Repurposing potential of posaconazole and grazoprevir as inhibitors of SARS-CoV-2 helicase

As the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic engulfs millions worldwide, the quest for vaccines or drugs against the virus continues. The helicase protein of SARS-CoV-2 represents an attractive target for drug discovery since inhibition of helicase activity can suppress viral replication. Using in silico approaches, we have identified drugs that interact with SARS-CoV-2 helicase based on the presence of amino acid arrangements matching binding sites of drugs in previously annotated protein structures. The drugs exhibiting an RMSD of ≤ 3.0 Å were further analyzed using molecular docking, molecular dynamics (MD) simulation, and post-MD analyses. Using these approaches, we found 12 drugs that showed strong interactions with SARS-CoV-2 helicase amino acids. The analyses were performed using the recently available SARS-CoV-2 helicase structure (PDB ID: 5RL6). Based on the MM-GBSA approach, out of the 12 drugs, two drugs, namely posaconazole and grazoprevir, showed the most favorable binding energy, - 54.8 and - 49.1 kcal/mol, respectively. Furthermore, of the amino acids found conserved among all human coronaviruses, 10/11 and 10/12 were targeted by, respectively, grazoprevir and posaconazole. These residues are part of the crucial DEAD-like helicase C and DEXXQc_Upf1-like/ DEAD-like helicase domains. Strong interactions of posaconazole and grazoprevir with conserved amino acids indicate that the drugs can be potent against SARS-CoV-2. Since the amino acids are conserved among the human coronaviruses, the virus is unlikely to develop resistance mutations against these drugs. Since these drugs are already in use, they may be immediately repurposed for SARS-CoV-2 therapy

Comparison of the Microwave-Heated Ziehl-Neelsen Stain and Conventional Ziehl-Neelsen Method in the Detection of Acid-Fast Bacilli in Lymph Node Biopsies

BACKGROUND: Tuberculosis is a chronic inflammatory disease with lymphadenopathy being the most common extra-pulmonary manifestation. The conventional Ziehl–Neelsen method plays an essential role in the diagnosis of tuberculosis; however, it has a low sensitivity in detecting acid-fast bacilli. AIM: The present study emphasises the role of the microwave-heated method (modified Ziehl–Neelsen) over conventional Ziehl-Neelsen stain and to set at the best condition for irradiation. MATERIAL AND METHODS: The study included 90 patients with clinically suspected tuberculous lymphadenopathy who were referred to the Department of Pathology at Omdurman Military Hospital, Sudan. Demographic data such as age, sex, and site of swelling were documented for each patient. Specimens were stained with conventional Ziehl-Neelsen, fluoresce and the modified methods. RESULTS: Patient’s age ranged from 20 to 70 year. Of the total 90 cases with clinically suspected tuberculous lymphadenopathy, 18 cases were positive for AFB in conventional Ziehl-Neelsen method giving a sensitivity of 13.3%, while in microwave-heated method 82 cases of TB were detected positive for AFB yielded sensitivity and specificity of 97.6% and 85.7%, respectively, and positive and negative predictive values of 98.8% and 75.0% respectively compared to fluorescence methods. CONCLUSION: In the present study, the microwave-heated Ziehl-Neelsen method, was found to have sensitivity and specificity of 97.6% and 85.7%, respectively which matches the fluorescence technique. It has specificity in detecting lymph node tuberculosis that makes it superior over all other modified methods. However, the availability and cost-effectiveness might limit the use of fluorescence in routine practice. Furthermore, the study set the best staining temperature is provided at power 1 level (60 w) for 1.5 minutes

Mucormycosis co-infection in COVID-19 patients: An update

Mucormycosis (MCM) is a rare fungal disorder that has recently been increased in parallel with novel COVID-19 infection. MCM with COVID-19 is extremely lethal, particularly in immunocompromised individuals. The collection of available scientific information helps in the management of this co-infection, but still, the main question on COVID-19, whether it is occasional, participatory, concurrent, or coincidental needs to be addressed. Several case reports of these co-infections have been explained as causal associations, but the direct contribution in immunocompromised individuals remains to be explored completely. This review aims to provide an update that serves as a guide for the diagnosis and treatment of MCM patients’ co-infection with COVID-19. The initial report has suggested that COVID-19 patients might be susceptible to developing invasive fungal infections by different species, including MCM as a co-infection. In spite of this, co-infection has been explored only in severe cases with common triangles: diabetes, diabetes ketoacidosis, and corticosteroids. Pathogenic mechanisms in the aggressiveness of MCM infection involves the reduction of phagocytic activity, attainable quantities of ferritin attributed with transferrin in diabetic ketoacidosis, and fungal heme oxygenase, which enhances iron absorption for its metabolism. Therefore, severe COVID-19 cases are associated with increased risk factors of invasive fungal co-infections. In addition, COVID-19 infection leads to reduction in cluster of differentiation, especially CD4+ and CD8+ T cell counts, which may be highly implicated in fungal co-infections. Thus, the progress in MCM management is dependent on a different strategy, including reduction or stopping of implicit predisposing factors, early intake of active antifungal drugs at appropriate doses, and complete elimination via surgical debridement of infected tissues

Aqueous Extract of <i>Artemisia annua</i> Shows In Vitro Antimicrobial Activity and an In Vivo Chemopreventive Effect in a Small-Cell Lung Cancer Model

Artemisia annua (A. annua) has been used as a medicinal plant in the treatment of several infectious and non-infectious diseases in the forms of tea and press juice since ancient times. The aim of this study was to evaluate the aqueous extract of A. annua (AAE) as an antimicrobial agent in vitro and to evaluate its chemopreventive efficacy in vivo in a small-cell lung cancer (SCLC) animal model. The dried powder of AAE was prepared using the Soxhlet extraction system from the leaves of Artemisia annua. The in vitro activity of AAE was determined against Candida albicans (C. albicans), Enterococcus faecalis (E. faecalis), Klebsiella pneumoniae (K. pneumoniae), and methicillin-resistant Staphylococcus aureus (MRSA) using the agar well diffusion method and propidium iodide (PI)-stained microbial death under a confocal microscope. The pretreatment of mice with AAE was initiated two weeks before the first dose of benzo[a]pyrene and continued for 21 weeks. The chemopreventive potential of the extract was evaluated by flow cytometry and biochemical and histopathological analyses of the tissues and serum accordingly, after sacrificing the mice. The data revealed the antimicrobial potential of AAE against all the species investigated, as it showed growth-inhibitory activity by MIC, as well as confocal microscopy. The pretreatment of AAE exhibited significant protection in carcinogen-modulated, average body weight (ABW), and relative organ weight (ROW) cancer biomarkers in the serum and antioxidants in the lungs. The hematoxylin and eosin (H&E) staining of the tissues revealed that AAE prevented malignancy in the lungs. AAE also induced apoptosis and decreased intracellular reactive oxygen species (ROS) in the lung cells analyzed by flow cytometry. The current findings demonstrated the use of AAE as an alternative medicine in the treatment of infectious disease and the chemoprevention of lung cancer. To our knowledge, this is the first study that summarizes the chemopreventive potential of AAE in a lung cancer model in vivo. However, further investigations are suggested to understand the role of AAE to potentiate the therapeutic index of the commercially available drugs that show multiple drug resistance against microbial growth and high toxicity during cancer chemotherapy

A Comprehensive Computer Aided Vaccine Design Approach to Propose a Multi-Epitopes Subunit Vaccine against Genus Klebsiella Using Pan-Genomics, Reverse Vaccinology, and Biophysical Techniques

Klebsiella is a genus of nosocomial bacterial pathogens and is placed in the most critical list of World Health Organization (WHO) for development of novel therapeutics. The pathogens of the genus are associated with high mortality and morbidity. Owing to their strong resistance profile against different classes of antibiotics and nonavailability of a licensed vaccine, urgent efforts are required to develop a novel vaccine candidate that can tackle all pathogenic species of the Klebsiella genus. The present study aims to design a broad-spectrum vaccine against all species of the Klebsiella genus with objectives to identify the core proteome of pathogen species, prioritize potential core vaccine proteins, analyze immunoinformatics of the vaccine proteins, construct a multi-epitopes vaccine, and provide its biophysical analysis. Herein, we investigated all reference species of the genus to reveal their core proteome. The core proteins were then subjected to multiple reverse vaccinology checks that are mandatory for the prioritization of potential vaccine candidates. Two proteins (TonB-dependent siderophore receptor and siderophore enterobactin receptor FepA) were found to fulfill all vaccine parameters. Both these proteins harbor several potent B-cell-derived T-cell epitopes that are antigenic, nonallergic, nontoxic, virulent, water soluble, IFN-γ producer, and efficient binder of DRB*0101 allele. The selected epitopes were modeled into a multi-epitope peptide comprising linkers and Cholera Toxin B adjuvant. For docking with innate immune and MHC receptors and afterward molecular dynamics simulations and binding free energy analysis, the vaccine structure was modeled for tertiary structure and refined for structural errors. To assess the binding affinity and presentation of the designed vaccine construct, binding mode and interactions analysis were performed using molecular docking and molecular dynamics simulation techniques. These biophysical approaches illustrated the vaccine as a good binder to the immune receptors and revealed robust interactions energies. The vaccine sequence was further translated to nucleotide sequence and cloned into an appropriate vector for expressing it at high rate in Escherichia coli K12 strain. In addition, the vaccine was illustrated to generate a good level of primary, secondary, and tertiary immune responses, proving good immunogenicity of the vaccine. Based on the reported results, the vaccine can be a good candidate to be evaluated for effectiveness in wet laboratory validation studies

Ajwa-Dates (Phoenix dactylifera)-Mediated Synthesis of Silver Nanoparticles and Their Anti-Bacterial, Anti-Biofilm, and Cytotoxic Potential

Green nanotechnology is the evolution of cost-effective and environmentally friendly processes for the production of metal-based nanoparticles due to medicinal importance and economic value. The aim of the present study was to biosynthesize and characterize silver nanoparticles (AgNPs) using the seed extract of Ajwa dates (Aw). The anti-bacteriostatic activity of biosynthesized Aw&ndash;AgNPs against Gram-positive and Gram-negative bacterial strains was evaluated. The anti-biofilm activity was examined by the tissue culture plate method. Lastly, the anti-cancer potential of Aw&ndash;AgNPs was investigated against the human breast cancer cell line HCC712. UV&ndash;visible absorption spectra exhibited the plasmon resonance peak at 430 nm, with the solution undergoing rapid color changes that verified the existence of biosynthesized silver nanoparticles in the solution. TEM and SEM images illustrated that the Aw&ndash;AgNPs were spherical and between 15 and 80 nm in diameter. The reduction and stabilization of Aw&ndash;AgNPs was due to the functional groups present in the biomolecules of the Ajwa seeds, as identified by FTIR. The Aw&ndash;AgNPs exhibited significant anti-bacterial activity against all the tested bacterial strains. Moreover, the Aw&ndash;AgNPs efficiently hampered the biofilm formation of the bacterial strains and exhibited cytotoxicity at various concentrations. Overall, these findings suggest that biosynthesized Aw&ndash;AgNPs may be used as a potential therapeutic formulation against bacterial infections and breast cancer

Enhanced activity of Ellagic acid in lipid nanoparticles (EA-liposomes) against Acinetobacter baumannii in immunosuppressed mice

Acinetobacter baumannii infections have come to the surface in huge numbers in the recent decades. Furthermore, A. baumannii has adopted great ability to nullify the majority of currently available antibiotics. With the purpose of finding a nontoxic and efficient therapeutic agent, we analyzed the activity of Ellagic acid (EA) against the multidrug-resistant A. baumannii. EA not only demonstrated its activity against A. baumannii, but also inhibited the biofilm formation. Since EA shows poor solubility in an aqueous environment, a lipid nanoparticle-based (liposomal) formulation of EA (EA-liposomes) was prepared and its effectiveness was assessed to treat bacterial infection in the immunocompromised murine model. Therapy with EA-liposomes imparted greater protection to infected mice by increasing the survival and decreasing the bacterial load in the lungs. A. baumannii infected mice treated with EA-liposomes (100 mg/kg) showed 60% survival rate as compared to 20% of those treated with free EA at the same dose. The bacterial load was found to be 32778 ± 12232 in the lungs of EA-liposomes (100 mg/kg)-treated mice, which was significantly lower to 165667 ± 53048 in the lung tissues of free EA treated mice. Likewise, EA-liposomes also restored the liver function (AST and ALT) and kidney function parameters (BUN and creatinine). The broncho-alveolar fluid (BALF) from infected mice contained greater quantities of IL-6, IL-1β and TNF-α, which were significantly alleviated in EA-liposomes treated mice. These findings together support the possible implication of EA-liposomes to treat A. baumannii infection, especially in immunocompromised mice

Glycosphingolipids (GSLs) from Sphingomonas paucimobilis Increase the Efficacy of Liposome-Based Nanovaccine against Acinetobacter baumannii-Associated Pneumonia in Immunocompetent and Immunocompromised Mice

Due to the high propensity of drug resistance in Acinetobacter baumannii, the number of currently available therapeutic drugs has become very limited. Thus, it becomes incredibly important to prepare an effective vaccine formulation capable of eliciting an effective immune response against A. baumannii. In this study, we prepared a liposomal vaccine formulation bearing glycosphingolipids (GSLs) from Sphingomonas paucimobilis and loaded with the whole cell antigen (WCAgs-GSLs-liposomes) of A. baumannii. The immune-stimulating potential and prophylactic efficacy of WCAgs-GSLs-liposomes were compared with those of WCAgs-liposomes (without GSLs) or free WCAgs in both immunocompetent and immunodeficient mice. The efficacy of vaccine formulations was determined by analyzing antibody titer, cytokine levels, and survival studies in the immunized mice. The findings revealed that vaccination with WCAgs-GSLs-liposomes stimulated a greater secretion of antibodies and cytokines, higher lymphocyte proliferation, and increased expression of the co-stimulatory molecules. Anti-sera from WCAgs-GSLs-liposomes-immunized mice remarkably reduced the biofilm formation by A. baumannii. Most importantly, WCAgs-GSLs-liposomes-vaccinated mice demonstrated a higher defiance against the pathogen, as compared to the immunizations with WCAgs-liposomes (without GSLs) or free WCAgs. Immunocompetent mice immunized with WCAgs-GSLs-liposomes showed a 100% survival rate, while those immunized with WCAgs-liposomes exhibited a 60% survival rate. The protective effect of WCAgs-GSLs-liposomes was also found to be higher in immunocompromised mice, as the immunized mice showed a 50% survival rate, which was greater than the 20% survival rate of those immunized with WCAgs-liposomes. The survival data was also supported by the findings of bacterial load and histological analysis that substantiated the greatest prophylactic potential of the WCAgs-GSLs-liposomes. These findings recommend that WCAgs-GSLs-liposomes may be reckoned as a prospective vaccine to protect the persons against A. baumannii infection

Antioxidative Capacity of Liver- and Adipose-Derived Mesenchymal Stem Cell-Conditioned Media and Their Applicability in Treatment of Type 2 Diabetic Rats

Type 2 diabetes mellitus (T2DM) is mainly characterized by insulin resistance and impaired insulin secretion, which cannot be reversed with existing therapeutic strategies. Using mesenchymal stem cells (MSCs), cell-based therapy has been demonstrated in displaying therapeutic effects in T2DM for their self-renewable, differentiation potential, and immunosuppressive properties and higher levels of angiogenic factors. Stem cell therapies are complicated and have a serious adverse effect including tumor formation and immunogenicity, while using mesenchymal stem cell-conditioned media (MSC-CM) significantly reduces stem cell risk, maintaining efficacy and showing significantly higher levels of growth factors, cytokines, and angiogenic factors that stimulate angiogenesis and promote fracture healing in diabetes. In the present study, we investigated the therapeutic potential of the liver and adipose MSC-CM in diabetic endothelial dysfunction compared with standard insulin therapy. Fifty adult male Sprague Dawley rats were divided equally into 5 groups as follows: control, diabetic, diabetic+insulin, diabetic+liver MSC-CM, and diabetic+adipose MSC-CM; all treatments continued for 4 weeks. Finally, we observed that liver MSC-CM therapy had the most apparent improvement in levels of blood glucose; HbA1c; AGEs; lipid panel (cholesterol, TG, LDL, HDL, and total lipids); renal function (urea, uric acid, creatinine, and total protein); liver function (AST, ALT, ALP, bilirubin, and albumin); CPK; C-peptide; HO-1; inflammatory markers including IL-6, TNF-α, and CRP; growth factors (liver and serum IGF-1); amylase; histopathological changes; pancreatic cell oxidative stress; and antioxidant markers (MDA, GSH, ROS, CAT, SOD, HO-1, and XO) toward the normal levels compared with insulin and adipose MSCs-CM. Moreover, both the liver and adipose MSC-CM relieved the hyperglycemic status by improving pancreatic islet β cell regeneration, promoting the conversion of alpha cells to beta cells, reducing insulin resistance, and protecting pancreatic tissues against oxidative stress-induced injury as well as possessing the ability to modulate immunity and angiogenesis. These results indicated that MSC-CM infusion has therapeutic effects in T2DM rats and may be a promising novel therapeutic target

Geospatial Assessment of Ground Water Quality and Associated Health Problems in the Western Region of India

Groundwater constitutes a significant component of freshwater resources in India being vital for its economy and domestic water security. The quantity, quality and accessibility of water resources forms the basis of balanced socio-economic development and its optimum utilization cannot be sustained unless its quality is assessed. The current study tries to access the quality and suitability of groundwater for drinking purposes in western drier parts of India in the state of Rajasthan. Based on collected data, selected hydro-geochemical parameters, the quality of water has been determined and Water Quality Index (WQI) have been prepared using GIS applications. Applying the Inverse Distance Weighting method, WQI values for 89 villages in the area have been computed, which ranged between 71.23 and 447.39. While 68% of the region had &ldquo;poor water quality&rdquo;, only 32% is sustained as &lsquo;good water&rsquo; for consumption. The fluoride content ranging between 1.66 and 8.60 mg/L and TDS &gt; 1000 mg/L with average pH levels &gt; 7 (8&ndash;9 pH) were found to be very high amongst all the 12 water quality parameters taken for the study. The northeastern region with a WQI value of &gt;250 had the worst water quality. Furthermore, the existing water quality is also examined for influencing two water borne diseases, i.e., gastroenteritis and fluorosis in the region. The study thus establishes that the majority of groundwater in the region is beyond the permissible safer consumption limits, and a large population of the region, which is directly dependent on groundwater sources, is prone to water borne health hazards. A significantly high correlation was observed between Specific Water Quality Parameters in the region and prevalence of gastroenteritis (and fluorosis diseases with R2 = 0.530 and R2 = 0.813, respectively)