Molecular characterization of rotavirus indicates predominance of G9P[4] genotype among children with acute gastroenteritis: First report after vaccine introduction in Pakistan.

Globally, Group A rotavirus (RVA) is the leading cause of acute gastroenteritis in children under 5 years old, with Pakistan having the highest rates of RVA-related morbidity and mortality. The current study aims to determine the genetic diversity of rotavirus and evaluate the impact of Rotarix-vaccine introduction on disease epidemiology in Pakistan. A total of 4749 children, hospitalized with acute gastroenteritis between 2018 and 2020, were tested at four hospitals in Lahore and Karachi. Of the total, 19.3% (918/4749) cases were tested positive for RVA antigen, with the positivity rate varying annually (2018 = 22.7%, 2019 = 14.4%, 2020 = 20.9%). Among RVA-positive children, 66.3% were under 1 year of age. Genotyping of 662 enzyme-linked immuno sorbent assay-positive samples revealed the predominant genotype as G9P[4] (21.4%), followed by G1P[8] (18.9%), G3P[8] (11.4%), G12P[6] (8.7%), G2P[4] (5.7%), G2P[6] (4.8%), and 10.8% had mixed genotypes. Among vaccinated children, genotypes G9P[4] and G12P[6] were more frequently detected, whereas a decline in G2P[4] was observed. Phylogenetic analysis confirmed the continued circulation of indigenous genotypes detected earlier in the country except G9 and P[6] strains. Our findings highlight the predominance of G9P[4] genotype after the vaccine introduction thus emphasizing continual surveillance to monitor the disease burden, viral diversity, and their impact on control of rotavirus gastroenteritis in children.

Employing computational tools to design a multi-epitope vaccine targeting human immunodeficiency virus-1 (HIV-1).

BACKGROUND: Despite being in the 21st century, the world has still not been able to vanquish the global AIDS epidemic, and the only foreseeable solution seems to be a safe and effective vaccine. Unfortunately, vaccine trials so far have returned unfruitful results, possibly due to their inability to induce effective cellular, humoral and innate immune responses. The current study aims to tackle these limitations and propose the desired vaccine utilizing immunoinformatic approaches that have returned promising results in designing vaccines against various rapidly mutating organisms. For this, all polyprotein and protein sequences of HIV-1 were retrieved from the LANL (Los Alamos National Laboratory) database. The consensus sequence was generated after alignment and used to predict epitopes. Conserved, antigenic, non-allergenic, T-cell inducing, B-cell inducing, IFN-É£ inducing, non-human homologous epitopes were selected and combined to propose two vaccine constructs i.e., HIV-1a (without adjuvant) and HIV-1b (with adjuvant). RESULTS: HIV-1a and HIV-1b were subjected to antigenicity, allergenicity, structural quality analysis, immune simulations, and MD (molecular dynamics) simulations. Both proposed multi-epitope vaccines were found to be antigenic, non-allergenic, stable, and induce cellular, humoral, and innate immune responses. TLR-3 docking and in-silico cloning of both constructs were also performed. CONCLUSION: Our results indicate HIV-1b to be more promising than HIV-1a; experimental validations can confirm the efficacy and safety of both constructs and in-vivo efficacy in animal models.

Genome mining, antimicrobial and plant growth-promoting potentials of halotolerant Bacillus paralicheniformis ES-1 isolated from salt mine.

Salinity severely affects crop yield by hindering nitrogen uptake and reducing plant growth. Plant growth-promoting bacteria (PGPB) are capable of providing cross-protection against biotic/abiotic stresses and facilitating plant growth. Genome-level knowledge of PGPB is necessary to translate the knowledge into a product as efficient biofertilizers and biocontrol agents. The current study aimed to isolate and characterize indigenous plant growth-promoting strains with the potential to promote plant growth under various stress conditions. In this regard, 72 bacterial strains were isolated from various saline-sodic soil/lakes; 19 exhibited multiple in vitro plant growth-promoting traits, including indole 3 acetic acid production, phosphate solubilization, siderophore synthesis, lytic enzymes production, biofilm formation, and antibacterial activities. To get an in-depth insight into genome composition and diversity, whole-genome sequence and genome mining of one promising Bacillus paralicheniformis strain ES-1 were performed. The strain ES-1 genome carries 12 biosynthetic gene clusters, at least six genomic islands, and four prophage regions. Genome mining identified plant growth-promoting conferring genes such as phosphate solubilization, nitrogen fixation, tryptophan production, siderophore, acetoin, butanediol, chitinase, hydrogen sulfate synthesis, chemotaxis, and motility. Comparative genome analysis indicates the region of genome plasticity which shapes the structure and function of B. paralicheniformis and plays a crucial role in habitat adaptation. The strain ES-1 has a relatively large accessory genome of 649 genes (~ 19%) and 180 unique genes. Overall, these results provide valuable insight into the bioactivity and genomic insight into B. paralicheniformis strain ES-1 with its potential use in sustainable agriculture.

Alocasia odora-mediated synthesis of silver nanoparticles, their cytotoxicity, and virucidal potential.

Silver nanoparticles (AgNPs) have various applications in the biomedical field and are considered excellent microbicidal agents. Moreover, biological synthesis of AgNPs using medicinal plants further improves the medicinal applicability of these plants. In this study, the aqueous extract of Alocasia odora rhizome (RE) and Alocasia odora stem (SE) were used to synthesize stem aqueous extract-AgNPs (SNP) and rhizome aqueous extract-AgNPs (RNP). Furthermore, RNP and SNP were evaluated for their virucidal potential. The synthesis of SNP and RNP was monitored using a UV spectrophotometer by observing their surface plasmon resonance peak. In addition, scanning electron microscopy (SEM) gave further insight into their morphology and particle size, whereas energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of silver ions. Interestingly, Fourier-transform infrared spectroscopy (FTIR) analysis of AgNPs revealed that phytomolecules acted as capping and stabilizing agents for SNP and RNP. The in vitro cytotoxicity of SNP and RNP was further analyzed using MTT assay on the U87-MG human glioblastoma cancer cell line and SNP found to be the most cytotoxic (43.40 µg/ml) among all. Besides that, SNP has also found to show the maximum cytopathic effects (CPE) against dengue virus type 2 (DENV-2) on Huh-7 cell line. As a result of the observations, it can be concluded that they can become a promising antiviral drug candidate and thus merit further testing. KEY POINTS: • AgNPs were successfully synthesized through Alocasia odora aqueous extract. • AgNPs were more cytotoxic on the U87-MG cell line than the extract alone. • AgNPs have shown significant reduction in the dengue viral infection than the extract alone.

Investigation of Newly Synthesized Bis-Acyl-Thiourea Derivatives of 4-Nitrobenzene-1,2-Diamine for Their DNA Binding, Urease Inhibition, and Anti-Brain-Tumor Activities.

Bis-acyl-thiourea derivatives, namely N,N'-(((4-nitro-1,2-phenylene)bis(azanediyl)) bis(carbonothioyl))bis(2,4-dichlorobenzamide) (UP-1), N,N'-(((4-nitro-1,2-phenylene) bis(azanediyl))bis(carbonothioyl))diheptanamide (UP-2), and N,N'-(((4-nitro-1,2-phenylene)bis(azanediyl))bis(carbonothioyl))dibutannamide (UP-3), were synthesized in two steps. The structural characterization of the derivatives was carried out by FTIR, 1H-NMR, and 13C-NMR, and then their DNA binding, anti-urease, and anticancer activities were explored. Both theoretical and experimental results, as obtained by density functional theory, molecular docking, UV-visible spectroscopy, fluorescence (Flu-)spectroscopy, cyclic voltammetry (CV), and viscometry, pointed towards compounds' interactions with DNA. However, the values of binding constant (Kb), binding site size (n), and negative Gibbs free energy change (ΔG) (as evaluated by docking, UV-vis, Flu-, and CV) indicated that all the derivatives exhibited binding interactions with the DNA in the order UP-3 > UP-2 > UP-1. The experimental findings from spectral and electrochemical analysis complemented each other and supported the theoretical analysis. The lower diffusion coefficient (Do) values, as obtained from CV responses of each compound after DNA addition at various scan rates, further confirmed the formation of a bulky compound-DNA complex that caused slow diffusion. The mixed binding mode of interaction as seen in docking was further verified by changes in DNA viscosity with varying compound concentrations. All compounds showed strong anti-urease activity, whereas UP-1 was found to have comparatively better inhibitory efficiency, with an IC50 value of 1.55 ± 0.0288 µM. The dose-dependent cytotoxicity of the synthesized derivatives against glioblastoma MG-U87 cells (a human brain cancer cell line) followed by HEK-293 cells (a normal human embryonic kidney cell line) indicated that UP-1 and UP-3 have greater cytotoxicity against both cancerous and healthy cell lines at 400 µM. However, dose-dependent responses of UP-2 showed cytotoxicity against cancerous cells, while it showed no cytotoxicity on the healthy cell line at a low concentration range of 40-120 µM.

Cannabinoid and endocannabinoid system: a promising therapeutic intervention for multiple sclerosis.

Multiple sclerosis (MS) is a chronic and complex neurodegenerative disease, distinguished by the presence of lesions in the central nervous system (CNS) due to exacerbated immunological responses that inflict oligodendrocytes and the myelin sheath of axons. In recent years, studies have focused on targeted therapeutics for MS that emphasize the role of G protein-coupled receptors (GPCRs), specifically cannabinoids receptors. Clinical studies have suggested the therapeutic potential of cannabinoids derived from Cannabis sativa in relieving pain, tremors and spasticity. Cannabinoids also appear to prevent exaggerated immune responses in CNS due to compromised blood-brain barrier. Both, endocannabinoid system (ECS) modulators and cannabinoid ligands actively promote oligodendrocyte survival by regulating signaling, migration and myelination of nerve cells. The cannabinoid receptors 1 (CB1) and 2 (CB2) of ECS are the main ones in focus for therapeutic intervention of MS. Various CB1/CB2 receptors agonists have been experimentally studied which showed anti-inflammatory properties and are considered to be effective as potential therapeutics for MS. In this review, we focused on the exacerbated immune attack on nerve cells and the role of the cannabinoids and its interaction with the ECS in CNS during MS pathology.

Investigations on Anticancer Potentials by DNA Binding and Cytotoxicity Studies for Newly Synthesized and Characterized Imidazolidine and Thiazolidine-Based Isatin Derivatives.

Imidazolidine and thiazolidine-based isatin derivatives (IST-01-04) were synthesized, characterized, and tested for their interactions with ds-DNA. Theoretical and experimental findings showed good compatibility and indicated compound-DNA binding by mixed mode of interactions. The evaluated binding parameters, i.e., binding constant (Kb), free energy change (ΔG), and binding site sizes (n), inferred comparatively greater and more spontaneous binding interactions of IST-02 and then IST-04 with the DNA, among all compounds tested under physiological pH and temperature (7.4, 37 °C). The cytotoxic activity of all compounds was assessed against HeLa (cervical carcinoma), MCF-7 (breast carcinoma), and HuH-7 (liver carcinoma), as well as normal HEK-293 (human embryonic kidney) cell lines. Among all compounds, IST-02 and 04 were found to be cytotoxic against HuH-7 cell lines with percentage cell toxicity of 75% and 66%, respectively, at 500 ng/µL dosage. Moreover, HEK-293 cells exhibit tolerance to the increasing drug concentration, suggesting these two compounds are less cytotoxic against normal cell lines compared to cancer cell lines. Hence, both DNA binding and cytotoxicity studies proved imidazolidine (IST-02) and thiazolidine (IST-04)-based isatin derivatives as potent anticancer drug candidates among which imidazolidine (IST-02) is comparatively the more promising.

Impact of pre-existing drug resistance on risk of virological failure in South Africa.

OBJECTIVES: There is conflicting evidence on the impact of pre-existing HIV drug resistance mutations (DRMs) in patients infected with non-B subtype virus. METHODS: We performed a case-cohort substudy of the AIDS Drug Resistance Surveillance Study, which enrolled South African patients initiating first-line efavirenz/emtricitabine/tenofovir. Pre-ART DRMs were detected by Illumina sequencing of HIV pol and DRMs present at <20% of the viral population were labelled as minority variants (MVs). Weighted Cox proportional hazards models estimated the association between pre-ART DRMs and risk of virological failure (VF), defined as confirmed HIV-1 RNA ≥1000 copies/mL after ≥5 months of ART. RESULTS: The evaluable population included 178 participants from a randomly selected subcohort (16 with VF, 162 without VF) and 83 additional participants with VF. In the subcohort, 16% of participants harboured ≥1 majority DRM. The presence of any majority DRM was associated with a 3-fold greater risk of VF (P = 0.002), which increased to 9.2-fold (P < 0.001) in those with <2 active drugs. Thirteen percent of participants harboured MV DRMs in the absence of majority DRMs. Presence of MVs alone had no significant impact on the risk of VF. Inclusion of pre-ART MVs with majority DRMs improved the sensitivity but reduced the specificity of predicting VF. CONCLUSIONS: In a South African cohort, the presence of majority DRMs increased the risk of VF, especially for participants receiving <2 active drugs. The detection of drug-resistant MVs alone did not predict an increased risk of VF, but their inclusion with majority DRMs affected the sensitivity/specificity of predicting VF.

Effect of oxidative stress and calcium deregulation on FAM26F (CALHM6) expression during hepatitis B virus infection.

BACKGROUND: Family with sequence similarity 26, member F (FAM26F) is an important innate immunity modulator playing a significant role in diverse immune responses, however, the association of FAM26F expression with HBV infection is not yet known. Thus, the current study aims to explore the differential expression of FAM26F in vitro in HepAD38 and HepG2 cell lines upon HBV infection, and in vivo in HBV infected individuals. The effects of antioxidant and calcium inhibitors on the regulation of FAM26F expression were also evaluated. The expression of FAM26F was simultaneously determined with well-established HBV infection markers: IRF3, and IFN-ß. METHODS: The expression of FAM26F and marker genes was analyzed through Real-time qPCR and western blot. RESULTS: Our results indicate that the differential expression of FAM26F followed the same trend as that of IRF3 and IFN-ß. The in vitro study revealed that, in both HBV infected cell lines, FAM26F expression was significantly down-regulated as compared to uninfected control cells. Treatment of cells with N-acetyl-L-cysteine (NAC), EGTA-AM, BAPTA-AM, and Ru360 significantly upregulated the expression of FAM26F in both the cell lines. Moreover, in in vivo study, FAM26F expression was significantly downregulated in all HBV infected groups as compared to controls (p = 0.0007). The expression was higher in the HBV recovered cases, probably due to the decrease in infection and increase in the immunity of these individuals. CONCLUSION: Our study is the first to show the association of FAM26F with HBV infection. It is proposed that FAM26F expression could be an early predictive marker for HBV infection, and thus is worthy of further investigation.

Synthesis, X-ray, Hirshfeld surface analysis, exploration of DNA binding, urease enzyme inhibition and anticancer activities of novel adamantane-naphthyl thiourea conjugate.

1-(adamantane-1-carbonyl-3-(1-naphthyl)) thiourea (C22H24N2OS (4), was synthesized by the reaction of freshly prepared adamantane-1-carbonyl chloride from corresponding acid (3) with ammonium thiocyanate in 1:1 M ratio in dry acetone to afford the adamantane-1-carbonyl isothiocyanate (2) in situ followed by treatment with 1-naphthyl amine (3). The structure was established by elemental analyses, FTIR, 1H, 13C NMR and mass spectroscopy. The molecular and crystal structure were determined by single crystal X-ray analysis. It belongs to triclinic system P - 1 space group with a = 6.7832(5) Å, b = 11.1810(8) Å, c = 13.6660(10) Å, α = 105.941(6)°, ß = 103.730(6)°, γ = 104.562(6)°, Z = 2, V = 910.82(11) Å3. The naphthyl group is almost planar. In the crystal structure, intermolecular CH···O hydrogen bonds link the molecules into centrosymmetric dimers, enclosing R22(14) ring motifs, while the intramolecular NH···O hydrogen bonds enclose S(6) ring motifs, in which they may be effective in the stabilization of the structure. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H … H (59.3%), H … C/C … H (19.8%) and H … S/S … H (10.1%) interactions. Hydrogen bonding and van der Waals interactions are the dominant interactions in the crystal packing. DFT, molecular docking and urease inhibition studies revealed stability and electron withdrawing nature of 4 as compared to DNA base pairs and residues of urease. The DNA binding results from docking, UV- visible spectroscopy, and viscosity studies indicated significant binding of 4 with the DNA via intercalation and groove binding. Further investigation of the compound was done on hepatocellular carcinoma; Huh-7 cell line as well as normal human embryonic kidney; Hek-293 cell line. The compound showed significant cytotoxic activity against Huh-7 cells in comparison to normal Hek-293 cells indicating selective cytotoxicity towards cancer cells.

Methylphenidate and Rosmarinus officinalis improves cognition and regulates inflammation and synaptic gene expression in AlCl3-induced neurotoxicity mouse model.

Methylphenidate (MPH), a psychotropic medication is commonly used for children with attention deficit hyperactivity disorder (ADHD). In this study we elucidated the neuroprotective and anti-inflammatory effects of MPH and Rosmarinus officinalis (rosemary) extract, an ancient aromatic herb with several applications in traditional medicine. Briefly, six groups of mice (n = 8 each group), were specified for the study and behavioral analysis was performed to analyze spatial memory followed by histological assessment and gene expression analysis of synaptic (Syn I, II and III) and inflammatory markers (IL-6, TNFα and GFAP) via qRT-PCR, in an AlCl3-induced mouse model for neurotoxicity. The behavioral analysis demonstrated significant cognitive decline, memory defects and altered gene expression in AlCl3-treated group. Rosemary extract significantly decreased the expression of inflammatory and synaptic markers to the similar levels as that of MPH. The present findings suggested the neuroprotective potential of Rosmarinus officinalis extract. However, further characterization of its anti-inflammatory and neuroprotective properties and MPH is required to strategize future treatments for several neurological and neurodegenerative disorders, including Alzheimer's disease.

Aroylthiourea derivatives of ciprofloxacin drug as DNA binder: Theoretical, spectroscopic and electrochemical studies along with cytotoxicity assessment.

Aroylthiourea derivatives of ciprofloxacin drug - [1-cyclopropyl-6-fluoro-7-(4-((4-methoxybenzoyl)carbamothioyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] ATU-1, [1-cyclopropyl-7-(4-((2,4-dibromobenzoyl)carbamothioyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] ATU-2, and [1-cyclopropyl-7-(4-((3,5-dinitrobenzoyl)carbamothioyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid] ATU-3 were synthesized, characterized and investigated for DNA binding at stomach pH (4.7) and at 37 °C. All findings by using DFT, molecular docking, spectroscopic (UV-, fluorescence; FL-), cyclic voltammetric (CV) and viscometric techniques revealed that these compounds have the potency to bind with DNA via a mixed mode of interaction. The binding affinity of ATU-1 was evaluated comparatively greater with Kb × 104/M-1 (docking; 5.55, UV-; 7.93, FL-; 5.62, CV; 6.06), ΔG/kJmol-1(docking; -27.07, UV-; -29.07, FL-; -28.18, CV; -28.38) and n (FL-; 1.20, CV; 2.72). Stern-Volmer quenching constant (Ksv) further pointed towards comparatively greater binding affinity of ATU-1 for DNA, while bimolecular quenching constant (Kq) values showed the involvement of static quenching mechanism in the compound - DNA interaction. Comparatively lesser IC50 (7.1 µM) value obtained from biological work on Huh-7 cancer cell line further confirmed the greater anticancer potential of ATU-1 than that of ATU-2&3.

Pre-infection transcript levels of FAM26F in peripheral blood mononuclear cells inform about overall plasma viral load in acute and post-acute phase after simian immunodeficiency virus infection.

CD8+ cells from simian immunodeficiency virus (SIV)-infected long-term non-progressors and some uninfected macaques can suppress viral replication in vitro without killing the infected cells. The aim of this study was to identify factors responsible for non-cytolytic viral suppression by transcriptional profiling and to investigate their potential impact on SIV replication. Results of microarray experiments and further validation with cells from infected and uninfected macaques revealed that FAM26F RNA levels distinguished CD8+ cells of controllers and non-controllers (P=0.001). However, FAM26F was also expressed in CD4+ T-cells and B-cells. FAM26F expression increased in lymphocytes after in vitro IFN-γ treatment on average 40-fold, and ex vivo FAM26F RNA levels in peripheral blood mononuclear cells correlated with plasma IFN-γ but not with IFN-α. Baseline FAM26F expression appeared to be stable for months, albeit the individual expression levels varied up to tenfold. Investigating its role in SIV-infection revealed that FAM26F was upregulated after infection (P<0.0008), but did not directly correlate with viral load in contrast to MX1 and CXCL10. However, pre-infection levels of FAM26F correlated inversely with overall plasma viral load (AUC) during the acute and post-acute phases of infection (e.g. AUC weeks post infection 0-8; no AIDS vaccine: P<0.0001, Spearman rank correlation coefficient (rs)=-0.89, n=16; immunized with an AIDS vaccine: P=0.033, rs=-0.43; n=25). FAM26F transcript levels prior to infection can provide information about the pace and strength of the antiviral immune response during the early stage of infection. FAM26F expression represented, in our experiments, one of the earliest prognostic markers, and could supplement major histocompatibility complex (MHC)-typing to predict disease progression before SIV-infection.

Can IFNL3 polymorphisms predict response to interferon/ribavirin treatment in hepatitis C patients with genotype 3?

Favourable genotypes of IFNL3 polymorphism CC for rs12979860 and TT for rs8099917 are strongly associated with the interferon/ribavirin treatment outcome in hepatitis C virus (HCV) patients with genotypes 1 and 4. Contrarily, conflicting results have been reported for patients with HCV genotypes 2 and 3. Therefore, we sought to investigate the association between IFNL3 with sustained virological response (SVR) after treatment to ascertain the predictive value of IFNL3 single-nucleotide polymorphisms (SNPs) in HCV patients with genotype 3. For this purpose, we genotyped five IFNL3 SNPs, rs12980275, rs12979860, rs9109886, rs8099917 and rs7248668, in HCV patients with genotype 3 and assessed its association with SVR, individually and in haplotype. Interestingly, we report that the IFNL3 SNPs we genotyped have shown no association with SVR following treatment, either individually or in haplotype, indicating that genotyping IFNL3 SNPs have limited predictive value in HCV patients with genotype 3. Therefore, we propose that IFNL3 genotyping can be excluded from a patient's pre-treatment workup for subsequent treatment choice. This will greatly reduce the economic burden for HCV patients with genotype 3 in resource-limited regions, especially South Asia where genotype 3 is predominant.

Increased BST2 expression during simian immunodeficiency virus infection is not a determinant of disease progression in rhesus monkeys.

BACKGROUND: Bone marrow stromal cell antigen 2 (BST2), also known as tetherin, HM1.24 or CD317 represents a type 2 integral membrane protein, which has been described to restrict the production of some enveloped viruses by inhibiting the virus release from the cell surface. This innate antiviral mechanism is counteracted by the HIV-1 viral factor Vpu, targeting BST2 for cellular degradation. Since antiviral BST2 activity has been mainly confirmed by in vitro data, we investigated its role in vivo on the disease progression using the SIV/macaque model for AIDS. We determined BST2 expression in PBMC and leukocyte subsets of uninfected and SIV-infected rhesus macaques by real-time PCR and flow cytometry and correlated it with disease progression and viral load. RESULTS: Compared to pre-infection levels, we found increased BST2 expression in PBMC, purified CD4(+) lymphocytes and CD14(+) monocytes of SIV-infected animals, which correlated with viral load. Highest BST2 levels were found in progressors and lowest levels comparable to uninfected macaques were observed in long-term non-progressors (LTNPs). During acute viremia, BST2 mRNA increased in parallel with MX1, a prototype interferon-stimulated gene. This association was maintained during the whole disease course. CONCLUSION: The detected relationship between BST2 expression and viral load as well as with MX1 indicate a common regulation by the interferon response and suggest rather limited influence of BST2 in vivo on the disease outcome.

Evolution of protein domain repertoires of CALHM6.

Calcium (Ca2 +) homeostasis is essential in conducting various cellular processes including nerve transmission, muscular movement, and immune response. Changes in Ca2 + concentration in the cytoplasm are significant in bringing about various immune responses such as pathogen clearance and apoptosis. Various key players are involved in calcium homeostasis such as calcium binders, pumps, and channels. Sequence-based evolutionary information has recently been exploited to predict the biophysical behaviors of proteins, giving critical clues about their functionality. Ion channels are reportedly the first channels developed during evolution. Calcium homeostasis modulator protein 6 (CALHM6) is one such channel. Comprised of a single domain called Ca_hom_mod, CALHM6 is a stable protein interacting with various other proteins in calcium regulation. No previous attempt has been made to trace the exact evolutionary events in the domain of CALHM6, leaving plenty of room for exploring its evolution across a wide range of organisms. The current study aims to answer the questions by employing a computational-based strategy that used profile Hidden Markov Models (HMMs) to scan for the CALHM6 domain, integrated the data with a time-calibrated phylogenetic tree using BEAST and Mesquite, and visualized through iTOL. Around 4,000 domains were identified, and 14,000 domain gain, loss, and duplication events were observed at the end which also included various protein domains other than CALHM6. The data were analyzed concerning CALHM6 evolution as well as the domain gain, loss, and duplication of its interacting partners: Calpain, Vinculin, protein S100-A7, Thioredoxin, Peroxiredoxin, and Calmodulin-like protein 5. Duplication events of CALHM6 near higher eukaryotes showed its increasing complexity in structure and function. This in-silico phylogenetic approach applied to trace the evolution of CALHM6 was an effective approach to get a better understanding of the protein CALHM6.

Fabrication of Bilayer Nanofibrous-Hydrogel Scaffold from Bacterial Cellulose, PVA, and Gelatin as Advanced Dressing for Wound Healing and Soft Tissue Engineering.

Tissue engineering is currently one of the fastest-growing areas of engineering, requiring the fabrication of advanced and multifunctional materials that can be used as scaffolds or dressings for tissue regeneration. In this work, we report a bilayer material prepared by electrospinning a hybrid material of poly(vinyl alcohol) (PVA) and bacterial cellulose (BC NFs) (top layer) over a highly interconnected porous 3D gelatin-PVA hydrogel obtained by a freeze-drying process (bottom layer). The techniques were combined to produce an advanced material with synergistic effects on the physical and biological properties of the two materials. The bilayer material was characterized using Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and a water contact measurement system (WCMS). Studies on swelling, degradability, porosity, drug release, cellular and antibacterial activities were performed using standardized procedures and assays. FTIR confirmed cross-linking of both the top and bottom layers, and SEM showed porous structure for the bottom layer, random deposition of NFs on the surface, and aligned NFs in the cross section. The water contact angle (WCA) showed a hydrophilic surface for the bilayer material. Swelling analysis showed high swelling, and degradation analysis showed good stability. The bilayer material released Ag-sulfadiazine in a sustained and controlled manner and showed good antibacterial activities against severe disease-causing gram + ive and -ive (Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa) bacterial strains. In vitro biological studies were performed on fibroblasts (3T3) and human embryonic kidneys (HEK-293), which showed desirable cell viability, proliferation, and adhesion to the bilayer. Thus, the synergistic effect of NFs and the hydrogel resulted in a potential wound dressing material for wound healing and soft tissue engineering.

Host immune players and their response to Hepatitis C therapies.

This study aimed to investigate alterations in the expression of four key cytokines (IL-7, IL-11, IL-15, and IL-27) and assess differential FAM26F expression in response to Hepatitis C virus (HCV) infection. Additionally, it sought to analyze changes in these cytokines after treatment in 244 chronic HCV patients and 28 controls undergoing various treatments, including standard interferon, pegylated interferon, and Direct Acting Antivirals (DAAs). The objective was to compare immune system regulation between treatment groups. The expression levels of FAM26F and the cytokines (IL-7, IL-11, IL-15, and IL-27) were evaluated using Real-time qPCR in PBMCs of treatment groups. Results revealed significant downregulation of IL-7 and IL-27 in infected individuals compared to healthy controls, persisting even after treatment. This suggests the crucial roles of these immune modulators in facilitating the necessary T-cell response for viral clearance. IL-11 expression also remained suppressed post-treatment, supporting viral clearance by restoring the Th1 response. The decrease in IL-11 levels during treatment indicates the restoration of the Th1 response, vital for viral clearance. IL-15, the key cytokine regulating cytotoxic cells (NKT and NK cells), displayed consistent expression across all sample groups, indicating maintained IL-15-induced cytotoxicity in both control and infected individuals. Additionally, FAM26F expression was reduced in the HCV-infected group compared to controls, but higher in HCV-recovered cases, potentially due to reduced infection and enhanced immunity. In conclusion, this research unveils the relationship between FAM26F and HCV infection, highlighting the virus's tendency to suppress cytokine and FAM26F expression. An effective treatment strategy for establishing an ideal host immune response may involve restoring FAM26F and cytokine expression to their normal levels.

Exploration of newly synthesized amantadine-thiourea conjugates for their DNA binding, anti-elastase, and anti-glioma potentials.

Three newly synthesized amantadine thiourea conjugates namely MS-1 N-(((3 s,5 s,7 s)-adamantan-1-yl)carbamothioyl)benzamide, MS-2 N-(((3 s,5 s,7 s)-adamantan-1-yl)carbamothioyl)-4-methylbenzamide and MS-3 N-((3 s,5 s,7 s)-adamantan-1-ylcarbamothioyl)-4-chlorobenzamide were investigated for their structures, bindings (DNA/ elastase), and for their impact on healthy and cancerous cells. Theoretical (DFT/docking) and experimental {UV-visible (UV-), fluorescence (Flu-), and cyclic voltammetry (CV)} studies indicated binding interactions of each conjugate with DNA and elastase enzyme. Theoretically and experimentally calculated binding parameters for conjugate - DNA interaction revealed MS-3 - DNA to have most significant binding with comparatively greater values of binding parameters {(Kb/M-1: docking, 3.8 × 105; UV-, 5.95 × 103; Flu-,1.55 × 105; CV, 1.52 × 104), (∆G/ kJmol-1: docking, -32.09; UV-, -22.40; Flu-,-30.81; CV, -24.82)}. The docked structures, greater bindings site size values (n), and the trend in DNA viscosity changes in the presence of each conjugate concentration confirmed a mixed binding mode of interaction among them. Conjugate - elastase binding by docking agreed with the experimental anti-elastase findings. Cytotoxicity studies of each tested conjugate demonstrated greater cytotoxicity for cancerous (MG-U87) cells in comparison to control, while for the normal (HEK-293) cells the cytotoxicity was found comparatively low. Overall exploration suggested that MS-3 is the most effective candidate for DNA binding, anti-elastase, and for anti-glioma activities.