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Viruses Jun 2024Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment...
Human alphaherpesvirus 1 (HSV-1) is a significantly widespread viral pathogen causing recurrent infections that are currently incurable despite available treatment protocols. Studies have highlighted the potential of antimicrobial peptides sourced from venom, particularly those belonging to the mastoparan family, as effective against HSV-1. This study aimed to demonstrate the antiviral properties of mastoparans, including mastoparan-L [I, R], mastoparan-MO, and [I, R] mastoparan, against HSV-1. Initially, Vero cell viability was assessed in the presence of these peptides, followed by the determination of antiviral activity, mechanism of action, and dose-response curves through plaque assays. Structural analyses via circular dichroism and nuclear magnetic resonance were conducted, along with evaluating membrane fluidity changes induced by [I, R] mastoparan using fluorescence-labeled lipid vesicles. Cytotoxic assays revealed high cell viability (>80%) at concentrations of 200 µg/mL for mastoparan-L and mastoparan-MO and 50 µg/mL for [I, R] mastoparan. Mastoparan-MO and [I, R] mastoparan exhibited over 80% HSV-1 inhibition, with up to 99% viral replication inhibition, particularly in the early infection stages. Structural analysis indicated an α-helical structure for [I, R] mastoparan, suggesting effective viral particle disruption before cell attachment. Mastoparans present promising prospects for HSV-1 infection control, although further investigation into their mechanisms is warranted.
Topics: Herpesvirus 1, Human; Antiviral Agents; Animals; Vero Cells; Chlorocebus aethiops; Peptides; Wasp Venoms; Intercellular Signaling Peptides and Proteins; Cell Survival; Humans; Virus Replication
PubMed: 38932240
DOI: 10.3390/v16060948 -
Viruses May 2024The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions...
The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV's evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (), white-tailed deer (), and lesser dog-like bat () in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.
Topics: Virus Replication; Animals; Hepatitis Delta Virus; Humans; Hepatitis B virus; Marmota; Cell Division; Chiroptera; Viral Envelope Proteins; Cell Line; Hepatitis B; Hepatitis B Surface Antigens; Genotype; HEK293 Cells; Hepatitis D; RNA, Viral
PubMed: 38932152
DOI: 10.3390/v16060859 -
Sensors (Basel, Switzerland) Jun 2024In the realm of offline handwritten text recognition, numerous normalization algorithms have been developed over the years to serve as preprocessing steps prior to...
In the realm of offline handwritten text recognition, numerous normalization algorithms have been developed over the years to serve as preprocessing steps prior to applying automatic recognition models to handwritten text scanned images. These algorithms have demonstrated effectiveness in enhancing the overall performance of recognition architectures. However, many of these methods rely heavily on heuristic strategies that are not seamlessly integrated with the recognition architecture itself. This paper introduces the use of a Pix2Pix trainable model, a specific type of conditional generative adversarial network, as the method to normalize handwritten text images. Also, this algorithm can be seamlessly integrated as the initial stage of any deep learning architecture designed for handwritten recognition tasks. All of this facilitates training the normalization and recognition components as a unified whole, while still maintaining some interpretability of each module. Our proposed normalization approach learns from a blend of heuristic transformations applied to text images, aiming to mitigate the impact of intra-personal handwriting variability among different writers. As a result, it achieves slope and slant normalizations, alongside other conventional preprocessing objectives, such as normalizing the size of text ascenders and descenders. We will demonstrate that the proposed architecture replicates, and in certain cases surpasses, the results of a widely used heuristic algorithm across two metrics and when integrated as the first step of a deep recognition architecture.
PubMed: 38931676
DOI: 10.3390/s24123892 -
Microorganisms Jun 2024The purpose of the research was to determine the effect of the use of a diet containing 30% triticale grain. In an experiment lasting 28 days, 180 one-day Ross-308...
The purpose of the research was to determine the effect of the use of a diet containing 30% triticale grain. In an experiment lasting 28 days, 180 one-day Ross-308 chickens (sex ratio 1:1) with an average initial body weight in treatment of 44.6 g were randomly assigned to 30 metabolic cages/replications, 6 birds in each. To compare the results between treatments, a one-way ANOVA was used with uneven replication numbers. The control group (I) received a standard diet containing maize and soybean meal. In the other treatments, 30% of different cereals were used: II-wheat, III-barley, and IV-triticale. Significant differences in body weight (BW) and feed conversion ratio (FCR) were observed on the 4th day of the life of broiler chickens ( < 0.05). Differences were determined between the control group (90.7 g BW and 1.32 kg of feed/kg BWG in the case of FCR) and birds receiving barley (93.0 g BW and 1.29 kg of feed/kg BWG in the case of FCR), compared to chickens fed diets with a 30% share of wheat grain (86.2 g BW and 1.53 kg feed/kg BWG in the case of FCR) and triticale (86.6 g BW and 1.53 kg feed/kg BWG in the case of FCR). Later, the differences in performance of birds between treatments did not occur ( > 0.05). In the nutrition of broiler chickens, control or 30% of the triticale diet caused a significant reduction ( < 0.01) of the number of () in the crop of broiler chickens (0 log cfu/g), compared to birds obtaining feed with 30% of wheat (1.78 log cfu/g). The diet containing triticale also reduced the number of ( < 0.05) within the ileum (0.78 log cfu/g) compared to chickens obtaining barley grain in the diet (2.12 log cfu/g). As a result of the use of triticale grain ( < 0.05), the total length of the bird intestines (199.64 cm) was compared to 30% of barley grain (209.76 cm). The increase in the length of the large intestine of broiler chickens in treatments was positively correlated (r = 0.613, < 0.05) with the number of sp. in the ileum. Triticale increased the pH in the crop of broilers chickens. The research results indicate that triticale, after longer storage, can be used in amounts of 30% of the diet without significant effect on the performance of broiler chickens, with a reduction in in crop in comparison with wheat and in ileum with barley.
PubMed: 38930621
DOI: 10.3390/microorganisms12061239 -
Microorganisms Jun 2024Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug...
Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug toxicity, is essential to eradicating HIV. This study investigated the potential of fractionated crude extracts of as immunomodulatory or anti-HIV drug candidates. Solid-phase extraction (SPE) was used to fractionate PO4PR2 using three different columns: MAX (Mixed-mode, strong Anion-eXchange), MCX (Mixed-mode, strong Cation-eXchange), and HLB (Hydrophilic-Lipophilic Balance) with methanol gradient methods (5%, 45%, and 95%). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to assess the cell viability and cytotoxicity of the fractionated crude extract PO4PR2 in the TZM-bl cell lines. This was followed by a luciferase-based antiviral assay to assess the antiviral activity of PO4PR2. A time of addition (TOA) assay was performed to ascertain the mechanism of inhibition employed by the fractionated crude extract of PO4PR2 in the HIV life cycle. The p24 titer was determined using an ELISA, while a luciferase-based antiviral assay was used to evaluate the HIV percentage inhibition for different HIV-1 replication cycles. The TOA assay was established using antiviral drugs that target different sites in the HIV replication cycle. These included maraviroc, azidothymidine, raltegravir, and amprenavir. The immunomodulatory effect of the fractionated crude extracts on CD4+ T cells was measured by a flow cytometric analysis, for which fluorochrome-labelled monoclonal antibodies were used as markers for activation (CD38 and HLA-DR) and exhaustion (PD-1). The MCX fraction demonstrated a more significant anti-HIV inhibition than that of the fractions generated in other columns, with an IC of 0.3619 µg/mL, an HIV inhibition of 77%, 5% HLB (IC: 0.7232 µg/mL; HIV inhibition of 64%), and 5% MAX (IC: 5.240 µg/mL; HIV inhibition of 67%). It was evident from the time of addition data that the crude extract and the 5% MCX fraction inhibited viral binding (68%), reverse transcription (75%), integration (98%), and proteolysis (77%). It was shown that (the MCX fraction) have a significant inhibitory effect on reverse transcription (75% HIV inhibition) and integration (100% HIV inhibition). The 5% MCX ( = 0.0062), 5% HLB ( = 0.0269), and 5% MAX ( = 0.0117) fractionated crude extracts had low levels of CD4+ T cell (CD38 + HLA-DR+) activation compared to those of the AZT treatment, while CD4+ T cell activation was insignificant. The 5% MAX and HLB fractions may possess immunomodulatory compounds with less anti-HIV-1 activity. could be a key source of innovative anti-HIV drugs with immunomodulatory characteristics.
PubMed: 38930532
DOI: 10.3390/microorganisms12061150 -
Microorganisms May 2024Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this...
Replication of the mitochondrial (mt) genome in filamentous fungi is under-studied, and knowledge is based mainly on data from yeasts and higher eukaryotes. In this study, the mitochondrial DNA polymerase γ (Mip1) of the entomopathogenic fungus is characterized and analyzed with disruption experiments and its in silico interactions with key proteins implicated in mt gene transcription, i.e., mt RNA polymerase Rpo41 and mt transcription factor Mtf1. Disruption of 1 gene and its partial expression influences cell growth, morphology, germination and stress tolerance. A putative in silico model of Mip1-Rpo41-Mtf1, which is known to be needed for the initiation of replication, was proposed and helped to identify potential amino acid residues of Mip1 that interact with the Rpo41-Mtf1 complex. Moreover, the reduced expression of 1 indicates that Mip1 is not required for efficient transcription but only for replication. Functional differences between the Mip1 and its counterparts from and higher eukaryotes are discussed.
PubMed: 38930434
DOI: 10.3390/microorganisms12061052 -
Life (Basel, Switzerland) May 2024Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the... (Review)
Review
Theoretical and experimental approaches have been applied to study the polymer physics underlying the compaction of DNA in the bacterial nucleoid. Knowledge of the compaction mechanism is necessary to obtain a mechanistic understanding of the segregation process of replicating chromosome arms (replichores) during the cell cycle. The first part of this review discusses light microscope observations demonstrating that the nucleoid has a lower refractive index and thus, a lower density than the cytoplasm. A polymer physics explanation for this phenomenon was given by a theory discussed at length in this review. By assuming a phase separation between the nucleoid and the cytoplasm and by imposing equal osmotic pressure and chemical potential between the two phases, a minimal energy situation is obtained, in which soluble proteins are depleted from the nucleoid, thus explaining its lower density. This theory is compared to recent views on DNA compaction that are based on the exclusion of polyribosomes from the nucleoid or on the transcriptional activity of the cell. These new views prompt the question of whether they can still explain the lower refractive index or density of the nucleoid. In the second part of this review, we discuss the question of how DNA segregation occurs in in the absence of the so-called active ParABS system, which is present in the majority of bacteria. How is the entanglement of nascent chromosome arms generated at the origin in the parental DNA network of the nucleoid prevented? Microscopic observations of the position of fluorescently-labeled genetic loci have indicated that the four nascent chromosome arms synthesized in the initial replication bubble segregate to opposite halves of the sister nucleoids. This implies that extensive intermingling of daughter strands does not occur. Based on the hypothesis that leading and lagging replichores synthesized in the replication bubble fold into microdomains that do not intermingle, a passive four-excluding-arms model for segregation is proposed. This model suggests that the key for segregation already exists in the structure of the replication bubble at the very start of DNA replication; it explains the different patterns of chromosome arms as well as the segregation distances between replicated loci, as experimentally observed.
PubMed: 38929644
DOI: 10.3390/life14060660 -
International Journal of Molecular... Jun 2024The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion...
The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% ( < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.
Topics: Humans; HIV-1; HEK293 Cells; Virion; Jurkat Cells; Fluorescence Resonance Energy Transfer; Virus Replication; Virus Assembly; HIV Infections
PubMed: 38928103
DOI: 10.3390/ijms25126396 -
International Journal of Molecular... Jun 2024In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing...
The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
Topics: Humans; Viral Nonstructural Proteins; Virus Replication; Autophagosomes; HeLa Cells; Phlebovirus; Autophagy; Tyrosine; Tryptophan; TOR Serine-Threonine Kinases; Mutation; Amino Acid Substitution; Severe Fever with Thrombocytopenia Syndrome; Lysosomes; Nucleoproteins
PubMed: 38928101
DOI: 10.3390/ijms25126394 -
Genes Jun 2024Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a...
Dysfunction in ion channels or processes involved in maintaining ionic homeostasis is thought to lower the threshold for cortical spreading depression (CSD), and plays a role in susceptibility to associated neurological disorders, including pathogenesis of a migraine. Rare pathogenic variants in specific ion channels have been implicated in monogenic migraine subtypes. In this study, we further examined the channelopathic nature of a migraine through the analysis of common genetic variants in three selected ion channel or transporter genes: , , and . Using the Agena MassARRAY platform, 28 single-nucleotide polymorphisms (SNPs) across the three candidate genes were genotyped in a case-control cohort comprised of 182 migraine cases and 179 matched controls. Initial results identified significant associations between migraine and rs3776578 ( = 0.04) and rs16903247 ( = 0.05) genotypes within the gene, which encodes the EAAT1 glutamate transporter. These SNPs were subsequently genotyped in an independent cohort of 258 migraine cases and 290 controls using a high-resolution melt assay, and association testing supported the replication of initial findings-rs3776578 ( = 0.0041) and rs16903247 ( = 0.0127). The polymorphisms are in linkage disequilibrium and localise within a putative intronic enhancer region of . The minor alleles of both SNPs show a protective effect on migraine risk, which may be conferred via influencing the expression of .
Topics: Humans; Polymorphism, Single Nucleotide; Migraine Disorders; Female; Male; Excitatory Amino Acid Transporter 1; Adult; Case-Control Studies; Genetic Predisposition to Disease; Middle Aged; Genetic Association Studies
PubMed: 38927733
DOI: 10.3390/genes15060797