A rare homozygous ALX4 mutation in a Bangladeshi girl with frontonasal dysplasia type-2 (FND2).

Background: Frontonasal dysplasia type-2(FND2), a rare phenotypically variable and heterogeneous developmental anomaly resulting from mutation of the ALX4 gene, is primarily characterized by malformation of the skull and facial skeleton. This study was designed to showcase a clinical, imaging, and genetic analysis of FND2 in a consanguineous family of Bangladeshi origin. Methodology: Clinical imaging and whole genome sequencing of mother, father and patient was done by using Nextera DNA flex library preparation kit (Illumina, USA) using Novaseq 6000 next generation sequencer to find out ALX4 mutation which causes FND2 in patient. Result: We report the clinical as well as molecular findings in an 8-year-old girl with FND2. The child presented with various characteristic features of skull and facial anomalies associated with FND 2 along with numerous other features many of which have not been described in previous literature. The parents also showed some key clinical, radiological, and genetic features of FND 2. The whole genome sequencing (WGS) revealed homozygosity for a 793C-T transition in the ALX4 gene, which resulted in premature termination at codon 265 (p.Arg265Ter). Both of her parents were heterozygous carriers of ALX4 mutation. Conclusions: This is the first report that associates clinical, imaging, and genomics analyses in a Bangladeshi patient for better understanding the disease FND2. These results will facilitate diagnosis and genetic counseling of the future FND2 patients.

In silico design of an epitope-based vaccine against PspC in Streptococcus pneumoniae using reverse vaccinology.

BACKGROUND: Streptococcus pneumoniae is a major pathogen that poses a significant hazard to global health, causing a variety of infections including pneumonia, meningitis, and sepsis. The emergence of antibiotic-resistant strains has increased the difficulty of conventional antibiotic treatment, highlighting the need for alternative therapies such as multi-epitope vaccines. In this study, immunoinformatics algorithms were used to identify potential vaccine candidates based on the extracellular immunogenic protein Pneumococcal surface protein C (PspC). METHOD: The protein sequence of PspC was retrieved from NCBI for the development of the multi-epitope vaccine (MEV), and potential B cell and T cell epitopes were identified. Linkers including EAAAK, AAY, and CPGPG were used to connect the epitopes. Through molecular docking, molecular dynamics, and immunological simulation, the affinity between MEV and Toll-like receptors was determined. After cloning the MEV construct into the PET28a ( +) vector, SnapGene was used to achieve expression in Escherichia coli. RESULT: The constructed MEV was discovered to be stable, non-allergenic, and antigenic. Microscopic interactions between ligand and receptor are confirmed by molecular docking and molecular dynamics simulation. The use of an in-silico cloning approach guarantees the optimal expression and translation efficiency of the vaccine within an expression vector. CONCLUSION: Our study demonstrates the potential of in silico approaches for designing effective multi-epitope vaccines against S. pneumoniae. The designated vaccine exhibits the required physicochemical, structural, and immunological characteristics of a successful vaccine against SPN. However, laboratory validation is required to confirm the safety and immunogenicity of the proposed vaccine design.

Assessment of antibacterial activity and cytotoxic effects of in vitro and in vivo plant parts of a medicinal plant Gynura procumbens (Lour.) Merr.

The goal of this study was to evaluate the antibacterial and cytotoxic effects of both the in vitro and in vivo plant part extracts of the medicinal plant Gynura procumbens. An effective protocol for regeneration and callus formation was developed using nodal segments and regenerated leaf explants, respectively. The highest fresh and dry weight calli were produced after four weeks of culture on Murashige and Skoog (MS) medium containing 2.0 mg/L BAP and 2.0 mg/L NAA, while the most shoots were produced on MS medium containing 1.0 mg/L BAP and 0.5 mg/L IAA. The in vitro shoots developed roots on MS media with 0.1 mg/L IBA. The antibacterial activity of extracts against various bacteria was examined to determine their significance (p < 0.05). The least significant difference (LSD) test results showed that the regenerated leaf extract had the highest antibacterial activity while the callus extract had the lowest. The minimum bactericidal concentration (MBC) and the minimum inhibitory concentration (MIC) were also established. Regenerated leaf extract had the highest toxicity and the lowest lethal concentration (LC50) value (1.21 ± 0.03 µg/mL) in a brine shrimp lethality bioassay. In contrast, callus extract had the lowest toxicity and the highest LC50 (11.09 ± 0.4 µg/mL). In addition, the in vitro cytotoxicity test results revealed that the callus and field leaf extracts had anti-cell-proliferative properties. The regenerated leaf and stem extract, however, could induce cell growth.

Emergence of SARS-CoV-2 variant of interest B.1.525 (Eta) in Bangladesh.

In the present study, we report the complete genome of five Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) from Bangladesh harboring mutations at Spike protein (E484K, Q677H, D614G, A67V, Q52R, Y144del, H69del, V70del, F888L) assigned to the B.1.525 lineage (Variant of interest). Mutations are also found in viral structural proteins other than spike region (E_L21F, M_I82F, N_A12G and N_T208I) and other mutations (NSP3_T1189I, NSP6_S106del, NSP6_F108del, NSP6_G107del, NSP12_P323F) from all of five B.1.525 SARS-CoV-2 variants of Bangladesh. We have also found four unique mutations from two of SARS-CoV-2 B.1.525 variant of Bangladesh. Among the four unique mutations two mutations (NS7a_L96H, NS7a_Y97D) obtained from strain BCSIR-NILMRC-718, one (NSP3_A1430V) from BCSIR-NILMRC-738 and two mutation including one spike protein mutation (NSP2_L444I, Spike_I68 M) present in BCSIR-AFIP-10 strain. The identification of new mutations will contribute to characterizing SARS-CoV-2, to continue tracking its spread and better understanding its biological and clinical features to take medical countermeasures and vaccines.

M gene targeted qRT-PCR approach for SARS-CoV-2 virus detection.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been established targeting different genes of the virus. Due to the high mutation rate of these genes, false negative results arise thus complicating the interpretation of the diagnosis and increasing the need of alternative targets. In this study, an alternative approach for the detection of SARS-CoV-2 viral RNA targeting the membrane (M) gene of the virus using qRT-PCR was described. Performance evaluation of this newly developed in-house assay against commercial qRT-PCR kits was done using clinical oropharyngeal specimens of COVID-19 positive patients. The limit of detection was determined using successive dilutions of known copies of SARS-CoV-2 pseudovirus. The M gene based assay was able to detect a minimum of 100 copies of virus/mL indicating its capacity to detect low viral load. The assay showed comparable accuracy, sensitivity and specificity with commercially available kits while detecting all the variants efficiently. The study concluded that the in-house M gene based assay might be an effective alternative for the currently available commercial qRT-PCR kits.

Spike protein mutations and structural insights of pangolin lineage B.1.1.25 with implications for viral pathogenicity and ACE2 binding affinity.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID -19, is constantly evolving, requiring continuous genomic surveillance. In this study, we used whole-genome sequencing to investigate the genetic epidemiology of SARS-CoV-2 in Bangladesh, with particular emphasis on identifying dominant variants and associated mutations. We used high-throughput next-generation sequencing (NGS) to obtain DNA sequences from COVID-19 patient samples and compared these sequences to the Wuhan SARS-CoV-2 reference genome using the Global Initiative for Sharing All Influenza Data (GISAID). Our phylogenetic and mutational analyzes revealed that the majority (88%) of the samples belonged to the pangolin lineage B.1.1.25, whereas the remaining 11% were assigned to the parental lineage B.1.1. Two main mutations, D614G and P681R, were identified in the spike protein sequences of the samples. The D614G mutation, which is the most common, decreases S1 domain flexibility, whereas the P681R mutation may increase the severity of viral infections by increasing the binding affinity between the spike protein and the ACE2 receptor. We employed molecular modeling techniques, including protein modeling, molecular docking, and quantum mechanics/molecular mechanics (QM/MM) geometry optimization, to build and validate three-dimensional models of the S_D614G-ACE2 and S_P681R-ACE2 complexes from the predominant strains. The description of the binding mode and intermolecular contacts of the referenced systems suggests that the P681R mutation may be associated with increased viral pathogenicity in Bangladeshi patients due to enhanced electrostatic interactions between the mutant spike protein and the human ACE2 receptor, underscoring the importance of continuous genomic surveillance in the fight against COVID -19. Finally, the binding profile of the S_D614G-ACE2 and S_P681R-ACE2 complexes offer valuable insights to deeply understand the binding site characteristics that could help to develop antiviral therapeutics that inhibit protein-protein interactions between SARS-CoV-2 spike protein and human ACE2 receptor.

Computational design of medicinal compounds to inhibit RBD-hACE2 interaction in the Omicron variant: unveiling a vulnerable target site.

The COVID-19 pandemic, caused by SARS-CoV-2, has globally affected both human health and economy. Several variants with a high potential for reinfection and the ability to evade immunity were detected shortly after the initial reported case of COVID-19. A total of 30 mutations in the spike protein (S) have been reported in the SARS-CoV-2 (BA.2) variant in India and South Africa, while half of these mutations are in the receptor-binding domain and have spread rapidly throughout the world. Drug repurposing offers potential advantages over the discovery of novel drugs, and one is that it can be delivered quickly without lengthy assessments and time-consuming clinical trials. In this study, computational drug design, such as pharmacophore-based virtual screening and MD simulation has been concentrated, in order to find a novel small molecular inhibitor that prevents hACE2 from binding to the receptor binding domain (RBD). three medicinal compound databases: North-East African, North African, and East African were screened and carried out a multi-step screening approach that identified three compounds, which are thymoquinol 2-O-beta-glucopyranoside (C1), lanneaflavonol (C2), and naringenin-4'-methoxy-7-O-Alpha-L-rhamnoside (C3), with excellent anti-viral properties against the RBD of the omicron variant. Furthermore, PAIN assay interference, computation bioactivity prediction, binding free energy, and dissociation constant were used to validate the top hits, which indicated good antiviral activity. The three compounds that were found may be useful against COVID-19, though more research is required. These findings could aid the development of novel therapeutic drugs against the emerging Omicron variant of SARS-CoV-2.

A multicentre study reveals dysbiosis in the microbial co-infection and antimicrobial resistance gene profile in the nasopharynx of COVID-19 patients.

The impact of SARS-CoV-2 infection on the nasopharyngeal microbiome has not been well characterised. We sequenced genetic material extracted from nasopharyngeal swabs of SARS-CoV-2-positive individuals who were asymptomatic (n = 14), had mild (n = 64) or severe symptoms (n = 11), as well as from SARS-CoV-2-negative individuals who had never-been infected (n = 5) or had recovered from infection (n = 7). Using robust filters, we identified 1345 taxa with approximately 0.1% or greater read abundance. Overall, the severe cohort microbiome was least diverse. Bacterial pathogens were found in all cohorts, but fungal species identifications were rare. Few taxa were common between cohorts suggesting a limited human nasopharynx core microbiome. Genes encoding resistance mechanisms to 10 antimicrobial classes (> 25% sequence coverages, 315 genes, 63 non-redundant) were identified, with ß-lactam resistance genes near ubiquitous. Patients infected with SARS-CoV-2 (asymptomatic and mild) had a greater incidence of antibiotic resistance genes and a greater microbial burden than the SARS-CoV-2-negative individuals. This should be considered when deciding how to treat COVID-19 related bacterial infections.

Transcriptome analysis reveals increased abundance and diversity of opportunistic fungal pathogens in nasopharyngeal tract of COVID-19 patients.

We previously reported that SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiomes (bacteria, archaea and commensal respiratory viruses) with inclusion of pathobionts. This study aimed to assess the possible changes in the abundance and diversity of resident mycobiome in the nasopharyngeal tract (NT) of humans due to SARS-CoV-2 infections. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 = 8, Recovered = 7, and Healthy = 7) were collected for RNA-sequencing followed by taxonomic profiling of mycobiome. Our analyses indicate that SARS-CoV-2 infection significantly increased (p < 0.05, Wilcoxon test) the population and diversity of fungi in the NT with inclusion of a high proportion of opportunistic pathogens. We detected 863 fungal species including 533, 445, and 188 species in COVID-19, Recovered, and Healthy individuals, respectively that indicate a distinct mycobiome dysbiosis due to the SARS-CoV-2 infection. Remarkably, 37% of the fungal species were exclusively associated with SARS-CoV-2 infection, where S. cerevisiae (88.62%) and Phaffia rhodozyma (10.30%) were two top abundant species. Likewise, Recovered humans NT samples were predominated by Aspergillus penicillioides (36.64%), A. keveii (23.36%), A. oryzae (10.05%) and A. pseudoglaucus (4.42%). Conversely, Nannochloropsis oceanica (47.93%), Saccharomyces pastorianus (34.42%), and S. cerevisiae (2.80%) were the top abundant fungal species in Healthy controls nasal swabs. Importantly, 16% commensal fungal species found in the Healthy controls were not detected in either COVID-19 patients or when they were cured from COVID-19 (Recovered). We also detected several altered metabolic pathways correlated with the dysbiosis of fungal mycobiota in COVID-19 patients. Our results suggest that SARS-CoV-2 infection causes significant dysbiosis of mycobiome and related metabolic functions possibly play a determining role in the progression of SARS-CoV-2 pathogenesis. These findings might be helpful for developing mycobiome-based diagnostics, and also devising appropriate therapeutic regimens including antifungal drugs for prevention and control of concurrent fungal coinfections in COVID-19 patients.

Immunoinformatics approach to epitope-based vaccine design against the SARS-CoV-2 in Bangladeshi patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic which has brought a great challenge to public health. After the first emergence of novel coronavirus SARS-CoV-2 in the city of Wuhan, China, in December 2019. As of March 2020, SARS-CoV-2 was first reported in Bangladesh and since then the country has experienced a steady rise in infections, resulting in 13,355,191 cases and 29,024 deaths as of 27 February 2022. Bioinformatics techniques are used to predict B cell and T cell epitopes from the new SARS-CoV-2 spike glycoprotein in order to build a unique multiple epitope vaccine. The immunogenicity, antigenicity scores, and toxicity of these epitopes were evaluated and chosen based on their capacity to elicit an immune response. RESULT: The best multi-epitope of the possible immunogenic property was created by combining epitopes. EAAAK, AAY, and GPGPG linkers were used to connect the epitopes. In several computer-based immune response analyses, this vaccine design was found to be efficient, as well as having high population coverage. CONCLUSION: This research is entirely reliant on the development of epitope-based vaccines, and these in silico findings would represent a major step forward in the development of a vaccine that might eradicate SARS-CoV-2 in Bangladeshi patients.

Differential gene expression profiling reveals potential biomarkers and pharmacological compounds against SARS-CoV-2: Insights from machine learning and bioinformatics approaches.

The COVID-19 pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has created an urgent global situation. Therefore, it is necessary to identify the differentially expressed genes (DEGs) in COVID-19 patients to understand disease pathogenesis and the genetic factor(s) responsible for inter-individual variability and disease comorbidities. The pandemic continues to spread worldwide, despite intense efforts to develop multiple vaccines and therapeutic options against COVID-19. However, the precise role of SARS-CoV-2 in the pathophysiology of the nasopharyngeal tract (NT) is still unfathomable. This study utilized machine learning approaches to analyze 22 RNA-seq data from COVID-19 patients (n = 8), recovered individuals (n = 7), and healthy individuals (n = 7) to find disease-related differentially expressed genes (DEGs). We compared dysregulated DEGs to detect critical pathways and gene ontology (GO) connected to COVID-19 comorbidities. We found 1960 and 153 DEG signatures in COVID-19 patients and recovered individuals compared to healthy controls. In COVID-19 patients, the DEG-miRNA, and DEG-transcription factors (TFs) interactions network analysis revealed that E2F1, MAX, EGR1, YY1, and SRF were the highly expressed TFs, whereas hsa-miR-19b, hsa-miR-495, hsa-miR-340, hsa-miR-101, and hsa-miR-19a were the overexpressed miRNAs. Three chemical agents (Valproic Acid, Alfatoxin B1, and Cyclosporine) were abundant in COVID-19 patients and recovered individuals. Mental retardation, mental deficit, intellectual disability, muscle hypotonia, micrognathism, and cleft palate were the significant diseases associated with COVID-19 by sharing DEGs. Finally, the detected DEGs mediated by TFs and miRNA expression indicated that SARS-CoV-2 infection might contribute to various comorbidities. Our results provide the common DEGs between COVID-19 patients and recovered humans, which suggests some crucial insights into the complex interplay between COVID-19 progression and the recovery stage, and offer some suggestions on therapeutic target identification in COVID-19 caused by the SARS-CoV-2.

SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiome with inclusion of pathobionts.

The microbiota of the nasopharyngeal tract (NT) play a role in host immunity against respiratory infectious diseases. However, scant information is available on interactions of SARS-CoV-2 with the nasopharyngeal microbiome. This study characterizes the effects of SARS-CoV-2 infection on human nasopharyngeal microbiomes and their relevant metabolic functions. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 patients = 8, recovered humans = 7, and healthy people = 7) were collected, and underwent to RNAseq-based metagenomic investigation. Our RNAseq data mapped to 2281 bacterial species (including 1477, 919 and 676 in healthy, COVID-19 and recovered metagenomes, respectively) indicating a distinct microbiome dysbiosis. The COVID-19 and recovered samples included 67% and 77% opportunistic bacterial species, respectively compared to healthy controls. Notably, 79% commensal bacterial species found in healthy controls were not detected in COVID-19 and recovered people. Similar dysbiosis was also found in viral and archaeal fraction of the nasopharyngeal microbiomes. We also detected several altered metabolic pathways and functional genes in the progression and pathophysiology of COVID-19. The nasopharyngeal microbiome dysbiosis and their genomic features determined by our RNAseq analyses shed light on early interactions of SARS-CoV-2 with the nasopharyngeal resident microbiota that might be helpful for developing microbiome-based diagnostics and therapeutics for this novel pandemic disease.

Genome Sequencing of the SARS-CoV-2 Delta (B.1.617.2) Variant of Concern Detected in Bangladesh.

We report the near-complete genome sequence and phylogenetic analysis of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant (B.1.617.2) strain. This variant is associated with increased transmission and immune evasion.

Genome Sequence of a SARS-CoV-2 P.1 Variant of Concern (20J/501Y.V3) from Bangladesh.

This study reports the coding-complete genome sequence, with variant identifications and phylogenetic analysis, of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) P.1 variant (20J/501Y.V3), obtained from an oropharyngeal swab specimen from a female Bangladeshi patient diagnosed with coronavirus disease 2019 (COVID-19) with no travel history.

Whole genome mapping and identification of single nucleotide polymorphisms of four Bangladeshi individuals and their functional significance.

OBJECTIVE: The major objective of the study was to sequence the whole genome of four Bangladeshi individuals and identify variants that are known to be associated with functional changes or disease states. We also carried out an ontology analysis to identify the functions and pathways most likely to be affected by these variants. RESULTS: We identified around 900,000 common variants and close to 5 million unique ones in all four of the individuals. This included over 11,500 variants that caused nonsynonymous changes in proteins. Heart function associated pathways were heavily implicated by the ontology analysis; corroborating previous studies that claimed the Bangladeshi population as highly susceptible to heart disorders. Two variants were found that have been previously identified as pathogenic factors in familial hypercholesteremia and structural disorders of the heart. Other pathogenic variants we found were associated with pseudoxanthoma elasticum, cancer progression, polyagglutinable erythrocyte syndrome, preeclampsia, and others.

Coding-Complete Genome Sequences of Three SARS-CoV-2 Strains from Bangladesh.

We report the sequencing of three severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from Bangladesh. We have identified a unique mutation (NSP2_V480I) in one of the sequenced genomes (isolate hCoV-19/Bangladesh/BCSIR-NILMRC-006/2020) compared to the sequences available in the Global Initiative on Sharing All Influenza Data (GISAID) database. The data from this analysis will contribute to advancing our understanding of the epidemiology of SARS-CoV-2 in Bangladesh as well as worldwide at the molecular level and will identify potential new targets for interventions.