Search

Understanding the early events in viral biology holds the key to the development of potent preventives. In this study, fluorescent hepatitis C virus pseudoparticles (HCVpp) have been generated where the envelope glycoprotein of Hepatitis C virus (HCV) has an EGFP tag. Using these pseudoparticles, entry assays were conducted where their entry was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 min of HCV entry. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent HCVpp which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.

Hepatitis C is a positive stranded enveloped RNA virus belonging to the Flaviviridae family. HCV infection leads to severe liver diseases, cirrhosis and hepatocellular carcinoma worldwide. Although treatments have been available for a while, due to its complexity and genetic diversity, only few are reported to be effective against all HCV genotypes. Here, we review the HCV life cycle and its immunogenic potential and various mechanisms via which the virus interferes in the signalling process. A comprehensive overview of current anti-HCV therapeutics, such as, Direct Acting Antiviral (DAA) as well as Host Targeting Agents (HTA), along with their scope, known mechanism of action and limitations are presented. Supplementary Information The online version contains supplementary material available at 10.1007/s13337-021-00697-0.

Coronavirus disease 2019 (COVID-19) triggered by a new viral pathogen, named severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2), is now a global health emergency. This debilitating viral pandemic not only paralyzed the normal daily life of the global community but also spread rapidly via global travel. To date there are no effective vaccines or specific treatments against this highly contagious virus; therefore, there is an urgent need to advocate novel prophylactic or therapeutic interventions for COVID-19. This brief opinion critically discusses the potential of Silymarin, a flavonolignan with diverse pharmacological activity having antiinflammatory, antioxidant, antiplatelet, and antiviral properties, with versatile immune-cytokine regulatory functions, that able to bind with transmembrane protease serine 2 (TMPRSS2) and induce endogenous antiviral cytokine interferon-stimulated gene 15, for the management of COVID-19. Silymarin inhibits the expression of host cell surface receptor TMPRSS2 with a docking binding energy corresponding to -1,350.61 kcal/mol and a full fitness score of -8.11. The binding affinity of silymarin with an impressive virtual score exhibits significant potential to interfere with SARS-CoV-2 replication. We propose in-depth pre-clinical and clinical review studies of silymarin for the development of anti-COVID-19 lead, based on its clinical manifestations of COVID-19 and multifaceted bioactivities.

Arsenic toxicity purportedly threats a broad spectrum of female reproductive functions. We investigated the remedial role of a casein- and pea protein-enriched high-protein diet (HPD) in combating the arsenic insult. Cyclic female rats maintained on standard diet (n = 6) or an isocaloric HPD (n = 6) were gavaged with As2O3 at 3 mg/kg BW/rat/day (n = 12) for 28 days. Vehicle-fed rats (n = 6) maintained on the standard diet served as the control. We monitored the estrus cycles and performed the histomorphometric analyses of the uterus and ovary. Uterine luminal epithelial (ULE) ultrastructure was appraised by scanning electron microscopy. Uterine oxidative stress was evaluated in the forms of ROS generation and activities of the ROS scavengers. The uterine apoptotic manifestation was blueprinted by Western blot analysis of caspase-3 and Bax expression. Arsenic treatment arrested the follicular maturation and disrupted the estrus cycles with a typical increase in the diestrus index. Shrunken endometrial glands and thinned microvilli density of the ULE reflected loss of cell polarity and mislaid uterine homeostasis. Increased ROS generation and attenuated activities of the ROS scavengers marked a state of uterine oxidative imbalance and loss of redox regulation. Superfluous expression of procaspase-3, cleaved caspase-3, and Bax mirrored an inflated state of uterine apoptosis. HPD supplementation, by and large, counteracted these arsenic impacts and maintained the frameworks close to the control levels. In conclusion, arsenic mediates its reproductive toxicity, at least in part, by upsetting the uterine ROS homeostasis and redox regulation. Pea proteins and casein-supplemented HPD can counteract the arsenic effects and maintain the reproductive functions.

Recent evidences indicate that change in cellular metabolic pathways can alter immune response and function of the host; emphasizing the role of metabolome in health and diseases. Human Herpes simplex virus type-1 (HSV-1) and type-2 (HSV-2) causes diseases from asymptomatic to highly prevalent oral and genital herpes, recurrent blisters or neurological complications. Immune responses against HSV are complex with delicate interplay between innate signaling pathways and adaptive immune responses. The innate response involves the induction of protective IFN-1; while Natural Killer (NK) cells and plasmacytoid Dendritic Cells (pDC) confer in vivo adaptive anti-HSV response along with humoral and cellular components in controlling infection and latency. Metabolic changes lead to up-/down-regulation of several cytokines and chemokines like IFN-γ, IL-2, IL-4, IL-10 and MIP1β in HSV infection and recurrences. Recently, the viral protein ICP0 has been identified as an attenuator of TLR signaling, that inhibit innate responses to HSV. This review will summarize the role of metabolome in innate and adaptive effectors in infection, pathogenesis and immune control of HSV, highlighting the delicate interplay between the metabolic changes and immunity.

Understanding the early events in viral biology holds the key to the development of preventives. In this study fluorescent Hepatitis C Virus pseudoparticles have been generated where the envelope glycoprotein has a GFP tag. Using these pseudoparticles entry assays were conducted where the entry of the pseudoparticles was tracked via confocal microscopy. Using this system, fusion of host and viral membranes is predicted to occur within 15 minutes of entry in HCV. Using cells with a knockdown for Rab1a, HCV trafficking was observed to be altered, indicating a role of Rab1a in HCV trafficking. In conclusion, this study reports the generation and use of fluorescent pseudoparticles which may be used to understand the early events of viral entry. This system may be adapted for the study of other enveloped viruses as well.HighlightsFluorescent HCV pseudoparticles have been created to study early entry events.HCV entry tracking via confocal microscopy reveals fusion within 15 minutes.Rab1a is important for HCV trafficking within the cell.

Hepatitis C Virus is a blood borne pathogen responsible for chronic hepatitis in more than 71 million people. Wide variations across strains and genotypes are one of the major hurdles in therapeutic development. While genotype 1 remains the most extensively studied and abundant strain, genotype 3 is more virulent and second most prevalent. This study aimed to compare differences in the glycoprotein E2 across HCV genotypes at nucleotide, protein and structural levels. Nucleotide sequences of E2 from 29 strains across genotypes 1a, 1b, 3a and 3b revealed a stark preference for C-richness which was attributed to a distinct bias for C-rich codons in genotype 1. Genotype 3 exhibited a similar preference to a lesser extent. Amino acid level comparison revealed majority of the changes at the C-terminal half of the proteins leaving the N-terminal region conspicuously conserved apart from the two hyper variable regions. Amino acid changes across genotypes were mostly polar-nonpolar alterations. In silico models of E2 glycoproteins and docking analysis with the energy minimized PDB-CD81 model revealed unique interacting residues in both E2 and CD81. While several CD81 binding residues were common for all four genotypes, number and composition of interacting residues varied. The interacting residues of E2 were however unique for each genotype. E2 of genotype 3a and CD81 had the strongest interaction. In conclusion this is the first comprehensive study comparing E2 sequences across genotypes 1a, 1b, 3a and 3b revealing stark genotype-specific differences which requires more extensive investigation.

A novel exopolysaccharide (EPS) was produced by a bacterium which was isolated from Psophocarpus tetragonolobus (L) D.C. and identified as 99% Rhizobium tropici SRA1 by 16S rDNA sequencing. The flocculating performances along with emulsifying activity began simultaneously with the growth and the production of EPS and reached its utmost at 28 h. EPS was purified via chilled ethanol precipitation followed by dialysis and lyophilization. The existence of hydroxyl, methoxyl, and carboxylic functional groups were confirmed by Fourier transform infrared (FT-IR) spectrum. EPS was found to be compose of 82.44% neutral sugar and 15.93% uronic acid. The average molecular weight of the exopolysaccharide was estimated as ~ 1.8 × 105. Gas–liquid chromatography indicated the presence of glucose and galactose at a molar ratio of 3:1 in EPS. In the pH range of 3–5 with EPS dosage of 15 mg/l at 30 °C, cation-independent flocculation greater than 90% was observed. Emulsification indices (E24) of EPS were observed as 86.66%, 83.33%, 76.66%, and 73.33% with olive oil, kerosene, toluene, and n-hexane respectively. Biosorption of Cu K [45.69 wt%], Cu L [05.67 wt%], Co K [15.58 wt%], and Co L [11.72 wt%] by EPS was confirmed by energy-dispersive X-ray spectroscopy (EDS). This report on the flocculating, emulsifying, and metal sorption properties of EPS produced by R. tropici SRA1 is unique in the literature.

Background:Chronic arsenic exposure via contaminated drinking water is a global environmental health problem associated with hematological, hepatic and many serious systemic disorders. This study on adult male rats evaluated the protective effects of vitamin E (VE) and vitamin C (VC) against arsenic-mediated hematological and hepatic toxicities.Methods:Arsenic was administered orally as arsenic trioxide (3 mg/kg body weight/day), as a single dose for 30 consecutive days or along with VC/ascorbic acid (200 mg/kg body weight/day dissolved in water) and VE/α-tocopherol (400 mg/kg body weight/day dissolved in olive oil) as supplements. Multiple hematological and hepatic parameters were assessed.Results:Arsenic exposure caused significant reduction of erythrocyte counts (pConclusions:The present investigation offers strong evidence regarding the protective efficacy of co-administration of VC and VE against hematotoxicity and hepatotoxicity in adult male rats caused by chronic arsenic exposure.

Assembly of herpes simplex viruses (HSV) is a poorly understood process involving multiple redundant interactions between large number of tegument and envelope proteins. We have previously shown (G. E. Lee, G. A. Church, and D. W. Wilson, J. Virol. 77: 2038-2045, 2003) that the virion host shutoff (Vhs) tegument protein is largely insoluble in HSV-infected cells and is also stably associated with membranes. Here we demonstrate that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore, we have found that the amino-terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a green fluorescent protein (GFP) reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. We conclude that the amino terminus of Vhs mediates targeting to sites of HSV assembly and to the viral tegument.

Trogocytosis was originally thought to be restricted to the interaction of cells of the immune system and interactions of these cells with cancer cells. Such membrane exchanges are probably a general process in cell biology, and membrane exchange has been demonstrated to occur between non-immune cells within an organism. Herein, we report that membrane and protein exchange, consistent with trogocytosis, between Trypanosoma cruzi (both the Brazil and Tulahuen strains) and the mammalian cells it infects. Transfer of labeled membrane patches was monitored by labeling of either parasites or host cells, i.e. human foreskin fibroblasts and rat myoblasts. Trypomastigotes and amastigotes transferred specific surface glycoproteins to the host cells along with membranes. Exchange of membranes between the parasite and host cells occurred during successful invasion. Extracellular amastigotes did not transfer membrane patches and heat killed trypomastigotes were did not transfer either membranes or proteins to the host cells. Membrane exchange was also found to occur between interacting epimastigotes in cell-free culture and may be important in parasite-parasite interactions as well. Further studies should provide new insights into pathogenesis and provide targets for therapeutic intervention.

We previously reported that Rab1a is associated with asialoorosomucoid (ASOR)-containing early endocytic vesicles, where it is required for their microtubule-based motility. In Rab1a knockdown (KD) cell lines, ASOR failed to segregate from its receptor and, consequently, did not reach lysosomes for degradation, indicating a defect in early endosome sorting. Although Rab1 is required for Golgi/endoplasmic reticulum trafficking, this process was unaffected, likely due to retained expression of Rab1b in these cells. The present study shows that Rab1a has a more general role in endocytic vesicle processing that extends to EGF and transferrin (Tfn) trafficking. Compared with results in control Huh7 cells, EGF accumulated in aggregates within Rab1a KD cells, failing to reach lysosomal compartments. Tfn, a prototypical example of recycling cargo, accumulated in a Rab11-mediated slow-recycling compartment in Rab1a KD cells, in contrast to control cells, which sort Tfn into a fast-recycling Rab4 compartment. These data indicate that Rab1a is an important regulator of early endosome sorting for multiple cargo species. The effectors and accessory proteins recruited by Rab1a to early endocytic vesicles include the minus-end-directed kinesin motor KifC1, while others remain to be discovered.

Trypanosoma cruzi, the etiological agent of Chagas disease, causes vasculopathy and cardiomyopathy in humans and is associated with elevated levels of several vasoactive molecules such as nitric oxide, endothelin-1 and thromboxane A 2 (TXA 2). Parasite derived TXA 2 modulates vasculopathy and other pathophysiological features of Chagasic cardiomyopathy. Previously, we demonstrated that in response to infection with T. cruzi, TXA 2 receptor (TP) null mice displayed increased parasitemia; mortality and cardiac pathology compared with wild type (WT) and TXA 2 synthase null mice. In order to further study the role of TXA 2-TP signaling in the development of Chagas disease, GeneChip microarrays were used to detect transcriptome changes in rat fat pad endothelial cells (RFP-ECs) which is incapable of TXA 2 signaling (TP null) to that of control (wild type) and RFP-EC with reconstituted TP expression. Genes that were significantly regulated due to infection were identified using a time course of 2, 18 and 48 hrs post infection. We identified several key genes such as suppressor of cytokine signaling (SOCS-5), several cytokines (CSF-1, CXCF ligands), and MAP kinases (MAPK-1, Janus kinase) that were upregulated in the absence of TP signaling. These data underscore the importance of the interaction of the parasite with mammalian TP and may explain the increased mortality and cardiovascular pathology observed in infected TP null mice.

Texas-Red–asialoorosomucoid (ASOR) fluorescence-sorted early and late endocytic vesicles from rat liver were subjected to proteomic analysis with the aim of identifying functionally important proteins. Several Rab GTPases, including Rab1a, were found. The present study immunolocalized Rab1a to early and late endocytic vesicles and examined its potential role in endocytosis. Huh7 cells with stable knockdown of Rab1a exhibited reduced endocytic processing of ASOR. This correlated with the finding that Rab1a antibody reduced microtubule-based motility of rat-liver-derived early but not late endocytic vesicles in vitro. The inhibitory effect of Rab1a antibody was observed to be specifically towards minus-end-directed motility. Total and minus-end-directed motility was also reduced in early endocytic vesicles prepared from Rab1a-knockdown cells. These results corresponded with virtual absence of the minus-end-directed kinesin Kifc1 from early endocytic vesicles in Rab1a knockdown cells and imply that Rab1a regulates minus-end-directed motility largely by recruiting Kifc1 to early endocytic vesicles.

1. Abstract2. Introduction3. Overview of the microarray technology3.1. The Principle of Microarray Technology3.2. Preparation of the microarray slide3.3. Synthesis of cDNA probes3.4. Synthesis of oligonucleotide probes3.5. Sample preparation and hybridization3.6. Confocal scanning3.7. Image analysis and normalization3.8. Verification of gene expression alterations4. Use of microarray in cardiovascular disease4.1. Myocardial ischemic and infarction4.2. Cardiac hypertrophy4.3. Dilated cardiomyopathy and congestive heart failure4.4. Pro-inflammatory molecules and reactive oxygen species in cardiac hypertrophy4.5. Hypertension and mechanical injury4.6. Aortic aneurysm5. Use of microarray in infectious heart diseases5.1. Viral and bacterial infections5.2. Parasitic Heart disease-Chagas’ disease6. Conclusions7. Acknowledgements8. References

Herpes simplex virus (HSV) capsids assemble, mature and package their viral genome in the nucleoplasm. They then exit the nucleus into the cytoplasm, where they acquire their final tegument and envelope. The molecular mechanism of cytoplasmic envelopment is unclear, but evidence suggests that the viral glycoprotein tails play an important role in the recruitment of tegument and capsids at the final envelopment site. However, due to redundancy in protein–protein interactions among the viral glycoproteins, genetic analysis of the role of individual glycoproteins in assembly has been difficult. To overcome this problem, a glutathione S-transferase fusion protein-binding assay was used in this study to test the interaction between the cytoplasmic tail of one specific viral glycoprotein, gD, and tegument proteins. The study demonstrated that the 38 kDa tegument protein VP22 bound specifically to the gD tail. This association was dependent on arginine and lysine residues at positions 5 and 6 in the gD tail. In addition, HSV-1 capsids bound the gD tail and exhibited a similar sequence dependence. It is concluded that VP22 may serve as a linker protein, mediating the interaction of the HSV capsid with gD.

Chagas disease, characterized by acute myocarditis and chronic cardiomyopathy, is caused by infection with the protozoan parasite, Trypanosoma cruzi. We sought to identify genes altered during the development of parasite-induced cardiomyopathy. Microarrays containing 27,400 sequence verified mouse cDNAs were used to analyze global gene expression changes in the myocardium of a murine model of chagasic cardiomyopathy. Changes in the gene expression were determined as the acute stage of infection developed into the chronic stage. This analysis was performed on the hearts of male CD-1 mice infected with trypomastigotes of T. cruzi (Brazil strain). At each interval we compared infected and uninfected mice and confirmed the microarray data with an oppositely labeled flip array. We identified eight district categories of mRNAs that were differentially regulated during infection and identified dysregulation of several key genes. These data may provide insight into the pathogenesis of Chagasic cardiomyopathy and provide new targets for intervention.