Detection of A2A2 genotype of beta casein protein (CSN2) gene in local, exotic and cross bred cattle in Pakistan.

Genetic variants of bovine Beta-casein protein (CSN2) gene especially A1 and A2 are the most important variants in dairy cattle. A1 milk protein is considered as risk factor for different disease and milk intolerance which release Beta-Casomorphin-7 during digestion which is a bioactive opioid but not released from A2 milk protein. This opioid is responsible for several human health problems like Coronary Heart disease, type 1 diabetics, milk intolerance and other neurological disorders. In present study, 360 blood sample were collected from Lohani, Achai, jersey, Holstein Friesian, Achai x jersey, Friesian x Sahiwal and Sahiwal x Friesian from different region of Khyber Pakhtunkhwa (KP) province. The polymerase chain reaction (PCR) amplicons were sequenced for the identification of polymorphism in exon 7 of Beta-casein protein (CSN2) gene. Sequencing analysis explored CSN2 genotype in exon 7 using the Genomic sequence from GenBank (X.71104) g.8101 C > A at codon 67. The allelic and genotypic frequencies of CSN2 gene were analyzed and observed that Holstein Friesian cattle exhibited A1A2 33%, A1A1 50% and A2A2 17%, Jersey cattle show 68% A1A1, 18% A1A2 and 14% A2A2, Sahiwal x Friesian 56% A1A1, 26% A1A2 and 18% A2A2, Jersey × Achai 78% A2A2, 15% A1A2 and 7% A1A1, Achai 100% A2A2 Lohani 100% A2A2. This is a preliminary study, conducted with meager resources, therefore, it is very difficult to make conclusion that which particular breed possess harmful alleles and which breed possess useful alleles of beta-casein gene. Therefore, a comprehensive molecular work is needed to be performed with greater number of samples sequencing.

SARS-CoV-2 nucleocapsid and Nsp3 binding: an in silico study.

Severe acute respiratory syndrome virus 2 (SARS-CoV-2) belongs to the single-stranded positive-sense RNA family. The virus contains a large genome that encodes four structural proteins, small envelope (E), matrix (M), nucleocapsid phosphoprotein (N), spike (S), and 16 nonstructural proteins (nsp1-16) that together, ensure replication of the virus in the host cell. Among these proteins, the interactions of N and Nsp3 are essential that links the viral genome for processing. The N proteins reside at CoV RNA synthesis sites known as the replication-transcription complexes (RTCs). The N-terminal of N has RNA-binding domain (N-NTD), capturing the RNA genome while the C-terminal domain (N-CTD) anchors the viral Nsp3, a component of RTCs. Although the structural information has been recently released, the residues involved in contacts between N-CTD with Nsp3 are still unknown. To find the residues involved in interactions between two proteins, three-dimensional structures of both proteins were retrieved and docked using HADDOCK. Residues at N-CTD were detected in interaction with L499, R500, K501, V502, P503, T504, D505, N506, Y507, I508, T509, K529, K530K532, S533 of Nsp3 and N-NTD to synthesize SARS-CoV-2 RNA. The interaction between Nsp3 and CTD of N protein may be a potential drug target. The current study provides information for better understanding the interaction between N protein and Nsp3 that could be a possible target for future inhibitors.

Mitochondrial phylogenetic and diversity analysis in Azi-Kheli buffalo.

NOVELTY STATEMENT: The present study was conducted for the first time in Pakistan to investigate Cytochrome C Oxidase Subunit 1 (CO1) gene and full-length Displacement Loop (D-loop) region of mitochondrial DNA in Azi-Kheli buffalo breed native to northern hilly areas of Khyber Pakhtunkhwa Province of Pakistan. The present study was designed to investigate phylogeny and diversity in Azi-Kheli buffalo, through two mitochondrial DNA regions, i.e., Cytochrome C Oxidase Subunit-I (CO1) and Displacement Loop (D-loop) region. Thirty (30) blood samples were taken from Azi-Kheli pure breed animals from original breeding tract, i.e., Khwazakhela, Swat. Polymerase chain reactions using gene-specific primers were carried out for amplifying 709-bp region of CO1 gene and 1159-bp region of D-Loop for identification, phylogeny, and diversity in Azi-Kheli buffalo, respectively. The sequences of CO1 gene revealed four (04) haplotypes, whereas D-loop sequences revealed five (05) haplotypes. Mean interspecific diversity with related species was 2.56%, and mean intraspecific diversity within Azi-Kheli buffalo was 0.25%, estimated via Kimura-2 parameter. Phylogenetic tree (maximum likelihood) revealed clustering of Azi-Kheli haplotypes with river buffalo and is distinct from swamp buffalo and other related species of genus Bubalus. Mean haplotype and nucleotide diversity of D-loop were Hd = 0.9601 ± SD = 0.096 and π = 0.01208 ± SD = 0.00182, respectively. Phylogenetic tree (neighbor-joining) revealed two main clades, i.e., river buffalo and swamp buffalo clade. The haplotypes of Azi-Kheli clustered with haplotypes of different river buffalo breeds at different positions. The current study suggests that Azi-Kheli has common origin with other river buffalo breeds; hence, it is river buffalo which harbors high genetic diversity.

Molecular characterization and antibiotic susceptibility of Shiga toxin- producing Escherichia coli (STEC) isolated from raw milk of dairy bovines in Khyber Pakhtunkhwa, Pakistan.

This study investigated the virulence potential and antibiotic susceptibility analysis of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups, which are significant cause of food borne diseases. A study collected 800 samples of dairy bovine raw milk through various sources, 500 from milk shops, 200 from dairy farms, 26 from milk collection centers, and 74 from street vendors. Using a standard method, E. coli was detected in 321 out of the 800 samples collected. Out of the 321 E. coli-positive samples isolated, 148 were identified as STEC using selective media, specifically Cefixime Tellurite Sorbitol MacConkey's Agar (CT-SMA). Out of the 148 positive samples, 40 were confirmed as STEC non-O157 strains using multiplex PCR, indicating a prevalence of 5% (40 out of 800 samples). STEC isolates were subjected to antimicrobial susceptibility testing, and all isolates were resistant to at least one or more antimicrobials tested through the disk diffusion method, revealed high resistance to Amoxicillin 100%, Ceftriaxone 50%, and Penicillin 44.5%, and notably 44% of the strains exhibited Streptomycin resistance, while Enrofloxacin 55%, Florfenicol 50% and Norfloxacin 44%, demonstrated the highest susceptibility. Out of 40 STEC non-O157, twelve were subjected to Multi Locus Sequence Typing (MLST) sequencing through Illumina Inc. MiSeq platform's next-generation sequencing technology, United States. The genome investigation evidenced the persistence of twelve serotypes H4:O82, H30:O9a, H4:O82, H16:O187, H9:O9, H16:O113, H30:O9, H32:O, H32:O, H32, H32, and H38:O187, linked to the potential infections in humans. Conclusion: STEC isolates showed resistance to multiple antimicrobials, raising concerns for both animal and public health due to widespread use of these drugs in treatment and prevention. The study contributes new insights into monitoring STEC in raw milk, emphasizing the critical role of whole genome sequencing (WGS) for genotyping and sequencing diverse isolates. Still a deficiency in understanding STEC pathogenesis mechanisms, ongoing surveillance is crucial for safeguarding human health and enhancing understanding of STEC genetic characteristics.

Marine natural compounds as potents inhibitors against the main protease of SARS-CoV-2-a molecular dynamic study.

Sever acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA (ssRNA) virus, responsible for severe acute respiratory disease (COVID-19). A large number of natural compounds are under trial for screening compounds, possessing potential inhibitory effect against the viral infection. Keeping in view the importance of marine compounds in antiviral activity, we investigated the potency of some marine natural products to target SARS-CoV-2 main protease (Mpro) (PDB ID 6MO3). The crystallographic structure of Mpro in an apo form was retrieved from Protein Data Bank and marine compounds from PubChem. These structures were prepared for docking and the complex with good docking score was subjected to molecular dynamic (MD) simulations for a period of 100 ns. To measure the stability, flexibility, and average distance between the target and compounds, root mean square deviations (RMSD), root mean square fluctuation (RMSF), and the distance matrix were calculated. Among five marine compounds, C-1 (PubChem CID 11170714) exhibited good activity, interacting with the active site and surrounding residues, forming many hydrogen and hydrophobic interactions. The C-1 also attained a stable dynamic behavior, and the average distance between compound and target remains constant. In conclusion, marine natural compounds may be used as a potential inhibitor against SARS-CoV-2 for better management of COVID-19.

Insight into the drug resistance whole genome of Mycobacterium tuberculosis isolates from Khyber Pakhtunkhwa, Pakistan.

Whole genome sequencing (WGS) is one of the most reliable methods for detection of drug resistance, genetic diversity in other virulence factor and also evolutionary dynamics of Mycobacterium tuberculosis complex (MTBC). First-line anti-tuberculosis drugs are the major weapons against Mycobacterium tuberculosis (MTB). However, the emergence of drug resistance remained a major obstacle towards global tuberculosis (TB) control program 2030, especially in high burden countries including Pakistan. To overcome the resistance and design potent drugs, genomic variations in drugs targets as well as in the virulence and evolutionary factors might be useful for better understanding and designing potential inhibitors. Here we aimed to find genomic variations in the first-line drugs targets, along with other virulence and evolutionary factors among the circulating isolates in Khyber Pakhtunkhwa, Pakistan. Samples were collected and drug susceptibility testing (DST) was performed as per WHO standard. The resistance samples were subjected to WGS. Among the five whole genome sequences, three samples (NCBI BioProject Accession: PRJNA629298, PRJNA629388) harbored 1997, 1162, and 2053 mutations. Some novel mutations have been detected in drugs targets. Similarly, numerous novel variants have also been detected in virulency and evolutionary factors, PE, PPE, and secretory system of MTB isolates. Exploring the genomic variations among the circulating isolates in geographical specific locations might be useful for future drug designing. To the best of our knowledge, this is the first study that provides useful data regarding the insight genomic variations in virulency, evolutionary factors including ESX and PE/PPE as well as drug targets, for better understanding and management of TB in a WHO declared high burden country.

Gibbs Free Energy Calculation of Mutation in PncA and RpsA Associated With Pyrazinamide Resistance.

A central approach for better understanding the forces involved in maintaining protein structures is to investigate the protein folding and thermodynamic properties. The effect of the folding process is often disturbed in mutated states. To explore the dynamic properties behind mutations, molecular dynamic (MD) simulations have been widely performed, especially in unveiling the mechanism of drug failure behind mutation. When comparing wild type (WT) and mutants (MTs), the structural changes along with solvation free energy (SFE), and Gibbs free energy (GFE) are calculated after the MD simulation, to measure the effect of mutations on protein structure. Pyrazinamide (PZA) is one of the first-line drugs, effective against latent Mycobacterium tuberculosis isolates, affecting the global TB control program 2030. Resistance to this drug emerges due to mutations in pncA and rpsA genes, encoding pyrazinamidase (PZase) and ribosomal protein S1 (RpsA) respectively. The question of how the GFE may be a measure of PZase and RpsA stabilities, has been addressed in the current review. The GFE and SFE of MTs have been compared with WT, which were already found to be PZA-resistant. WT structures attained a more stable state in comparison with MTs. The physiological effect of a mutation in PZase and RpsA may be due to the difference in energies. This difference between WT and MTs, depicted through GFE plots, might be useful in predicting the stability and PZA-resistance behind mutation. This study provides useful information for better management of drug resistance, to control the global TB problem.

Prevalence of Pyrazinamide Resistance in Khyber Pakhtunkhwa, Pakistan.

AIMS: Pyrazinamide (PZA) is an important component of first-line tuberculosis (TB) treatment because of its distinctive capability to kill subpopulations of persister Mycobacterium tuberculosis (MTB). The significance of PZA can be understood by its inclusion in the most recent World Health Organization-recommended multidrug-resistant (MDR) TB regimen. Very little information is available about the prevalence of PZA-resistant TB from geographically distinct regions of high burden countries, including Khyber Pakhtunkhwa (KPK), Pakistan, because drug susceptibility testing (DST) of PZA is not regularly performed due to the complexity. In this study, we aimed to find the prevalence of PZA resistance in geographically distinct, Pashtun-dominant KPK Province of Pakistan and its correlation with other first- and second-line drug resistance. MATERIALS AND METHODS: In this study, DST of PZA was performed through an automated BACTEC MGIT 960 system (BD Diagnostic Systems). The resistant samples were further subjected to DST of isoniazid (INH), rifampicin (RIF), ethambutol (EMB), streptomycin (SM), moxicillin (MOX), amikacin (AMK), ofloxacin (OFX), kanamycin (KM), and capreomycin (CAP). RESULTS: Out of 1,075 MTB-positive isolates, 83 (7.7%) were found to be resistant to PZA. Among the PZA-resistant isolates, 76 (90-91.6%) and 67 (80-80.7%) were found to be resistant to INH and RIF, respectively, whereas 63 (76%) were resistant to both first-line drugs, INH and RIF (MDR-TB). The resistance level of EMB, OFX, and SM was also significantly high in PZA resistance, 35 (42%), 40 (48%), and 41 (49-50%) respectively. CONCLUSION: PZA resistance is significantly associated with other first- and second-line drug resistance. A significant number of PZA-resistant isolates are MDR cases. Therefore, DST of PZA should regularly be performed along with other drugs for better management of treatment of MDR and extensively drug resistant (XDR), to avoid side effects in patients.