Search Results (35)

Phase-separated membraneless biomolecular condensates in the cytoplasm and nucleus are now recognized to play a major role in modulating diverse functions in mammalian cells, and contribute to cancer pathogenesis through dysregulated function of condensates of transcription factors such as STAT3 and fusion oncoproteins. Oral cancer, the sixth most prevalent malignancy worldwide, in the absence of overt causes such as tobacco or alcohol, most frequently occurs in a U-shaped zone (floor of mouth, side of tongue, anterior fauces and retromolar region) reflecting the path of liquid transit through the mouth. The cellular basis for this “high-risk” zone and the biochemical mechanisms used by oral cells to combat repetitive tonicity and temperature stresses are incompletely understood. We had previously observed that at 37 °C, in OECM1 oral carcinoma cells, cytoplasmic condensates of antiviral human GFP-MxA GTPase disassembled within 1–2 min of exposure of cells to saliva-like one-third hypotonicity, and underwent “spontaneous” reassembly in the next 5–7 min. Moreover, hypotonic beverages (water, tea, coffee), investigated at 37 °C, triggered this condensate cycling. In the present studies we investigated whether this process was temperature sensitive, representative of cold vs. warm drinks. We observed a slowing of this cycle at 5 °C, and speeding up at 50 °C. The involvement in this disassembly/reassembly process of WNK-SPAK/OSR1 serine-threonine kinase pathway, best studied for regulation of water and Na, K and Cl influx and efflux in kidney tubule cells, was evaluated by us in oral cells using pathway inhibitors WNK463, WNK-IN-11 and closantel. The pan-WNK inhibitor WNK463 inhibited hypotonicity-driven condensate disassembly, while the SPAK/OSR1 inhibitor closantel markedly slowed reassembly. Unexpectedly, the WNK1-selective inhibitor (WNK-IN-11), triggered a dramatic and rapid (within 1 h) spheroid to fibril transition of GFP-MxA condensates in live cells, but without affecting MxA antiviral function. The new data suggest a novel hypothesis for the anatomic localization of oral cancer in the U-shaped “high-risk” zone in the mouth: dysfunction of biomolecular condensates in oral cells along the beverage transit pathway through the mouth due to repetitive tonicity and temperature stresses that might underlie a prooncogenic progression. Full article

The growing risk of contracting viral infections due to high-density populations and ecological disruptions, such as climate change and increased population mobility, has highlighted the necessity for effective antiviral treatment and preventive measures against Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, there has been increasing attention on the use of probiotics as a potential antiviral option to reduce virus infections. The present study aimed to assess the immunomodulatory effects of heat-killed Lactococcus lactis strain plasma (LC-Plasma) on peripheral blood mononuclear cells (PBMCs) and its subsequent antiviral response against DENV, CHIKV, and ZIKV. To evaluate the immunomodulatory effects of LC-Plasma on PBMCs isolated from healthy individuals, PBMCs were cultured at a density of 2 × 10⁵ cells/well and stimulated with 10 µg/mL of LC-Plasma. LC-plasma-stimulated PBMCs demonstrated elevated interferon-alpha (IFN-α) production and cluster of differentiation 86 (CD86) and human leukocyte antigen-DR isotype (HLA-DR) upregulation, potentially linked to plasmacytoid dendritic cell (pDC) activation. The replication of DENV, CHIKV, and ZIKV was dose-dependently inhibited when Huh-7 cells were stimulated with LC-Plasma-stimulated PBMC supernatant (LCP Sup). IFN-stimulated gene (ISG) expression, including IFN-stimulated gene 15 (ISG15), IFN-stimulated exonuclease gene 20 (ISG20), IFN-induced transmembrane protein 1 (IFITM-1), myxovirus resistance protein A (MxA), and radical S-adenosyl methionine domain-containing protein 2 (RSAD2), was significantly upregulated in LCP Sup-stimulated Huh-7 cells. Findings from this study indicate that LC-Plasma has the potential to induce IFN-α production, leading to an enhancement in the expression of ISGs and contributing to a broad-spectrum antiviral response. Thus, LC-Plasma may serve as a rational adjunctive treatment to ameliorate viral diseases, warranting future clinical trials. Full article

The oral mucosa represents a defensive barrier between the external environment and the rest of the body. Oral mucosal cells are constantly bathed in hypotonic saliva (normally one-third tonicity compared to plasma) and are repeatedly exposed to environmental stresses of tonicity, temperature, and pH by the drinks we imbibe (e.g., hypotonic: water, tea, and coffee; hypertonic: assorted fruit juices, and red wines). In the mouth, the broad-spectrum antiviral mediator MxA (a dynamin-family large GTPase) is constitutively expressed in healthy periodontal tissues and induced by Type III interferons (e.g., IFN-λ1/IL-29). Endogenously induced human MxA and exogenously expressed human GFP-MxA formed membraneless biomolecular condensates in the cytoplasm of oral carcinoma cells (OECM1 cell line). These condensates likely represent storage granules in equilibrium with antivirally active dispersed MxA. Remarkably, cytoplasmic MxA condensates were exquisitely sensitive sensors of hypotonicity—the condensates in oral epithelium disassembled within 1–2 min of exposure of cells to saliva-like one-third hypotonicity, and spontaneously reassembled in the next 4–7 min. Water, tea, and coffee enhanced this disassembly. Fluorescence changes in OECM1 cells preloaded with calcein-AM (a reporter of cytosolic “macromolecular crowding”) confirmed that this process involved macromolecular uncrowding and subsequent recrowding secondary to changes in cell volume. However, hypertonicity had little effect on MxA condensates. The spontaneous reassembly of GFP-MxA condensates in oral epithelial cells, even under continuous saliva-like hypotonicity, was slowed by the protein-phosphatase-inhibitor cyclosporin A (CsA) and by the K-channel-blocker tetraethylammonium chloride (TEA); this is suggestive of the involvement of the volume-sensitive WNK kinase-protein phosphatase (PTP)-K-Cl cotransporter (KCC) pathway in the regulated volume decrease (RVD) during condensate reassembly in oral cells. The present study identifies a novel subcellular consequence of hypotonic stress in oral epithelial cells, in terms of the rapid and dynamic changes in the structure of one class of phase-separated biomolecular condensates in the cytoplasm—the antiviral MxA condensates. More generally, the data raise the possibility that hypotonicity-driven stresses likely affect other intracellular functions involving liquid–liquid phase separation (LLPS) in cells of the oral mucosa. Full article

FebriDx is a rapid point-of-care test combining qualitative measurements of C-reactive protein (CRP) and Myxovirus Resistance Protein A (MxA) using a disposable test device to detect and differentiate acute bacterial from viral respiratory tract infections. The goal of this study was to investigate the diagnostic accuracy of FebriDx in patients with suspected respiratory tract infections in the emergency department (ED). This was an observational cohort study, performed in the ED of an academic hospital. Patients were included if they had a suspected infection. The primary outcome was the presence of a bacterial or viral infection, determined by clinical adjudication by an expert panel. The sensitivity, specificity, and positive and negative predictive value of FebriDx for the presence of bacterial versus non-bacterial infections, and viral versus non-viral infections were calculated. Between March 2019 and November 2020, 244 patients were included. A bacterial infection was present in 41%, viral infection was present in 24%, and 4% of the patients had both viral and bacterial pathogens. FebriDx demonstrated high sensitivity in the detection of bacterial infection (87%), high NPV (91%) to rule out bacterial infection, and high specificity (94%) for viral infection in patients with a suspected infection in the ED. Full article

The proper supplementation of boron, an essential trace element, can enhance animal immune function. We utilized the method of TMT peptide labeling in conjunction with LC-MS/MS quantitative proteomics for the purpose of examining the effects of boric acid on a rat model and analyzing proteins from the duodenum. In total, 5594 proteins were obtained from the 0, 10, and 320 mg/L boron treatment groups. Two hundred eighty-four proteins that exhibit differential expression were detected. Among the comparison, groups of 0 vs. 10 mg/L, 0 vs. 320 mg/L, and 10 vs. 320 mg/L of boron, 110, 32, and 179 proteins, respectively, demonstrated differential expression. The results revealed that these differential expression proteins (DEPs) mainly clustered into two profiles. GO annotations suggested that most of the DEPs played a role in the immune system process, in which 2′-5′-oligoadenylate synthetase-like, myxovirus resistance 1, myxovirus resistance 2, dynein cytoplasmic 1 intermediate chain 1, and coiled-coil domain containing 88B showed differential expression. The DEPs had demonstrated an augmentation in the signaling pathways, which primarily include phagosome, antigen processing, and presentation, as well as cell adhesion molecules (CAMs). Our study found that immune responses in the duodenum were enhanced by lower doses of boron and that this effect is likely mediated by changes in protein expression patterns in related signaling pathways. It offers an in-depth understanding of the underlying molecular mechanisms that lead to immune modulation in rats subjected to dietary boron treatment. Full article

The objective of this study was to investigate the effect of puerarin supplementation on the growth performance and intestinal function of piglets challenged with enterotoxigenic Escherichia coli (ETEC) K88. Twenty-four ternary crossbred piglets were randomly assigned to three treatment groups: control group, ETEC group (challenged with ETEC K88 on day 8), and ETEC + Puerarin group (supplemented with 5 mg/kg puerarin and challenged with ETEC K88 on day 8). All piglets were orally administered D-xylose (0.1 g/kg body weight) on day 10, and blood samples were collected after 1 h. Subsequently, piglets were killed and intestinal samples were collected for further analysis. The results showed that puerarin supplementation significantly decreased the adverse effects of ETEC K88-challenged piglets; significantly improved growth performance; increased the number of Bifidobacterium in the colon and Lactobacillus in the jejunum, cecum and colon; decreased the number of Escherichia coli in the jejunum and cecum; reduced the hydrogen peroxide content in the jejunum and myeloperoxidase activity in the jejunum and ileum; and increased the activities of catalase and superoxide dismutase in the jejunum and ileum. In addition, puerarin supplementation alleviated ETEC K88-induced intestinal injury in piglets, significantly downregulated the mRNA level of Interleukin-1β and upregulated the mRNA levels of intercellular cell adhesion molecule-1, myxovirus resistance protein 1, myxovirus resistance protein 2, and guanylate-binding protein-1 in the small intestine of piglets. In conclusion, dietary supplementation with puerarin could attenuate ETEC K88-induced intestinal injury by increasing the antioxidant and anti-inflammatory capacity and the number of beneficial intestinal bacteria in piglets. Full article

Myxovirus resistance (MX) proteins are pivotal players in the innate immune response to viral infections. Less than 10 years ago, three independent groups simultaneously showed that human MX2 is an interferon (IFN)-stimulated gene (ISG) with potent anti-human immunodeficiency virus 1 (HIV-1) activity. Thenceforth, multiple research works have been published highlighting the ability of MX2 to inhibit RNA and DNA viruses. These growing bodies of evidence have identified some of the key determinants regulating its antiviral activity. Therefore, the importance of the protein amino-terminal domain, the oligomerization state, or the ability to interact with viral components is now well recognized. Nonetheless, there are still several unknown aspects of MX2 antiviral activity asking for further research, such as the role of cellular localization or the effect of post-translational modifications. This work aims to provide a comprehensive review of our current knowledge on the molecular determinants governing the antiviral activity of this versatile ISG, using human MX2 and HIV-1 inhibition as a reference, but drawing parallelisms and noting divergent mechanisms with other proteins and viruses when necessary. Full article

Because of endotoxemia during sepsis (a severe life-threatening infection), lipopolysaccharide (LPS) tolerance (the reduced responses to the repeated LPS stimulation) might be one of the causes of sepsis-induced immune exhaustion (the increased susceptibility to secondary infection and/or viral reactivation). In LPS tolerance macrophage (twice-stimulated LPS, LPS/LPS) compared with a single LPS stimulation (N/LPS), there was (i) reduced energy of the cell in both glycolysis and mitochondrial activities (extracellular flux analysis), (ii) decreased abundance of the following proteins (proteomic analysis): (a) complex I and II of the mitochondrial electron transport chain, (b) most of the glycolysis enzymes, (c) anti-viral responses with Myxovirus resistance protein 1 (Mx1) and Ubiquitin-like protein ISG15 (Isg15), (d) antigen presentation pathways, and (iii) the down-regulated anti-viral genes, such as Mx1 and Isg15 (polymerase chain reaction). To test the correlation between LPS tolerance and viral reactivation, asymptomatic mice with and without murine norovirus (MNV) infection as determined in feces were tested. In MNV-positive mice, MNV abundance in the cecum, but not in feces, of LPS/LPS mice was higher than that in N/LPS and control groups, while MNV abundance of N/LPS and control were similar. Additionally, the down-regulated Mx1 and Isg15 were also demonstrated in the cecum, liver, and spleen in LPS/LPS-activated mice, regardless of MNV infection, while N/LPS more prominently upregulated these genes in the cecum of MNV-positive mice compared with the MNV-negative group. In conclusion, defects in anti-viral responses after LPS tolerance, perhaps through the reduced energy status of macrophages, might partly be responsible for the viral reactivation. More studies on patients are of interest. Full article

Utilizing agro-industrial waste and herbal products to create a circular bioeconomy is becoming increasingly popular. Terminalia arjuna is a significant ethnomedicinal plant that has not yet been exploited in animal feed. In the present study, nutritional Terminalia arjuna bark powder-based fish feed was created and supplied to a candidate fish species Labeo rohita at varied levels: 0% (0 g/kg), 0.5% (5 g/kg), 1% (10 g/kg), and 1.5% (15 g/kg). These treatment groups are denoted as CT, T1, T2, and T3, respectively. Utilizing a contemporary comprehensive biomarker response strategy, the study clarified the genomic influence of dietary herb inclusion. In response to bacterial infection, the immunogenic genes, STAT 1 (signal transducer and activator of transcription 1), ISG 15 (interferon stimulating gene), and Mx “myxovirus resistance gene”, were shown to be elevated. The results of densitometry demonstrated a dose-dependent increase in STAT 1 and ISG 15, with Mx exhibiting maximal values at 1 g/kg TABP (Terminalia arjuna bark powder-based feed). This is the first study to identify TABP as an immunomodulator in fish and established the IBR (Integrated Bio-marker Response) as a reliable marker in evaluating the impact of multiple drivers in a holistic manner. Thus, the present study cleared the path for TABP to be utilized as an effective feed additive which enhances the specific adaptive immune system of the fish for the production of the Green fish product for a sustainable circular bioeconomy. Full article

The diagnosis of serious bacterial infection (SBI) in young febrile children remains challenging. This prospective, multicentre, observational study aimed to identify new protein marker combinations that can differentiate a bacterial infection from a viral infection in 983 children, aged 7 days–36 months, presenting with a suspected SBI at three French paediatric emergency departments. The blood levels of seven protein markers (CRP, PCT, IL-6, NGAL, MxA, TRAIL, IP-10) were measured at enrolment. The patients received the standard of care, blinded to the biomarker results. An independent adjudication committee assigned a bacterial vs. viral infection diagnosis based on clinical data, blinded to the biomarker results. Computational modelling was applied to the blood levels of the biomarkers using independent training and validation cohorts. Model performances (area under the curve (AUC), positive and negative likelihood ratios (LR+ and LR–)) were calculated and compared to those of the routine biomarkers CRP and PCT. The targeted performance for added value over CRP or PCT was LR+ ≥ 5.67 and LR− ≤ 0.5. Out of 652 analysed patients, several marker combinations outperformed CRP and PCT, although none achieved the targeted performance criteria in the 7 days–36 months population. The models seemed to perform better in younger (7–91 day-old) patients, with the CRP/MxA/TRAIL combination performing best (AUC 0.895, LR+ 10.46, LR− 0.16). Although computational modelling using combinations of bacterial- and viral-induced host-protein markers is promising, further optimisation is necessary to improve SBI diagnosis in young febrile children. Full article

Avian influenza viruses (AIVs) pose threats to animal and human health. Outbreaks from the highly pathogenic avian influenza virus (HPAIV) in indigenous chickens in Bangladesh are infrequent. This could be attributed to the Myxovirus resistance (Mx) gene. To determine the impact of Mx gene diversity on AIV infections in chicken, we assessed the Mx genes, AIVs, and anti-AIV antibodies. DNA from blood cells, serum, and cloacal swab samples was isolated from non-vaccinated indigenous chickens and vaccinated commercial chickens. Possible relationships were assessed using the general linear model (GLM) procedure. Three genotypes of the Mx gene were detected (the resistant AA type, the sensitive GG type, and the heterozygous AG type). The AA genotype (0.48) was more prevalent than the GG (0.19) and the AG (0.33) genotypes. The AA genotype was more prevalent in indigenous than in commercial chickens. A total of 17 hemagglutinating viruses were isolated from the 512 swab samples. AIVs were detected in two samples (2/512; 0.39%) and subtyped as H1N1, whereas Newcastle disease virus (NDV) was detected in the remaining samples. The viral infections did not lead to apparent symptoms. Anti-AIV antibodies were detected in 44.92% of the samples with levels ranging from 27.37% to 67.65% in indigenous chickens and from 26% to 87.5% in commercial chickens. The anti-AIV antibody was detected in 40.16%, 65.98%, and 39.77% of chickens with resistant, sensitive, and heterozygous genotypes, respectively. The genotypes showed significant association (p < 0.001) with the anti-AIV antibodies. The low AIV isolation rates and high antibody prevalence rates could indicate seroconversion resulting from exposure to the virus as it circulates. Results indicate that the resistant genotype of the Mx gene might not offer anti-AIV protection for chickens. Full article

The interferon-induced myxovirus resistance protein A (MxA) is a potent restriction factor that prevents zoonotic infection from influenza A virus (IAV) subtype H7N9. Individuals expressing antivirally inactive MxA variants are highly susceptible to these infections. However, human-adapted IAVs have acquired specific mutations in the viral nucleoprotein (NP) that allow escape from MxA-mediated restriction but that have not been observed in MxA-sensitive, human H7N9 isolates. To date, it is unknown whether H7N9 can adapt to escape MxA-mediated restriction. To study this, we infected Rag2-knockout (Rag2^−/−) mice with a defect in T and B cell maturation carrying a human MxA transgene (MxA^tg/−Rag2^−/−). In these mice, the virus could replicate for several weeks facilitating host adaptation. In MxA^tg/−Rag2^−/−, but not in Rag2^−/− mice, the well-described mammalian adaptation E627K in the viral polymerase subunit PB2 was acquired, but no variants with MxA escape mutations in NP were detected. Utilizing reverse genetics, we could show that acquisition of PB2 E627K allowed partial evasion from MxA restriction in MxA^tg/tg mice. However, pretreatment with type I interferon decreased viral replication in these mice, suggesting that PB2 E627K is not a true MxA escape mutation. Based on these results, we speculate that it might be difficult for H7N9 to acquire MxA escape mutations in the viral NP. This is consistent with previous findings showing that MxA escape mutations cause severe attenuation of IAVs of avian origin. Full article

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1–2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40–50 mOsm). Both reassembled into new structures within 1–2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90–175 mOsm) first rapidly disassembled within 1–3 min, and then, in most cells, spontaneously reassembled 7–15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress. Full article

Amphibians, including Andrias davidianus, are declining worldwide partly due to infectious diseases. The Myxovirus resistance (Mx) gene is a typical interferon (IFN)-stimulated gene (ISG) involved in the antiviral immunity. Therefore, knowledge regarding the antiviral immunity of A. davidianus can be used for improved reproduction in captivity and protection in the wild. In this study, we amplified and characterized four different A. davidianus Mx genes (adMx) and generated temporal mRNA expression profiles in healthy and Chinese giant salamander iridovirus (GSIV) infected A. davidianus by qualitative real-time PCR (qPCR). The four adMx genes ranged in length from 2008 to 2840 bp. The sequences revealed conserved protein domains including the dynamin superfamily signature motif and the tripartite guanosine-5-triphosphate (GTP)-binding motif. Gene and deduced amino acid sequence alignment revealed relatively high sequence identity with the Mx genes and proteins of other vertebrates. In phylogenetic analysis, the adMx genes clustered together, but also clustered closely with those of fish species. The four adMx genes were broadly expressed in healthy A. davidianus, but were differentially expressed in the spleen during the GSIV infection. Our results show that the adMx genes share major structural features with their homologs, suggesting similar functions to those in other species. Full article

Human platelet lysate (hPL) contains abundant growth factors for inducing human cell proliferation and may be a suitable alternative to fetal bovine serum (FBS) as a culture medium supplement. However, the application of hPL in virological research remains blank. Parechovirus type-A3 (PeV-A3) belongs to Picornaviridae, which causes meningoencephalitis in infants and young children. To understand the suitability of hPL-cultured cells for PeV-A3 infection, the infection of PeV-A3 in both FBS- and hPL-cultured glioblastoma (GBM) cells were compared. Results showed reduced PeV-A3 infection in hPL-cultured cells compared with FBS-maintained cells. Mechanistic analysis revealed hPL stimulating type I interferon (IFN) antiviral pathway, through which phospho-signal transducer and activator of transcription 1 (STAT1), STAT2, interferon regulatory factor 3 (IRF3) were activated and antiviral genes, such as IFN-α, IFN-β, and Myxovirus resistance protein 1 (MxA), were also detected. In addition, an enhanced PeV-A3 replication was detected in the hPL-cultured GBM cells treated with STAT-1 inhibitor (fludarabine) and STAT1 shRNA. These results in vitro suggested an unexpected effect of hPL-activated type I IFN pathway response to restrict virus replication and that hPL may be a potential antiviral bioreagent. Full article