Stress granule formation,disassembly, and composition are regulated by alphavirus ADP-ribosylhydrolase activity

While biomolecular condensates have emerged as an important biological phenomenon, mechanisms regulating their composition and the ways that viruses hijack these mechanisms remain unclear. The mosquito-borne alphaviruses cause a range of diseases from rashes and arthritis to encephalitis, and no licensed drugs are available for treatment or vaccines for prevention. The alphavirus virulence factor nonstructural protein 3 (nsP3) suppresses the formation of stress granules (SGs)—a class of cytoplasmic condensates enriched with translation initiation factors and formed during the early stage of infection. nsP3 has a conserved N-terminal macrodomain that hydrolyzes ADP-ribose from ADP-ribosylated proteins and a C-terminal hypervariable domain that binds the essential SG component G3BP1. Here, we show that macrodomain hydrolase activity reduces the ADP-ribosylation of G3BP1, disassembles virus-induced SGs, and suppresses SG formation. Expression of nsP3 results in the formation of a distinct class of condensates that lack translation initiation factors but contain G3BP1 and other SG-associated RNA-binding proteins. Expression of ADP-ribosylhydrolase–deficient nsP3 results in condensates that retain translation initiation factors as well as RNA-binding proteins, similar to SGs. Therefore, our data reveal that ADP-ribosylation controls the composition of biomolecular condensates, specifically the localization of translation initiation factors, during alphavirus infection.

Biomolecular condensates are prevalent in cells and critical for a range of cellular functions, including RNA metabolism, embryonic cell fate specification, and neuronal activity (1–3). While condensates often dynamically exchange components with the surrounding milieu, the overall composition of these cellular structures remains distinct (4). How cells control the specific composition of these condensates remains unclear. Stress granules (SGs), one of the best characterized biomolecular condensates, are RNA–protein assemblies formed in response to a variety of environmental cues (1). While SG composition can vary with the type of stress cue (5), certain common components, such as Ras GTP-activating protein-binding proteins G3BP1/2, are essential for formation of SGs (6, 7). Dysregulation of SG formation and disassembly is implicated in the pathogenesis of diseases, including viral infection, cancer, and neurodegeneration (2, 8–10).SG formation and disassembly are tightly regulated during viral infection, often reflecting cellular translation status (11–14). In the early phase of many viral infections, the presence of double-stranded viral RNAs (vRNAs) activate protein kinase R (PKR), resulting in eIF2α phosphorylation, messenger RNA (mRNA) translation inhibition, and formation of SGs enriched with translation initiation factors such as eIF3b. However, in later infection stages, many viruses instead suppress SG formation or disassemble SGs altogether. The mechanisms underlying this switch, and its physiological function, remain unclear.SG formation and disassembly are regulated by posttranslational modifications of proteins, including those that conjugate simple chemical groups, attach polypeptides, and add nucleotides as in the case of ADP-ribosylation (15–21). ADP-ribosylation refers to the addition of one or more ADP-ribose units onto proteins (22–24). In humans, ADP-ribosylation is accomplished primarily by a family of 17 ADP-ribosyltransferases, commonly known as poly(ADP-ribose) polymerases (PARPs). SG components are specifically ADP-ribosylated, and ADP-ribose polymers [i.e., poly(ADP-ribose) or PAR], five PARPs and two isoforms of the degradative enzyme PAR glycohydrolase (PARG) have been localized to these condensates (17, 25–27). Overexpression of these PARPs and PARG isoforms induces and suppresses SG formation, respectively, while PARG knockdown delays SG disassembly (17, 26). The noncovalent interaction between PAR and proteins facilitates SG targeting (25–27). For example, PAR-mediated targeting regulates TDP-43 localization to SGs and prevents the formation of pathological aggregates in amyotrophic lateral sclerosis (26, 27).The mosquito-borne alphaviruses, which cause a range of diseases from rashes and arthritis to encephalitis, induce SG formation early in infection and later initiate SG disassembly (11, 14, 28, 29). Previous studies have identified the alphaviral nonstructural protein 3 (nsP3), a key factor for virus replication and virulence (30–32), as able to suppress SG formation (28, 33–35). The alphaviral nsP3 is a tripartite protein composed of a highly conserved macrodomain (MD) in the N terminus, a central zinc-binding domain (ZBD), and a C-terminal hypervariable domain (HVD; ref. 30). Recent studies indicate that the HVD, which is of low complexity, directs alphaviral nsP3 binding to host SG proteins (30, 36). For example, the HVD of chikungunya virus (CHIKV) binds the essential SG components G3BP1 and G3BP2 (33, 37). Given that nsP3 expression increases over the course of viral infection, it has been proposed that nsP3 sequesters G3BP1/2, resulting in the suppression of SG formation during the late phase of infection (28, 29, 34).Here, we report that the expression of the G3BP-binding HVD alone does not suppress SG formation; rather, expression of the N-terminal MD alone can trigger the suppression of this biomolecular condensate. The structural integrity of SGs is dependent on ADP-ribosylation (17), and we and others recently found that the viral MD can remove single ADP-ribose groups, and possibly PAR, from ADP-ribosylated proteins (31, 38–40). We therefore hypothesized that MD ADP-ribosylhydrolase activity is required to suppress SG formation across stress conditions, with G3BP1 being a key target substrate. Indeed, we find that MD ADP-ribosylhydrolase activity is critical for disassembling SGs formed by G3BP1 expression and during viral infection. Consistent with this premise, live cell imaging revealed that SGs persist in cells infected with a hydrolase-deficient recombinant CHIKV. ADP-ribosylhydrolase activity is required for altering the composition of biomolecular condensates in nsP3-expressing or virus-infected cells and specifically regulates translation factor localization. Together, these data argue that nsP3 ADP-ribosylhydrolase activity modulates SG formation, disassembly, and composition.     

Anti-neuroinflammatory activity of Kamebakaurin from Isodon japonicus via inhibition of c-Jun NH₂-terminal kinase and p38 mitogen-activated protein kinase pathway in activated microglial cells

Compelling evidence supports the notion that the majority of neurodegenerative diseases are associated with microglia-mediated neuroinflammation. Therefore, quelling of microglial activation may lead to neuronal cell survival. The present study investigated the effects of Kamebakaurin (KMBK), a kaurane diterpene isolated from Isodon japonicus HARA (Labiatae), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated cytotoxicity in rat primary microglial cultures and the BV-2 cell line. KMBK significantly inhibited the LPS-induced production of nitric oxide (NO) in a concentration-dependent fashion in activated microglial cells. The mRNA and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxycenase-2 (COX-2) were also decreased dose-dependently. Furthermore KMBK inhibited the JNK and p38 mitogen-activated protein kinases (MAPKs) in LPS-stimulated BV-2 microglial cells. Considering the results obtained, the present study authenticated the potential benefits of KMBK as a therapeutic target in ameliorating microglia-mediated neuroinflammatory diseases.     

Adaptogenic and nootropic activities of aqueous extract of Vitis vinifera (grape seed): an experimental study in rat model

Background  

The aerial parts of Vitis vinifera (common grape or European grape) have been widely used in Ayurveda to treat a variety of common and stress related disorders. In the present investigation, the seed extract of V. vinifera was evaluated for antistress activity in normal and stress induced rats. Furthermore, the extract was studied for nootropic activity in rats and in-vitro antioxidant potential to correlate its antistress activity.     

Thermal Shock Resistance and Thermal Insulation Capability of Laser-Glazed Functionally Graded Lanthanum Magnesium Hexaluminate/Yttria-Stabilised Zirconia Thermal Barrier Coating

In this work, functionally graded lanthanum magnesium hexaluminate (LaMgAl₁₁O₁₉)/yttria-stabilised zirconia (YSZ) thermal barrier coating (FG-TBC), in as-sprayed and laser-glazed conditions, were investigated for their thermal shock resistance and thermal insulation properties. Results were compared with those of a dual-layered coating of LaMgAl₁₁O₁₉ and YSZ (DC-TBC). Thermal shock tests at 1100 °C revealed that the as-sprayed FG-TBC had improved thermal stability, i.e., higher cycle lifetime than the as-sprayed DC-TBC due to its gradient architecture, which minimised stress concentration across its thickness. In contrast, DC-TBC spalled at the interface due to the difference in the coefficient of thermal expansion between the LaMgAl₁₁O₁₉ and YSZ layers. Laser glazing improved cycle lifetimes of both the types of coatings. Microstructural changes, mainly the formation of segmentation cracks in the laser-glazed surfaces, provided strain tolerance during thermal cycles. Infrared rapid heating of the coatings up to 1000 °C showed that the laser-glazed FG-TBC had better thermal insulation capability, as interlamellar pores entrapped gas and constrained heat transfer across its thickness. From the investigation, it is inferred that (i) FG-TBC has better thermal shock resistance and thermal insulation capability than DC-TBC and (ii) laser glazing can significantly enhance the overall thermal performance of the coatings. Laser-glazed FG-TBC provides the best heat management, and has good potential for applications that require effective heat management, such as in gas turbines.     

Molecular effects of activated BV-2 microglia by mitochondrial toxin 1-methyl-4-phenylpyridinium

Microglia plays an important role in inflammation-mediated neurodegeneration. Compelling evidence supports the hypothesis that microglial activation contributes to the pathogenesis of various neurodegenerative diseases. However, little is known about the molecular outcome of activated microglia. In this report, we investigate the molecular consequences of MPP(+) toxin-induced activated BV-2 microglia. Intoxication of specific mitochondrial toxin methyl-4-phenylpyridinium iodide ion (MPP(+)) to BV-2 cells induced significant mitochondrial dysfunction and increased the reactive oxygen species generation, caspase-3 activation, and poly ADP ribose polymerase proteolysis. Further, MAC-1 immunostaining in the midbrain of mice revealed a decrease in activated microglia at day 4 after intoxication with MPP(+). From this study, it was confirmed that BV-2 microglia respond to the mitochondrial toxin MPP(+) which may lead to apoptotic cell death. Understanding of the mechanistic basis of apoptotic elimination of activated microglia may help to develop new strategies for the treatment of neurodegenerative diseases.     

Assessing the quality of clinical and administrative data extracted from hospitals: the General Medicine Inpatient Initiative (GEMINI) experience

ObjectiveLarge clinical databases are increasingly used for research and quality improvement. We describe an approach to data quality assessment from the General Medicine Inpatient Initiative (GEMINI), which collects and standardizes administrative and clinical data from hospitals.MethodsThe GEMINI database contained 245 559 patient admissions at 7 hospitals in Ontario, Canada from 2010 to 2017. We performed 7 computational data quality checks and iteratively re-extracted data from hospitals to correct problems. Thereafter, GEMINI data were compared to data that were manually abstracted from the hospital’s electronic medical record for 23 419 selected data points on a sample of 7488 patients.ResultsComputational checks flagged 103 potential data quality issues, which were either corrected or documented to inform future analysis. For example, we identified the inclusion of canceled radiology tests, a time shift of transfusion data, and mistakenly processing the chemical symbol for sodium (“Na”) as a missing value. Manual validation identified 1 important data quality issue that was not detected by computational checks: transfusion dates and times at 1 site were unreliable. Apart from that single issue, across all data tables, GEMINI data had high overall accuracy (ranging from 98%–100%), sensitivity (95%–100%), specificity (99%–100%), positive predictive value (93%–100%), and negative predictive value (99%–100%) compared to the gold standard.Discussion and ConclusionComputational data quality checks with iterative re-extraction facilitated reliable data collection from hospitals but missed 1 critical quality issue. Combining computational and manual approaches may be optimal for assessing the quality of large multisite clinical databases.     

Potential Nutrients from Natural and Synthetic Sources Targeting Inflammaging—A Review of Literature,Clinical Data and Patents

Inflammaging, the steady development of the inflammatory state over age is an attributable characteristic of aging that potentiates the initiation of pathogenesis in many age-related disorders (ARDs) including neurodegenerative diseases, arthritis, cancer, atherosclerosis, type 2 diabetes, and osteoporosis. Inflammaging is characterized by subclinical chronic, low grade, steady inflammatory states and is considered a crucial underlying cause behind the high mortality and morbidity rate associated with ARDs. Although a coherent set of studies detailed the underlying pathomechanisms of inflammaging, the potential benefits from non-toxic nutrients from natural and synthetic sources in modulating or delaying inflammaging processes was not discussed. In this review, the available literature and recent updates of natural and synthetic nutrients that help in controlling inflammaging process was explored. Also, we discussed the clinical trial reports and patent claims on potential nutrients demonstrating therapeutic benefits in controlling inflammaging and inflammation-associated ARDs.     

Identification of common polymorphisms in the coding sequence of the human MSH receptor (MCIR) with possible biological effects

The extension locus has been identified in many mammalian species as a gene that determines the relative amounts of eumelanin and phaeomelanin pigments in hair and skin. In at least three species, this locus has been demonstrated to encode the melanocyte-stimulating hormone receptor (MC1-R), and functionally variant alleles have been demonstrated to cause a broad range of pigmentation phenotypes. To test for MC1-R allelic variation in man, genomic DNA was extracted from skin samples collected from patients with different skin types (I–VI), and eye and hair color. A PCR-based approach was used to amplify the full-length coding sequence of the MC1-R and the resulting products were sequenced. Two polymorphic alleles were identified with single point mutations in the coding sequence: a valine-to-methionine substitution at position 92 (V92M), and an aspartic acid-to-glutamic acid substitution at position 84 (D84E). RFLP analysis demonstrated the presence of the V92M allele in 4 out of 60 (6.6%) of individuals examined, predominantly those with blue eyes and blond hair. This polymorphism was found in both heterozygous and homozygous states in individuals with type I skin. The D84E allele was found in one individual with skin type I; this person also has the V92 M allele and thus is a compound heterozygote. © 1997 Wiley-Liss, Inc.