Immunoinformatics-Based Identification of B and T Cell Epitopes in RNA-Dependent RNA Polymerase of SARS-CoV-2.

INTRODUCTION: The ongoing coronavirus disease 2019 (COVID-19), which emerged in December 2019, is a serious health concern throughout the world. Despite massive COVID-19 vaccination on a global scale, there is a rising need to develop more effective vaccines and drugs to curb the spread of coronavirus. METHODOLOGY: In this study, we screened the amino acid sequence of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 (the causative agent of COVID-19) for the identification of B and T cell epitopes using various immunoinformatic tools. These identified potent B and T cell epitopes with high antigenicity scores were linked together to design the multi-epitope vaccine construct. The physicochemical properties, overall quality, and stability of the designed vaccine construct were confirmed by suitable bioinformatic tools. RESULTS: After proper in silico prediction and screening, we identified 3 B cell, 18 CTL, and 10 HTL epitopes from the RdRp protein sequence. The screened epitopes were non-toxic, non-allergenic, and highly antigenic in nature as revealed by appropriate servers. Molecular docking revealed stable interactions of the designed multi-epitope vaccine with human TLR3. Moreover, in silico immune simulations showed a substantial immunogenic response of the designed vaccine. CONCLUSIONS: These findings suggest that our designed multi-epitope vaccine possessing intrinsic T cell and B cell epitopes with high antigenicity scores could be considered for the ongoing development of peptide-based novel vaccines against COVID-19. However, further in vitro and in vivo studies need to be performed to confirm our in silico observations.

Assessment of IL-1ß, IL-6, TNF-α, IL-8, and CCL 5 levels in newly diagnosed Saudi patients with rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systemic inflammatory disorder which mainly affects small joints, occurs most commonly in middle-aged adults, and can be fatal in severe cases. The exact etiology of RA remains unknown. However, uncontrolled expression of pro-inflammatory cytokines and chemokines can contribute to the pathogenesis of RA. AIM: In the current study, we assessed the potential of serum concentrations of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, IL-8, and C-C motif chemokine ligand (CCL)5 as early predictive markers for RA. METHODS: In addition to clinical examination, blood samples were collected from 100 Saudi patients recently diagnosed with early RA for basic and serological tests, including rheumatoid factor (RF), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Sera of 32 healthy individuals were used as controls. Specific enzyme-linked immunosorbent assay was used to quantify the serum IL-1ß, IL-6, TNF-α, IL-8, and CCL5 levels in the samples. RESULTS: Our results indicated that RF, CRP, and ESR levels were higher in RA patients compared to controls. Furthermore, serum levels of IL-1ß, IL-6, IL-8, and CCL5, but not TNF-α, significantly increased in RA patients compared to controls. CONCLUSION: Overall, the findings suggested that IL-1ß, IL-6, IL-8, and CCL5 can be used as biomarkers in the early diagnosis of RA.

Identification of SARS-CoV-2 RNA-dependent RNA polymerase inhibitors from the major phytochemicals of Nigella sativa: An in silico approach.

The coronavirus disease 2019 (COVID-19), which emerged in December 2019, continues to be a serious health concern worldwide. There is an urgent need to develop effective drugs and vaccines to control the spread of this disease. In the current study, the main phytochemical compounds of Nigella sativa were screened for their binding affinity for the active site of the RNA-dependent RNA polymerase (RdRp) enzyme of the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The binding affinity was investigated using molecular docking methods, and the interaction of phytochemicals with the RdRp active site was analyzed and visualized using suitable software. Out of the nine phytochemicals of N. sativa screened in this study, a significant docking score was observed for four compounds, namely α-hederin, dithymoquinone, nigellicine, and nigellidine. Based on the findings of our study, we report that α-hederin, which was found to possess the lowest binding energy (-8.6 kcal/mol) and hence the best binding affinity, is the best inhibitor of RdRp of SARS-CoV-2, among all the compounds screened here. Our results prove that the top four potential phytochemical molecules of N. sativa, especially α-hederin, could be considered for ongoing drug development strategies against SARS-CoV-2. However, further in vitro and in vivo testing are required to confirm the findings of this study.

Embilica officinalis L. inhibits the growth and proliferation of Leishmania donovani through the induction of ultrastructural changes, mitochondrial dysfunction, oxidative stress and apoptosis-like cell death.

Visceral leishmaniasis (VL) is caused by a protozoan parasite, Leishmania donovani (L. donovani). It affects around 1-2 million people around the world annually. There is an urgent need to investigate new medicament of it due to difficult method of drug administration, long period of treatment, high cost of the drug, adverse side-effects, low efficacy and development of parasite resistance to the available drugs. Medicinal plants have also been used for the treatment of different diseases in traditional system of medicines due to their holistic effects. The Drugs for Neglected Diseases initiative (DNDi), Geneva, Switzerland has already started the program for identification of potential medicinal plant and plant products having antileishmanial potential. Keeping all these in consideration, we planned to study the antileishmanial activity of one of the medicinal plant, Embilica officinalis L. (EO) fruit extract. EO fruit extract inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes in dose-dependent manner. EO fruit extract induced morphological and ultrastructural changes in parasites as observed under Electron Microscope. It also induced the oxidative stress, mitochondrial dysfunction, DNA laddering and apotosis-like cell death in parasites. Here, we for the first time reported such a detailed mechanism of action of antileishmanial activity of EO fruit extract. Our results suggested that EO fruit extract could be used for the development of new phytomedicine against leishmaniasis.

Virtual screening of natural compounds for potential inhibitors of Sterol C-24 methyltransferase of Leishmania donovani to overcome leishmaniasis.

Leishmaniasis is a neglected tropical disease caused by trypanosomatid parasite belonging to the genera Leishmania. Leishmaniasis is transmitted from one human to other through the bite of sandflies. It is endemic in around 98 countries including tropical and subtropical regions of Asia, Africa, Southern America, and the Mediterranean region. Sterol C-24 methyltransferase (LdSMT) of Leishmania donovani (L. donovani) mediates the transfer of CH3-group from S-adenosyl methionine to C-24 position of sterol side chain which makes the ergosterol different from cholesterol. Absence of ortholog in human made it potential druggable target. Here, we performed virtual screening of library of natural compounds against LdSMT to identify the potential inhibitor for it and to fight leishmaniasis. Gigantol, flavan-3-ol, and parthenolide showed the best binding affinity towards LdSMT. Further, based on absorption, distribution, metabolism, and excretion properties and biological activity prediction, gigantol showed the best lead-likeness and drug-likeness properties. Therefore, we further elucidated its antileishmanial properties. We found that gigantol inhibited the growth and proliferation of promastigotes as well as intra-macrophagic amastigotes. Gigantol exerted its antileishmanial action through the induction of reactive oxygen species in dose-dependent manner. Our study, suggested the possible use of gigantol as antileishmanial drug after further validations to overcome leishmaniasis.

Assessment of the Antileishmanial Potential of Cassia fistula Leaf Extract.

Cassia fistula has a wide array of biologically active and therapeutically important class of compounds. Leishmania donovani important drug targets, sterol 24-c methyltransferase (LdSMT), trypanothione reductase (LdTR), pteridine reductase (LdPTR1), and nucleoside hydrolase (LdNH), were modelled, and molecular docking was performed against the abundant phytochemicals of its leaf extract. Molecular docking results provided the significant prima facie evidence of the leaf extract to have antileishmanial potential. To confirm this, we performed in vitro antileishmanial and cytotoxicity assays. Methanolic extract of C. fistula leaves showed growth inhibition and proliferation of L. donovani promastigote with an IC50 value of 43.31 ± 4.202 µg/mL. It also inhibited the growth of intra-macrophagic amastigotes with an IC50 value of 80.76 ± 3.626 µg/mL. C. fistula extract was found cytotoxic at a very high concentration on human macrophages (CC50 = 626 ± 39 µg/mL). Annexin V/propidium iodide (PI) staining assay suggested partial apoptosis induction in parasites by C. fistula to exert its antileishmanial activity. Here, for the first time, we have shown the antileishmanial potential of C. fistula leaves. Overall, our results could open new insight for an affordable and natural antileishmanial with high efficacy and less toxicity.

Fungal Keratitis: Epidemiology, Rapid Detection, and Antifungal Susceptibilities of Fusarium and Aspergillus Isolates from Corneal Scrapings.

Fungal aetiology of keratitis/corneal ulcer is considered to be one of the leading causes of ocular morbidity, particularly in developing countries including India. More importantly, Fusarium and Aspergillus are reported commonly implicating corneal ulcer and against this background the present work was undertaken so as to understand the current epidemiological trend of the two fungal keratitis. During the project period, a total of 500 corneal scrapings were collected from suspected mycotic keratitis patients, of which 411 (82.2%) were culture positive for bacteria, fungi, and parasites. Among fungal aetiologies, Fusarium (216, 52.5% of 411) and Aspergillus (68, 16.5% of 411) were predominantly determined. While the study revealed a male preponderance with both the fungal keratitis , it further brought out that polyene compounds (natamycin and amphotericin B) and azoles were active, respectively, against Fusarium spp. and Aspergillus spp. Additionally, 94.1% of culture proven Fusarium keratitis and, respectively, 100% and 63.6% of A. flavus and A. fumigatus were confirmed by multiplex PCR. The sensitivity of the PCR employed in the present study was noted to be 10 fg/µl, 1 pg/µl, and 300 pg/µl of DNA, respectively, for Fusarium, A. flavus, and A. fumigatus. Alarming fact was that Fusarium and Aspergillus regionally remained to be the common cause of mycotic keratitis and the Fusarium isolates had a higher antifungal resistance than Aspergillus strains against most of the test drugs.

Significance of HBV NAT Among HBs Antigen-Negative Blood Donors in Saudi Arabia.

BACKGROUND: Bloodborne hepatitis B virus (HBV) transmission from asymptomatic donors with acute HBV infections who have undetectable surface antigen of HBV (HBsAg), or from donors with chronic infections in whom serological markers were not detected, could cause residual infections leading to relevant transfusion-transmitted infections (RTTIs). HBV nucleic acid testing (NAT) can detect HBV DNA in the HBsAg-negative and total hepatitis B core antibody (anti-HBc)-negative window period of infection and in chronic cases. OBJECTIVE: To assess the presence or absence of HBV DNA in blood donors with HBsAg negativity. METHODS: We collected 3014 blood specimens from volunteer blood donors at the blood bank of King Khalid University Hospital in Riyadh, Saudia Arabia. Specimens from each donor were tested for HBsAg, anti-HBc, and hepatitis B surface antibody (anti-HBs) by commercial immunoassays and for qualitative assessments of HBV-DNA by HBV-NAT testing. RESULTS: Of the 3014 donors, 7 (0.23%) tested positive for HBsAg and anti-HBc, 1 for HBsAg (0.03%) only, and of those 264 donors (8.8%) for anti-HBc. Of these last, 6.9% also tested positive for anti-HBs and 1.9% tested negative for anti-HBs. HBV-NAT testing was reactive in 75.0% of subjects who tested HBsAg positive, and nonreactive in 100% of subjects who tested anti-HBc positive/HBsAg negative (with or without anti-HBs). Among 2742 donors who tested seronegative, 1 specimen was determined to be reactive via HBV-NAT testing. CONCLUSIONS: The frequency of HBV DNA in blood donors who tested seronegative was low. This finding may indicate the significance of the HBV NAT technique in reducing the residual risk of transfusion-transmitted HBV infection.

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study.

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/µL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.

Multidrug-Resistant Bacteria Associated with Cell Phones of Healthcare Professionals in Selected Hospitals in Saudi Arabia.

Cell phones may be an ideal habitat for colonization by bacterial pathogens, especially in hot climates, and may be a reservoir or vehicle in transmitting nosocomial infections. We investigated bacterial contamination on cell phones of healthcare workers in three hospitals in Saudi Arabia and determined antibacterial resistance of selected bacteria. A questionnaire was submitted to 285 healthcare workers in three hospitals, and information was collected on cell phone usage at the work area and in the toilet, cell phone cleaning and sharing, and awareness of cell phones being a source of infection. Screening on the Vitek 2 Compact system (bioMérieux Inc., USA) was done to characterize bacterial isolates. Of the 60 samples collected from three hospitals, 38 (63.3%) were positive with 38 bacterial isolates (4 Gram-negative and 34 Gram-positive bacteria). We found 38.3% of cell phones were contaminated with coagulase-negative staphylococci, particularly Staphylococcus epidermidis (10 isolates). Other bacterial agents identified were S. aureus, S. hominis, Alloiococcus otitis, Vibrio fluvialis, and Pseudomonas stutzeri. Antimicrobial susceptibility testing showed that most coagulase-negative staphylococci were resistant to benzylpenicillin, erythromycin, and rifampicin. Eight isolates were resistant to oxacillin, specifically S. epidermidis (3), S. hominis (2), and S. warneri (2). A. otitis, a cause of acute otitis media showed multidrug resistance. One isolate, a confirmed hetero-vancomycin intermediate-resistant S. aureus, was resistant to antibiotics, commonly used to treat skin infection. There was a significant correlation between the level of contamination and usage of cell phone at toilet and sharing. Our findings emphasize the importance of hygiene practices in cell phone usage among healthcare workers in preventing the transmission of multidrug-resistant microbes.

PPM1A Methylation Is Associated With Vascular Recurrence in Aspirin-Treated Patients.

BACKGROUND AND PURPOSE: Despite great efforts by pharmacogenetic studies, the causes of aspirin failure to prevent the recurrence of ischemic events remain unclear. Our aim was to study whether epigenetics could be associated with the risk of vascular recurrence in aspirin-treated stroke patients. METHODS: We performed an epigenetic joint analysis study in 327 patients treated with aspirin. In the discovery stage, we performed a nested case-control study in 38 matched ischemic stroke patients in whom 450 000 methylation sites were analyzed. Nineteen patients presented vascular recurrence after stroke, and 19 matched patients did not present vascular recurrence during the first year of follow-up. In a second stage, 289 new patients were analyzed by EpiTYPER. RESULTS: The following 3 differentially methylated candidate CpG sites, were identified in the discovery stage and analyzed in the second stage: cg26039762 (P=9.69×10(-06), RAF1), cg04985020 (P=3.47×10(-03), PPM1A), and cg08419850 (P=3.47×10(-03), KCNQ1). Joint analysis identified an epigenome-wide association for cg04985020 (PPM1A; P=1.78×10(-07)), with vascular recurrence in patients treated with aspirin. CONCLUSIONS: The pattern of differential methylation in PPM1A is associated with vascular recurrence in aspirin-treated stroke patients.

Novel presenilin 1 mutation associated with early-onset Alzheimer`s disease in a Saudi patient.

We report a 60-year-old Saudi patient with the clinical diagnosis of Alzheimer`s disease (AD) and a novel mutation in the presenilin gene. We investigated mutations in the presenilin-1 gene in Saudi patients with AD using polymerase chain reaction and direct DNA sequencing methods. We extracted genomic DNA from the whole blood of both patients and normal control individuals. We sequenced and compared amplicons with the sequences of the respective exons of normal individuals as well as data available in GenBank. We detected a homozygous mutation (g-c) in exon 12, resulting in the missense mutation (Arg377Thr), in the DNA of a 60-year-old patient. We located this mutation in the cytoplasmic loop near the transmembrane domain 7.

Molecular cloning and sequencing of rabbit presenilin-1 cDNA fragment.

Molecular cloning and sequencing of a cDNA encoding rabbit presenilin-1 (Ps1) fragment was performed by reverse transcription polymerase chain reaction (RT-PCR) using primers: 5'-GGA TGA GCA GCT AAT CTA TAC C-3' and 5'-TCC ATT CAG GGA GGT ACT TGA TA-3'. The cDNA fragment revealed 402 nucleotides. The sequence was well conserved and found to be 91, 90, 88, 87 and 78% homologous to that of human, lemur, rat, mouse and chicken, respectively. The cDNA translated into a 130 amino-acid protein fragment. The deduced amino-acid sequence was also well conserved in various species and exhibited 98% similarities with those of rat, lemur and human homologues. However, differences were noticed at residues 145, 168 and 212. This cDNA fragment is quite significant because it is the most conserved portion of Ps1 in various animals and encodes four transmembrane regions (TM2, 3, 4, 5) as defined in human Ps1. Moreover, it includes more than 50% of the sites at which substitutions have been reported in familial Alzheimer's disease (FAD). Therefore, it is suggested that the rabbit can be used as an experimental model for future studies on Ps1 and its physiological functions to work out possible pathways leading to FAD linked neurodegeneration.