of intervention studies targeted at improvement of clinical management, treatment and prevention of the disease. We report the viral agents, clinical outcome and prognostic factors of mortality of encephalitis in children admitted to a referral hospital for children in southern Vietnam. We show that about one third of the enrolled patients die acutely, and that mortality is independently associated with patient age and Glasgow Coma Scale on admission. Japanese encephalitis, dengue virus and enterovirus (including enterovirus 71) are the major viruses detected in our patients. However, more than half of the patients remain undiagnosed, while mortality in this group is as high as in the diagnosed group. This study will benefit clinicians and public health in terms of clinical management and prevention of childhood encephalitis in Vietnam

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Herpesvirus lytic infection-induced mitophagy via viral interferon regulatory factor 1

ABSTRACTViral control of mitochondria via mitophagy has a dampening effect on mitochondrion-mediated innate immune responses. We previously found that human herpesvirus 8 (HHV-8) could activate mitophagy via its lytic gene product vIRF-1 (viral interferon regulatory factor 1). Mechanistically, we previously demonstrated that vIRF-1 interacts with the mitophagic proteins BNIP3L (BCL2 interacting protein 3 like) and TUFM (Tu translation elongation factor, mitochondrial). Despite these significant findings, however, the precise molecular mechanisms underlying vIRF-1-activated mitophagy, particularly with core components of the autophagy machinery, remained to be fully elucidated. We recently reported that vIRF-1 binds preferentially and directly to GABARAPL1 (GABA type A receptor associated protein like 1) in a noncanonical manner, and this interaction is essential for virus-productive replication. Furthermore, we found that BNIP3L is a crucial factor that promotes vIRF-1 oligomerization and associated mitophagy activation, including GABARAPL1 interaction with vIRF-1 and TUFM dimerization. Together, our findings deepen our understanding of lytic infection-induced mitophagy and provide the key protein-protein interactions involved in vIRF-1-mediated mitophagy.Abbreviations: ATG8, autophagy-related gene 8; BNIP3L, BCL2 interacting protein 3 like; HHV-8, human herpesvirus 8; GABARAP, GABA type A receptor associated protein; GABARAPL1, GABARAP-like 1; LC3, microtubule-associated protein 1 light chain 3; LIR, LC3-interacting region; Nix, NIP3-like protein X; TUFM, Tu translation elongation factor, mitochondria; vIRF-1, viral interferon regulatory factor 1; UIM, ubiquitin-interacting motif; VIR, vIRF-1-interacting region

NIX promotes the generation of vIRF-1 proteins with higher molecular weight.

(A) vIRF-1’s nuclear localization signal (NLS) and its mutation (NLSX). (B-C) Mitophagy (B) and immunoblot (C) analyses in HeLa.Kyotomito-mCE cells transfected with WT or NLSX vIRF-1 vector along with or without V5-NIX for 24 h. (D) IFA analysis of vIRF-1 and vIRF-1.NLSX in HeLa.Kyoto cells co-transfected with or without V5-NIX plasmid. Cells were fixed (Fix) before or after permeabilization (Per). Arrows indicate the co-localization of vIRF-1 and NIX in mitochondria. Scale bar, 10 μm. (E) Immunoblot analysis of the mitochondrial extracts derived from HeLa.Kyoto cells transfected with the indicated plasmids. (F) NanoBiT assay. 293T cells were transfected with the indicated NanoBiT plasmids with or without V5-NIX. Data are presented as the mean ± SD of six independent wells per condition. The one-way ANOVA test assessed the statistical significance of differences between groups, and the t-test was used for post hoc pairwise comparisons. **, p p < 0.05. Immunoblots of the cell extracts are shown below the chart.</p

vIRF-1 binds to GABARAPL1 in a manner different from cellular autophagy receptors.

(A) The schematic structure of GABARAPL1 and its variants, VIRX, HPX, and G116A, are defective in binding to vIRF-1, LIR-containing proteins, and in phosphatidylethanolamine-lipidation, respectively. GIR, GABARAPL1-interacting region; VIR, vIRF-1-interacting region; HP, hydrophobic pockets. (B-C) GST pull-down assays with the GST-fused GABARAPL1 variants and purified recombinant vIRF-1-T7 (B) and p62/SQSTM1 (C). (D) Co-IP assays for assessment of the intracellular interactions of the Flag-tagged mitophagy proteins vIRF-1, p62/SQSTM1, and NIX with V5-GABARAPL1 (WT and variants). 293T cells were transfected with the indicated proteins for 24 h. A longer exposure time was necessary to detect input Flag-NIX.</p

Analysis of the protein and mRNA expression of ATG8 proteins in latent and lytic iBCBL-1 cells.

(A) Immunoblots of extracts derived from iBCBL-1 cells treated with Dox for 0 to 3 days. Relative expression of each ATG8 protein was determined by dividing by the band intensity at 0 day and depicted in the bar graphs (right). (B) RT-qPCR analysis of the mRNA expression of ATG8 genes. iBCBL-1 cells were treated with or without Dox for 2 days. *, p (TIF)</p

GABARAPL1 plays an important role in NIX/vIRF-1-mediated mitophagy via the VIR, HP, and G116 regions.

Mitophagy assays in control and GABARAPL1 KO HeLa.Kyoto cells co-transfected with V5-NIX, vIRF-1-Flag, or HA-GABARAPL1 WT and variants (VIRX, HPX, and G116A) along with the reporter mito-mCE. More than 50 cells per sample were counted. The one-way ANOVA test was used to assess the statistical significance of differences between groups, and the t-test was used for post hoc pairwise comparisons. *, p p p (TIF)</p

vIRF-1 binds directly to GABARAPL1 in an LIR-independent manner.

(A) Schematic structure of vIRF-1:PD, Proline-rich domain; DBD, DNA-binding domain; and IAD, IRF-association domain. LC3-interacting regions (LIRs) were searched for using a position-specific scoring matrix (PSSM) via the web-based software iLIR, and a conserved LIR of vIRF-1 with the highest PSSM score was predicted at the N-terminal junction of the IAD, as noted. (B) GST pull-down assay to assess in vitro binding between vIRF-1 and ATG8 proteins (LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2). Purified recombinant T7-tagged vIRF-1 (vIRF-1-T7) was pulled down with 1μg of GST-fused ATG8 proteins immobilized on glutathione beads. GST, GST-SUMO, and GST-Ub were used as controls. (C) Co-immunoprecipitation (co-IP) assays for assessment of the intracellular interactions between vIRF-1 and ATG8 proteins in virus-infected cells. iBCBL-1 cells were reactivated by treatment with 1 μg/ml doxycycline (Dox) for 2 days and fractionated into the nuclear and cytoplasmic fractions. The fractions were immunoprecipitated with normal rabbit IgG (nIgG) and rabbit anti-vIRF-1 antibody, and the immunoprecipitated complex was analyzed by immunoblotting with the indicated antibodies. Lamin B1 and lactate dehydrogenase (LDH) were used as nuclear and cytoplasmic fraction markers, respectively. (D) Immunofluorescence assay of the co-localization of vIRF-1 and GABARAPL1 in mitochondria (TOM20) in lytically virus-infected cells. iBCBL-1 cells were reactivated by treatment with Dox for 2 days and were fixed (Fix) with 4% paraformaldehyde before permeabilization (Per) with 0.5% Triton X-100 or after permeabilization with 25 μg/ml of saponin. A rat anti-vIRF-1 antibody was used. Scale bar, 10 μm. (E) Mutation of the core residues in the predicted LIR of vIRF-1: both tryptophan at position 255 (W255) and leucine at position 258 (L258) were replaced with alanine. (F-G) GST pull-down assays for assessment of in vitro interactions of wild-type (WT) and A255/A258 vIRF-1 with GST-LC3B and GST-GABARAPL1. GST alone was used as a control. The whole-cell lysates of 293T cells expressing Flag-tagged vIRF-1 (vIRF-1-Flag) were used as input for the pull-down assay. (G) The intensities of co-precipitated vIRF-1 proteins relative to input were determined, and the data represent the mean ± SD of three independent experiments. ‘ns’, not significant. (H) Co-IP assay. 293T cells were co-transfected with vIRF-1(WT and A255/A258)-Flag with V5-LC3B or V5-GABARAPL1 and immunoprecipitated with anti-Flag antibody.</p