Ebola virus replication is regulated by the phosphorylation of viral protein VP35

Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation.     

Analyses of putative anti-cancer potential of three STAT3 signaling inhibitory compounds derived from Salvia officinalis

The extract of Salvia officinalis (Common Sage) exhibited inhibitory activity of STAT3 signal after screening of several plants extracts using the STAT3-responsive reporter system. Cirsiliol, luteolin, and carnosol were identified from the methanol extract of Silvia officinalis as inhibitors of STAT3 signaling and the effects of these three compounds on STAT3 protein or growth inhibition on cancer cells was compared. Luteolin at the dose of 90 μM clearly suppressed the phosphorylation of STAT3 induced by IL-6, while carnosol was prone to decrease total STAT3 proteins at high doses (>90 μM). Cirsiliol had almost no effect. Since the three compounds exhibited similar concentration-dependent suppression patterns in the reporter assay except for cirsiliol became plateau beyond 30 μM, these compounds appeared to function as STAT3 inhibitory factors in different ways. The direct anti-proliferative activity of three compounds was examined with or without the anti-cancer drug gefitinib using HepG2 and A549 cells. The anti-proliferative effect of the three compounds was additively enhanced by gefitinib. At the doses of 3.6 μM, statistically significant suppression of proliferation was observed in HepG2 cells only by cirsiliol among the three compounds in the absence of gefitinib but all three compounds were prone to suppress the proliferation of HepG2 cells and A549 cells dose-dependently although cirsiliol showed a modest dose-dependency and this suppression of proliferation was enhanced by the addition of gefitinib. Cirsiliol, a dimethyoxylated flavone, activated the natural killer activity of KHYG-1 cells against erythroleukemia K562 cells like a hexamethoxylated flavone, nobiletin, suggesting that it may also have an indirect anti-cancer potential through activation of NK cells. These results shed light on the putative anti-cancer potential of Salvia officinalis.     

Pretreatment of outer membrane vesicle and subsequent infection with influenza virus induces a long-lasting adaptive immune response against broad subtypes of influenza virus

Influenza is an acute respiratory disease and a global health problem. Although influenza vaccines are commercially available, frequent antigenic changes in hemagglutinin might render them less effective or unavailable. We previously reported that modified outer membrane vesicle (fmOMV) provided immediate and robust protective immunity against various subtypes of influenza virus. However, the effect was transient because it was innate immunity-dependent. In this study, we investigated the effects of consecutive administration of fmOMV and influenza virus on the adaptive immune response and long-term protective immunity against influenza virus. When the mice were pretreated with fmOMV and subsequently infected with influenza virus, strong influenza-specific antibody and T cell responses were induced in both systemic and lung mucosal compartments without pathogenic symptoms. Upon the secondary viral challenge at week 4, the mice given fmOMV and influenza virus exhibited almost complete protection against homologous and heterologous viral challenge. More importantly, this strong protective immunity lasted up to 18 weeks after the first infection. These results show that pretreatment with fmOMV and subsequent infection with influenza virus efficiently induces broad and long-lasting protective immunity against various virus subtypes, suggesting a novel antiviral strategy against newly-emerging viral diseases without suitable vaccines or therapeutics.     

K562 cell-derived exosomes suppress the adhesive function of bone marrow mesenchymal stem cells via delivery of miR-711

A failure of bone marrow mesenchymal stem cells (BM-MSCs) to adhere to hematopoietic cells is an essential cause of the progression of chronic myelogenous leukemia and is also a cause of failure of bone marrow (BM) transplantation, but the exact mechanisms of this have not been fully elucidated. Recent studies have indicated that microRNAs (miRNAs) are contained in leukemia-derived exosomes and are involved in modulating the BM microenvironment. In this study, we found that K562 cell-derived exosomes transfer miR-711 to BM-MSCs and suppress the adhesive function of BM-MSCs. Using qRT-PCR, we also confirmed a significantly higher level of miR-711 in exosomes derived from K562 cells than in exosomes derived from parental cells. The BM-MSCs co-cultured with exosomes derived from K562 cells showed a lower adhesion rate than did controls. We further demonstrated that exosomal transfer of miR-711 induced decreased adhesive abilities by inhibiting expression of adhesion molecule CD44 in BM-MSCs. In conclusion, our study reveals that K562 cell-derived exosomal miR-711 can be transferred to BM-MSCs and weaken adhesive abilities by silencing the expression of the adhesion molecule CD44.     

A current view on Tau protein phosphorylation in Alzheimer's disease

The functions of the neuronal microtubule-associated protein Tau in the central nervous system are regulated by manifold posttranslational modifications at more than 50 sites. Tau in healthy neurons carries multiple phosphate groups, mostly in its microtubule assembly domain. Elevated phosphorylation and aggregation of Tau are widely considered pathological hallmarks in Alzheimer’s disease (AD) and other tauopathies, triggering the quest for Tau posttranslational modifications in the disease context. However, the phosphorylation patterns of physiological and pathological Tau are surprisingly similar and heterogenous, making it difficult to identify specific modifications as therapeutic targets and biomarkers for AD. We present a concise summary of - and view on - important previous and recent advances in Tau phosphorylation analysis in the context of AD.     

Comparison of autophagy inducibility in various tyrosine kinase inhibitors and their enhanced cytotoxicity via inhibition of autophagy in cancer cells in combined treatment with azithromycin

Tyrosine kinase inhibitors (TKIs) induce autophagy in many types of cancer cells. We previously reported that gefitinib (GEF) and imatinib (IMA) induce autophagy in epidermal growth factor receptor (EGFR) knock-out A549 and non-BCR-ABL-expressing leukemia cell lines, respectively. This evidence suggests that TKI-induced autophagy is independent of the original target molecules. The present study compared the autophagy-inducing abilities of various TKIs, regardless of their targets, by quantitative autophagy flux assay. We established stable clones expressing the GFP-LC3-mCherry-LC3ΔG plasmid in A549, PC-9, and CAL 27 cell lines and assessed autophagy inducibility by monitoring the fluorescent ratios of GFP-LC3 to mCherry-LC3ΔG using an IncuCyte live cell imaging system during exposure to TKIs viz; GEF, osimertinib (OSI), lapatinib (LAP), lenvatinib (LEN), sorafenib (SOR), IMA, dasatinib (DAS), and tivantinib (TIV). Among these TKIs, DAS, GEF, and SOR exhibited prominent autophagy induction in A549 and PC-9 cells. In CAL 27 cells, IMA, SOR, and LEN, but not GEF, TIV, or OSI, exhibited autophagy induction. In the presence of azithromycin (AZM), which showed an inhibitory effect on autophagy flux, TKIs with prominent autophagy inducibility exhibited enhanced cytotoxicity via non-apoptotic cell death relative to effects of TKI alone. Therefore, autophagy inducibility of TKIs differed in the context of cancer cells. However, once induced, they appeared to have cytoprotective functions. Thus, blocking TKI-induced autophagy with AZM may improve the therapeutic effect of TKIs in cancer cells.     

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

Proteinaceous cysteine residues act as privileged sensors of oxidative stress. As reactive oxygen and nitrogen species have been implicated in numerous pathophysiological processes, deciphering which cysteines are sensitive to oxidative modification and the specific nature of these modifications is essential to understanding protein and cellular function in health and disease. While established mass spectrometry-based proteomic platforms have improved our understanding of the redox proteome, the widespread adoption of these methods is often hindered by complex sample preparation workflows, prohibitive cost of isotopic labeling reagents, and requirements for custom data analysis workflows. Here, we present the SP3-Rox redox proteomics method that combines tailored low cost isotopically labeled capture reagents with SP3 sample cleanup to achieve high throughput and high coverage proteome-wide identification of redox-sensitive cysteines. By implementing a customized workflow in the free FragPipe computational pipeline, we achieve accurate MS1-based quantitation, including for peptides containing multiple cysteine residues. Application of the SP3-Rox method to cellular proteomes identified cysteines sensitive to the oxidative stressor GSNO and cysteine oxidation state changes that occur during T cell activation.     

Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein—SFTPB (pulmonary surfactant protein B)—predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12–4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker—and perhaps mediator—of CVD risk in patients with T1DM.     

First report of computational protein–ligand docking to evaluate susceptibility to HIV integrase inhibitors in HIV-infected Iranian patients

BackgroundIran has recently included integrase (INT) inhibitors (INTIs) in the first‐line treatment regimen in human immunodeficiency virus (HIV)-infected patients. However, there is no bioinformatics data to elaborate the impact of resistance-associated mutations (RAMs) and naturally occurring polymorphisms (NOPs) on INTIs treatment outcome in Iranian patients.MethodIn this cross-sectional survey, 850 HIV-1-infected patients enrolled; of them, 78 samples had successful sequencing results for INT gene. Several analyses were performed including docking screening, genotypic resistance, secondary/tertiary structures, post-translational modification (PTM), immune epitopes, etc.ResultThe average docking energy (E value) of different samples with elvitegravir (EVG) and raltegravir (RAL) was more than other INTIs. Phylogenetic tree analysis and Stanford HIV Subtyping program revealed HIV-1 CRF35-AD was the predominant subtype (94.9%) in our cases; in any event, online subtyping tools confirmed A1 as the most frequent subtype. For the first time, CRF-01B and BF were identified as new subtypes in Iran. Decreased CD4 count was associated with several factors: poor or unstable adherence, naïve treatment, and drug user status.ConclusionAs the first bioinformatic report on HIV-integrase from Iran, this study indicates that EVG and RAL are the optimal INTIs in first-line antiretroviral therapy (ART) in Iranian patients. Some conserved motifs and specific amino acids in INT-protein binding sites have characterized that mutation(s) in them may disrupt INT-drugs interaction and cause a significant loss in susceptibility to INTIs. Good adherence, treatment of naïve patients, and monitoring injection drug users are fundamental factors to control HIV infection in Iran effectively.     

Biochemical characterization of TRIM72 E3 ligase and its interaction with the insulin receptor substrate 1

TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction.     

Natural COA water inhibits mitochondrial ROS-mediated apoptosis through Plk3 downregulation under STZ diabetic stress in pancreatic β-cell lines

Diabetes from pancreatic β cell death and insulin resistance is a serious metabolic disease in the world. Although the overproduction of mitochondrial reactive oxygen species (ROS) plays an important role in the pathogenesis of diabetes, its specific molecular mechanism remains unclear. Here, we show that the natural Charisma of Aqua (COA) water plays a role in Streptozotocin (STZ) diabetic stress-induced cell death inhibition. STZ induces mitochondrial ROS by increasing Polo-like kinase 3 (Plk3), a major mitotic regulator, in both Beta TC-6 and Beta TC-tet mouse islet cells and leads to apoptosis. Overexpression of Plk3 regulates an increase in mitochondrial ROS as well as cell death, also these events were inhibited by Plk3 gene knockdown in STZ diabetic stimulated-Beta TC-6 cells. Interestingly, we found that natural COA water blocks mitochondrial ROS generation through the reduction of Plk3 and prevents apoptosis in STZ-treated beta cells. Furthermore, using the 3D organoid (ex vivo) system, we confirmed that the insulin secretion of the supernatant medium under STZ treated pancreatic β-cells is protected by the natural COA water. These findings demonstrate that the natural water COA has a beneficial role in maintaining β cell function through the inhibition of mitochondrial ROS-mediated cell death, and it might be introduced as a potential insulin stabilizer.     

Semaphorin 3A potentiates the profibrotic effects of transforming growth factor-β1 in the cornea

Corneal scarring is a major cause of blindness worldwide with few effective therapeutic options. Finding a treatment would be of tremendous public health benefit, but requires a thorough understanding of the complex interactions that underlie this phenomenon. Here, we tested the hypothesis that the large increase in expression of Semaphorin 3A (SEMA3A) in corneal wounds contributes to the development of stromal fibrosis. We first verified this increased expression in vivo, in a cat model of photorefractive keratectomy-induced corneal wounding. We then examined the impact of adding exogenous SEMA3A to cultured corneal fibroblasts, and assessed how this affected the ability of transforming growth factor-beta1 (TGF-β1) to induce their differentiation into myofibroblasts. Finally, we examined how siRNA knockdown of endogenous SEMA3A affected these same phenomena. We found exogenous SEMA3A to significantly potentiate TGF-β1’s profibrotic effects, with only a minimal contribution from cell-intrinsic SEMA3A. Our results suggest a previously unrecognized interaction between SEMA3A and TGF-β1 in the wounded cornea, and a possible contribution of SEMA3A to the regulation of tissue fibrosis and remodeling in this transparent organ.     

Discovery and optimization of novel pyridines as highly potent and selective glycogen synthase kinase 3 inhibitors

Glycogen synthase kinase-3 plays an essential role in multiple biochemical pathways in the cell, particularly in regards to energy regulation. As such, Glycogen synthase kinase-3 is an attractive target for pharmacological intervention in a variety of disease states, particularly non-insulin dependent diabetes mellitus. However, due to homology with other crucial kinases, such as the cyclin-dependent protein kinase CDC2, developing compounds that are both potent and selective is challenging. A novel series of derivatives of 5-nitro-N2-(2-(pyridine-2ylamino)ethyl)pyridine-2,6-diamine were synthesized and have been shown to potently inhibit glycogen synthase kinase-3 (GSK3). Potency in the low nanomolar range was obtained along with remarkable selectivity. The compounds activate glycogen synthase in insulin receptor-expressing CHO-IR cells and in primary rat hepatocytes, and have acceptable pharmacokinetics and pharmacodynamics to allow for oral dosing. The X-ray co-crystal structure of human GSK3-β in complex with compound 2 is reported and provides insights into the structural determinants of the series responsible for its potency and selectivity.     

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A₂, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a “class switch” in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.