Biology subjects

Saif, R.

Publications and source records attributed to Saif, R..

At least 19 recordsLinked to original sources

bioRxiv2025

Microbial Scl1 Activates TGFBR1 receptor kinase signalling to Drive Fibrosis-Inflammation Axis in Alcohol-Associated Liver Disease

Background and AimsAlcohol consumption alters gut microbiota, which can affect metabolism, immune regulation, and signalling pathways and lead to alcohol-associated liver disease (ALD). We investigated how alcohol-associated gut microbiota (AGMs) modulate liver kinome signalling to drive inflammation and fibrosis in ALD. MethodLiver kinome and metaproteome changes were studied in rats (n=6/group) colonized with stool of severe alcohol-related hepatitis (SAH) patients (SAH[->]healthy-rats) or healthy human donors (HD[->]ALD-rats). AGM-associated liver kinome changes were cross-correlated with bacterial genera. A bacterial protein; Streptococcal collagen-like protein 1 (Scl1) was identified with affinity for TGF{beta}R1. This was validated by molecular docking and immunoprecipitation-LCMS assay. Expression of Streptococcus pneumonia and pyogenes was done in stool of SAH patients (n=10). Also, Scl1 was quantified in patient stool (n=24) and liver tissue (n=19) samples. Validation was performed by assessing Scl1 level in plasma and Streptococcus level in stool samples before and after fecal microbiota transplantation (FMT) in SAH patients. ResultsStool metaproteomics showed significant increase in 10 bacterial genera (Streptococcus, Staphylococcus, and Clostridium) in SAH[->]Healthy-rats, mirroring changes seen in ALD-rats (FC>1.5, p<0.05). Clusters of Orthologous Groups analysis indicated increased post-translational modifications (PTMs) and decreased lipid metabolism in ALD-rats and SAH[->]Healthy-rats. Liver kinome profiling showed 85 upregulated kinases in SAH[->]Healthy-rats, with 34 overlapping with ALD-rats, associated with inflammation (Mapk14, Map3k10 and others), fibrosis (Tgfbr1, Igf1r, and others), lipid metabolism (Cdk14, Cdk18) and regeneration (Met, Bmpr1a and others). FMT from healthy human-donors to ALD-rats reversed the expression of Streptococcus (10-fold), Staphylococcus (3-fold), and Clostridium (5-fold), along with 18 kinases associated with inflammation (Btk, Camk4, Nuak1) and fibrosis (Tgfbr1, Col4a1, Fgfr2 and others). Strong association (r{superscript 2}>0.9, p<0.05) was seen between Streptococcus abundance and fibrosis and inflammation-related kinases. Level of Scl1 was significantly high in SAH and ALD-rat stool samples (FC>2, p<0.05). It showed strong affinity for TGF{beta}R1, as validated by molecular docking (>86%confidence) and immunoprecipitation assays (>100FC,p<0.05), indicating it as a potential ligand for TGF{beta}R1. Concordantly, expression of Scl1 was highest amongst all the known ligands for TGF{beta}R1 (p<0.05) suggesting Scl1 is a major contributor for TGF{beta}R1 activation and its downstream signalling in these patients. Following fecal microbiota transplantation, expression of Scl1 in plasma and Streptococcus levels in stool were significantly reduced by 2.1-folds and 1.9 folds respectively in SAH patients. Further, Scl1 levels SAH plasma is capable of predicting poor therapeutic response and 30-day mortality with high accuracy (AUC=0.92, cutoff >70 normalized abundance). ConclusionAlcohol induced gut dysbiosis alters the liver kinome, promoting inflammation, fibrosis, and lipid dysregulation. Streptococcal Scl1 is identified as a bacterial protein mimicking human collagen and capable of activating fibrotic signalling pathway through TGF{beta}R1in liver and is capable of predicting poor response in SAH. FMT from healthy donor restores microbial imbalance and harmful microbial-host interactions.

microbiology↗

bioRxiv2025

Liver Kinome Profiling Identifies PS1145 as a Potential Therapeutic molecule for amelioration of Systemic Inflammation in Alcohol-related Liver Disease

Background and aimsAlcohol-related liver disease (ALD) has high mortality due to systemic inflammation. We analysed liver and monocyte kinome profiles in a chronic ethanol-fed pre-clinical rat model to identify therapeutic targets that could mitigate inflammation in ALD. MethodKinome profile was performed in liver and circulating monocytes at baseline, and after 8,12,16,20 and 24-weeks of 40% ethanol administration. Pathway-specific inhibitors, including PS1145 (IKK-phosphorylation inhibitor), PH-797804 (MAPK14 inhibitor), resveratrol and prednisolone were tested for their anti-inflammatory effects in ALD-rats, PBMC from patients and NIAAA mouse model. ResultsKinome profiling identified 497 liver and 345 monocyte kinases in ALD rats (FDR<0.01). A time-dependent increase in MAPK14-associated kinases was observed in both tissues (FC>1.5, p<0.05). By 24 weeks, 172 liver and 48 monocyte kinases were significantly upregulated, particularly those linked to MyD88-TLR4, PI3K-Akt, TNF, TGF{beta}, and cellular senescence pathways. Key contributors included TGF{beta}R1, ROS-generating kinases, and IL-1-driven MAPK14 and IKK phosphorylation. Targeting this axis, PS1145 (an IKK inhibitor) suppressed NF{kappa}B activation and inflammation in THP1 and HepG2 cells, as well as PBMCs from healthy and SAH patients, outperforming PH797804, resveratrol, and prednisolone (p<0.05, FC>1.5). PS1145 significantly reduced IL-6, TNF, and NF{kappa}B, while increasing IL-10. In vivo, PS1145 treatment in the NIAAA mouse model markedly reduced hepatic steatosis, cellular stress and inflammatory pathways (cytokine signalling, IL-36 signalling and others) more effectively than standard therapies, Notably, it also downregulated IL36R, impairing TLR receptor dimerization, suggesting a dual mechanism of action (p<0.05) highlighting its therapeutic potential in ameliorating systemic inflammation in ALD. ConclusionOur study highlights the key role of MAPK14 and MYD88-TLR4 pathway kinases in driving systemic inflammation in SAH. The IKK inhibitor PS1145, by blocking both IKK phosphorylation and TLR dimerization, effectively suppresses inflammatory signalling and improves liver pathology, positioning it as a promising targeted therapy for ALD.

pharmacology and toxicology↗

bioRxiv2024

Mathematical modeling using box-behnken response surface design for optimal DNA yield with variable parameters of time, temperature, and proteinase K concentration

The current research aims to explore an enzyme-assisted inorganic DNA extraction protocol to obtain maximum yield from whole blood. We considered various operating conditions such as incubation temperature and time along with proteinase K enzyme concentration. To model and simulate the extraction process, Box-Behnken Design (BBD) was employed with three factors and its five levels, coupled with the desired functional response surface methodology (RSM). RSM entails fitting a mathematical model for experimental data to optimize a response variable with Box-Behnken Design. The best simulation settings yielded the maximum DNA at an incubation temperature of 56{degrees}C for 10 hours and proteinase K enzyme concentration of 10{micro}L. Utilizing these parameters, a yield of 300{micro}L was obtained. Furthermore, other DNA extraction parameters may also be added for a more robust design for obtaining the maximum DNA yield.

molecular biology↗

bioRxiv2023

Optimizing PCR using mtDNA Universal Primers for Detecting Indigenous Domestic Animal Species from Pakistan

The prevalence of specific animal meat consumption is integral to human nutrition but is jeopardized by the widespread issue of meat adulteration, posing threats to both the industry and consumer health. Escalating demand has led to fraudulent practices, necessitating reliable techniques for authentic source identification, aligning with ethical and religious principles. PCR has proven effective in addressing this concern by examining twelve domestic animal species in Pakistan, differentiating between halal and haram species. The method employs universal primers for 16S rRNA gene amplification, generating species-specific DNA fragments. This sophisticated approach, producing distinct fragments for cow, camel, goat, sheep, chicken, cat, dog, donkey, duck, rabbit, horse, and human with 507, 385, 787, 763, 470, 700, 370, 600, 500, 750, 800 and 300bp holds promise for identifying and tracing these species in commercial meat products. By ensuring robust food traceability, this methodology combats contemporary challenges in adulteration, preserving the integrity of the food supply chain.

genetics↗

bioRxiv2023

Genetic Association of CYP1A2 Gene Variant (rs762551) with Caffeine Induced-Hypertension Susceptibility and Cytochrome P450 1A2 Protein Analyses

High blood pressure is one of the most common illnesses affecting the Pakistani population due to, but not limited to, dietary habits, sedentary lifestyle and socio-economic aspects which have devastating effects on human health and general well-being. Caffeine is metabolized by CYP1A2 which results in lowered blood pressure while its retention in case of low metabolism may lead to hypertension. This phenomenon occurs because caffeine and its metabolites block A1 receptors in the kidney lowering the function of adenosine in blood pressure regulation. Current research aimed to demonstrate the genetic association of CYP1A2 gene variant 15:74749576C>A (C allele lowering CYP1A2 activity) within caffeine induced hypertensive individuals of Pakistani origin using ARMS-PCR. This pilot scale study revealed that overall, 8%, 56% and 36% sampled population (n=50) is homozygous wild-type (C/C), heterozygous (C/A) and homozygous mutant (A/A) respectively. Similarly, alternative allele frequency is 0.28 and 0.48 in cases and controls. Chi-square ({chi}2) association test using PLINK data analysis toolset was applied which showed significant results of {chi}2(2, N = 50) = 4.244, p = 0.039. Hardy Weinberg Equilibrium analysis was also applied which establishes that sampled population is obeying the principle with p-value of 0.241. Moreover, odds-ratio depicts that the mutant allele is 0.42 times less prevalent in cases vs. controls. A few bioinformatics tools were employed e.g., ProtParam, PsiPred, PDB-RCSB, Motif finder, CTU-TMHMM-2.0, ScanProsite, GPS PAIL2.0, PRmePRed, NetOGlyc4.0, NetPhos3, SIFT analysis and STRING database in order to predict physicochemical properties, secondary structure, 3-dimensional structure, conserved motifs, transmembrane structure, post-translational modifications, protein variants impact on its function and protein-protein interactions respectively. The current endeavor attempted to provide the genetic architecture of the aforementioned variant in this case-control study in Pakistani individuals, which can pave paths of preventive medicine initiatives through genetic counsel of the masses.

genetics↗

bioRxiv2023

Evolutionary patterns and heterogeneity of Dengue Virus serotypes in Pakistan

Comprehensive and systematic examination of Dengue virus (DENV) evolution is essential in the context of Pakistan as the virus presents a significant public health challenge with the ability to adapt and evolve. To shed light on intricate evolutionary patterns of all four DENV serotypes, we analyzed complete genome sequences (n=43) and envelope (E) gene sequences (n=44) of all four DENV serotypes collected in Pakistan from 1994 to 2023 providing a holistic view of their genetic evolution. Our findings revealed that all four serotypes of DENV co-circulate in Pakistan with a close evolutionary relationship between DENV-1 and DENV-3. Genetically distinct serotypes DENV-2 and DENV-4 indicate that DENV-4 stands out as the most genetically different, while DENV-2 exhibits greater complexity due to the presence of multiple genotypes and the possibility of temporal fluctuations in genotype prevalence. Selective pressure analysis in Envelope (E) gene revealed heterogeneity among sequences (n=44) highlighting 46 codons in the genome experiencing selective pressure, characterized by a bias towards balancing selection indicating genetic stability of the virus. Furthermore, our study suggested an intriguing evolutionary shift of DENV-4 towards the DENV-2 clade, potentially influenced by antibodies with cross-reactivity to multiple serotypes providing a critical insight into the complex factors shaping DENV evolution and contributing to the emergence of new serotype. Author SummaryThe emergence of the fifth serotype of dengue virus heightened our interest in investigating its presence in Pakistan. In our quest to understand the evolving landscape of dengue in Pakistan, we conducted a comprehensive analysis, comparing whole genome sequences and E gene sequences. Notably, we focused on the E gene recognized as the most mutable component and a key determinant of dengues virulence. The phylogenetic analysis unveiled fascinating findings, demonstrating a strong genetic affinity between serotypes 1 and 3. Substantially signifying its implications for vaccine development and understanding of cross-immunity dynamics within serotypes. We delved into the genetic dynamics of dengue by subjecting the whole genome of DENV and E gene to neutrality tests. The outcomes of these tests unveiled a critical aspect of dengue virus evolution: the genome is not evolving neutrally. Instead, the E gene experiences selective pressure, indicating a bias towards balancing selection. The finding underscores the complex interplay of factors shaping the genetic diversity of dengue in Pakistan and provides valuable insights into the viruss adaptive strategies.

evolutionary biology↗

bioRxiv2023

Genetic Association of SLC47A1 Gene Variant (17:19571562C >T) and Bioinformatics Analyses of MATE1 Protein in Chronic Kidney Disease Patients of Pakistani Origin

Chronic Kidney Disease (CKD) is a serious human threat worldwide which is associated with a number of environmental, clinical and genetic factors that affect serum creatinine (SCr) and glomerular filtration rate (eGFR). One of the best biochemical and genetic markers to study renal functioning is SLC47A1 which encodes MATE1 protein and can be a promising target to study its genetic causes, subject protein is a cationic transporter involved in the regulation of creatinine and urea levels in the blood which may cause toxicity and renal disorders. Hence, the current study focused to interrogate the putative association of SLC47A1 gene variant (17:19571562C>T) in 75 individuals (cases=50, controls=25) of Pakistani origin using ARMS-PCR genotyping, out of which 10 were found heterozygous, 57 homozygous wild-type and 08 homozygous mutants. PLINK data analysis toolset was used which manifested that sampled population complies the Hardy-Weinberg Equilibrium by {chi}2 (2, N = 75) =, p = 4.756 x 10-005, similarly the Chi-square statistics p = 0.03274 along with odds-ratio of 3.244 showing a significant genotypic association with the subject phenotype indicating that mutant allele is almost 3-times more prevalent in cases vs control cohorts with an alternative allele frequency of 0.22 and 0.08 in CKD patients and healthy controls respectively. Besides, few of the bioinformatics tools i.e., ProtParam, PsiPred, PDB-RCSB, Motif Scan, CTU-TMHMM-2.0, ScanProsite, PRmePRed, GPS PAIL2.0, NetOGlyc4.0, NetPhos3, SIFT, Polyphen-2 and STRING were also used for the prediction of different parameters of both the wild-type and mutant MATE1 proteins such as physiochemical properties, secondary structure prediction, 3-D structure, protein conserved domains, transmembrane structures, posttranslational modifications, SNP prediction/amino acid substitution, pathogenicity and protein-protein interaction networks. Current preliminary genotyping provided an insight into the association of the aforementioned genetic variant with CKD to estimate the genetic risks along with prediction of its functional effects in the system biology context which may be considered for developing new targeted drugs along with genetic counselling initiatives to evade this peril in the population.

genetics↗

bioRxiv2023

Genetic Association of PLAG1 Gene Variant 14:25015640G>T with Wither-Height in Pakistani Cattle

The cow is one of the most valuable domesticated animals, producing milk, meat, fiber, hide and manure for agricultural purposes to serve humanity. Particularly, first two production traits are significantly linked with the physical characteristics of the animal e.g., wither-height, large body-size and skeletal frame are positively correlated with these production traits. The PLAG1 is one of the many genes that has been associated with the aforementioned trait in many livestock and human species. So, the current study was conducted on a total of 50 cattles using ARMS-PCR genotyping technique followed by genetic association testing of the 14:25015640G>T variant using PLINK data analysis toolset. Our findings showed that 24% of the sampled Pakistani cow population are homozygous wild-type (GG), 12% homozygous mutant (TT), while 64% found heterozygous (GT). Sampled cows were obeying Hardy Weinberg Equilibrium (HWE) with{chi} 2 (2, N = 50) = 10.39, p = 0.049. Similarly, Chi-square variant association was also observed significant with p-value of 1.267x10-3 having minor-allele frequency of 0.60 and 0.28 in heighted (cases) and control cohorts respectively. Additionally, a positive odds-ratio of 3.85 is also evident that the subject variant is under-selection and showing the tendency of the mutant allele almost 4-times higher in cases vs control groups. This pilot scale study would be helpful to gain insight of variability of the subject SNP in the sampled cow population but further functional studies on larger sample size may be conducted for validation and subsequent results can be disseminated to improve this valued trait of the indigenous cows for gaining maximum milk and meat production from this esteemed species.

genetics↗

bioRxiv2023

In-silico Screening of Origanum vulgare Phytocompounds as Potential Drug Agents Against Vp35 Protein of the Ebola Virus

The leading cause of the Ebola virus outbreak during 2013-16 in Western Africa was a lack of targeted anti- viral drug choices, a fast rate of mutations and the unavailability of many of the structural proteins and annotations within its genome. The surroundings of the Ebola River in DR-Congo fail to get rid of this endemic, the reason behind this was believed to be its origin from non-human primates, which made its risk assessment and tracing difficult. The Vp35 is a multifunctional protein with innate immune antagonistic properties and is considered one of the most suitable drug targets within this virus. The main motive of this study is to discover a potential anti-viral drug against the Ebola virus by targeting the aforementioned protein with different phytocompounds of oregano that have the lowest binding energies and qualifies over different simulation parameters, so firstly, molecular docking was performed on its 28 compounds using PyRx to get the best complexes with minimum binding energies e.g., -8.9Kcal/mol. Ligands with the best docking scores were gone through Lipinskis rule of five for drug likeliness potential. For the drug affirmation, molecular dynamic simulation was also performed with the best two docked complexes using NAMD/VMD to find out their conformational stability through RMSD, RMSF, Rg, SASA and H-bond analyses. Current computer-generated prediction suggested that Ursolic acid and Oleanolic acid possess potential inhibitory effects against virus replication. Furthermore, paradigm shifts of usage of natural and herbal products for treating infectious diseases are being encouraged here, However, further wet-lab experiments and clinical trials are still needed to determine the robustness of these virtually tested phytocompounds against the Vp35 protein of the Ebola virus.

bioinformatics↗

bioRxiv2023

De novo Genome Assembly, Functional Annotation and SSR Mining of Citrus reticulata (Kinnow) from Pakistan

Citrus reticulata (Blanco) fruit is native to South East Asia which owns many of the nutritional, medicinal and economic advantages, locally known as "Kinnow" and one of the priced mandarin varieties (Dancy, Fuetrells Early and Honey) of Citrus genera renowned for its exclusive taste, vitamin richness, thin peel, long shelf-life and seedless characteristics in Pakistan. However, genetic improvement and breeding strategies of this valued variety are lacking due to the in-housed insufficient genomic and technical resources. Therefore, the current research was initiated to provide the base-line de-novo genome assembly of C. reticulata (seedless kinnow) at a depth of 151x with Illumina paired-end short-read sequencing technology using HiSeq 2500. Whole-genome sequencing resulted in 139,436,350 raw reads ([~]20.09 GB) of data, however, after removing the low-quality reads (1.08%), duplicated sequences (10.5%) and Illumina adaptors, 137,901,462 clean reads were obtained with ([~]18.87 GB) of clean data which was further used for downstream variant calling analysis. In total, 348,861 scaffolds were generated with N50 value of 4827 which constitute 263,018,9 contigs ranging from 71-36,213 with total of 179,984,763 nucleotides. The GC content of the final draft assembly at 71-mer was 34.1%. Moreover, annotation was performed with "Hayai-Annotation Plants" tool which marked the whole-genome mapping with three main functional databases of interpro, Pfam and gene ontology. Additionally, in-silico identification of 111,032 Simple Sequence Repeats (SSR) was also accomplished with the help of GMATA tool, which may be used for further screening and genetic improvement of the citrus varieties by means of this current assembly as a resource of local reference genome.

bioinformatics↗

bioRxiv2023

Mathematical Modeling of PCR with Variable Concentrations of DNA and Taq Polymerase

Polymerase Chain Reaction (PCR) is one of the important techniques in molecular biology for many diagnostics and research applications. In this current theoretical PCR optimization research, out of its seven recipe reagents, two of the important constituents e.g., DNA which might be very rare in forensic cases and polymerase enzyme due to its high cost were mathematically modeled and computationally simulated using MATLAB programming platform. Chemical equations of PCR stages were established using the law of mass action followed by differential equations derivations from each of the DNA amplification phases. Firstly, a mathematical model of Taq polymerase enzyme with its variable concentrations ranging from 0.05-0.35 IU/L was formulated which showed the best PCR product of 0.61 ng/L obtained with 0.2 IU/L of polymerase. Similarly, by fixing the optimal polymerase concentration obtained in the previous simulation, the prime-DNA variable concentrations ranging from 0.5-3.5 ng/L were further simulated which showed the best PCR product of 0.94 ng/L at 2.0 ng/L of DNA. PCR wet-lab experimentation and gel-electrophoresis visualization validated mathematically simulated optimized concentrations of the aforementioned two reagents which vary the PCR product considerably. Current findings indicate that mathematically optimized concentrations of Taq-polymerase and template-DNA may reduce the PCR cost, time and energy, but other factors of target-sequence specificity, purity/concentrations of the other reagents, working hygiene, technical intricacies, biosafety and biosecurity factors may also contribute the desired yield of the amplicon.

bioengineering↗

bioRxiv2022

Whole-Genome Sequencing and Variant Discovery of Citrus reticulata 'Kinnow' from Pakistan

Citrus is a source of many nutritional and medicinal advantages, which is cultivated worldwide with major citrus groups of sweet oranges, mandarins, grapefruits, kumquats, lemons and limes. Pakistan produces all of its major citrus groups with mandarin (Citrus reticulata) being the prominent group that includes local commercial cultivars such as Feutrals Early, Dancy, Honey and Kinnow. The present study was designed to understand the genetic architecture of this unique variety of Citrus reticulata -Kinnow. The whole-genome resequencing and variant calling was performed to map the genomic variability that might be responsible for its particular characteristics like taste, seededness, juice content, thickness of peel and its shelf-life. A total of 139,436,350 raw sequence reads using Illumina platform were generated with 20.9 Gb data in Fastq format having 98% effectiveness and 0.2% base call error rate. Overall, a total of 3,503,033 SNPs, 176,949 MNPs, 323,287 INS and 333,083 DEL were identified using GATK4 variant calling pipeline against Citrus clementina as a reference genome. Further, g:Profiler bioinformatics tool was applied for annotating the newly found variants, harbor genes/transcripts and their involved pathways. A total of 73,864 transcripts harbors 4,336,352 variants, most of the observed variants were predicted in non-coding regions and 1,009 transcripts were found well annotated by different databases. Out of total aforementioned transcripts, 588 involved in biological processes, 234 in molecular functions and 167 transcripts involved in cellular components in Citrus reticulata. In a nutshell, 18,153 high-impact variants and 216 genic-variants found in the current study which may be used for marker assisted breeding programs of Kinnow to identify this particular variety among others and to propagate its valued traits to improve the contemporary citrus varieties as well. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/519411v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1568ae1org.highwire.dtl.DTLVardef@b7d20eorg.highwire.dtl.DTLVardef@3fc3a9org.highwire.dtl.DTLVardef@46ba89_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2022

Genomic Selection Pressure Discovery using Site-Frequency Spectrum & Reduced Local- Variability Statistics in Pakistani Dera-Din-Panah Goat

Population geneticists have long sought to comprehend various selection traces present in the goat genome due to natural or human-driven selection and breeding practices. As a step forward to pinpoint the selection signals in the Pakistani Dera-Din-Panah (DDP) goat breed, whole-genome pooled-sequencing (n=12) was performed and 618,236,192 clean paired-end reads were mapped against ARS1 reference goat assembly. Five different selection signal statistics were applied here using four Site-Frequency Spectrum (SFS) methods (Tajimas D (TD), Fay & Wus H (H), Zengs E (E), Pool - HMM) and one Reduced Local-Variability approach named pooled-heterozygosity (Hp). The under-selection regions were annotated with significant threshold values of -ZTD[≥]4.7, -ZH[≥]6, -ZE[≥]2.5, Pool-HMM[≥]12, and -ZHp[≥]5, which resulted in accumulative 364 candidate gene hits, while the highest signals were observed on Chr. 4, 6, 10, 12, 15, 16, 18, 20, 27 harbor ADAMTS6, CWC27 genes associated with body-height, RELN, MYCBP2, FGF14, STIM1, CFAP74, GNB1, CALML6, TMEM52, FAM149A, NADK, MMP23B, OPN3 with body-weight/meat production, FH, MFHAS1, KLKB1 with milk production, RRM1, KMO, SPEF2, F11 with fecundity rate/reproduction, ATP8B4 with immunity, KIT, KMO with pigmentation, ERI1 with olfaction and RHOG with wool production traits. Furthermore, we accentuate to highlight the putative windows that were captured commonly by any of the five statistical methods applied which harbor meat production, immunity and reproduction-associated genes validating the genotype-phenotype relationship of aforementioned traits private to this goat breed. Current insight into the genomic architecture of DDP goat provides a better understanding to improve its genetic potential and other vested traits of large body size and fiber production by updating the breeding strategies to boost the livestock-based agricultural economy of the country.

genetics↗

bioRxiv2022

Association Analysis of LCORL Genetic Variant (rs657074013A>AT) with Body-Height and Skeletal Frame in Pakistani Goats

Goat is one of the most valued and cost-effective household animals serving humanity by providing its meat, milk, fiber, skin and manure. Particularly, meat and milk production depends on the physical traits of the animals e.g., large body-size/skeletal frame. Previous studies has shown that the LCORL gene is associated with the subject traits in humans and many other animal species. So, the current study is aimed to investigate the genetic variability/association of rs657074013A>AT variant within diverse goats from Pakistan. By using ARMS-PCR, a total of 51 samples were genotyped with allele-specific primer-set, results of the current study depicted that, 27% population is homozygous wild-type (A/A), 24% homozygous-mutant (AT/AT) and 49% is heterozygous (A/AT). Hardy Weinberg Equilibrium (HWE) analysis revealed that our overall sampled population obeys this principle with{chi} 2(2, N = 51) = 0.046, p = 0.9730. Similarly, a significant genetic association was also envisioned by Chi-square statistics p = 9.60 x 10-5 using the PLINK data analysis toolset. Furthermore, alternative allele frequencies of 0.68 and 0.28 were also calculated within cases (n=25) and control (n=26) cohorts respectively along with an odds ratio of 5.242. As a nutshell, this pilot study improved our genomic insight by observing the variability and association of the subject LCORL genetic variant (c.828_829insA) in the Pakistani goat population, which may be used to improve this economically important trait by adopting marker-assisted breeding strategies and further investigated within other livestock species as well.

genetics↗

bioRxiv2022

Evaluation of Association between CSN2 Genetic Marker 7:32016718C>A with A1/A2 Milk in Pakistani Buffaloes

Milk is an essential part of the diet that plays an important role in human growth and development due to the presence of different components like calcium, fats and proteins. Among six major proteins of the milk, {beta}-casein is one of them which is being investigated here due to the A1/A2 milk genetic variants and its associated gene of CSN2 in one of the valued species of Bubalus bubalis in Pakistan. Fifty buffalo samples were genotyped using the ARMS-PCR technique. CSN2 gene locus 32016718C>A is located on Chr.7 with CDS position c.350C>A on its 7th exon as per (GenBank transcript ID: XM_006071124.3). In {beta}-casein, 117th amino acid position where histidine (CAT) is responsible for A1 and proline (CCT) for A2 milk phenotype. A2 milk is considered to be healthy for human health due to the absence of bioactive peptide {beta}-casomorphin-7 (BCM7) in comparison with A1 milk. The current results showed that overall, 100% buffalo population is homozygous wild-type (CC) which means no mutant allele is detected in all studied samples therefore, Pakistani buffalo is exclusively producing A2 milk. Furthermore, this work may also be conducted on different buffalo breeds in Pakistan as well as other species of cattle, goat, sheep and camel to explore their genomic architecture for this valued trait.

genetics↗

bioRxiv2022

Genetic Association of CSN2 Variant 6:85451298C>A with A1/A2 Milk Phenotype in Pakistani Cows

The cow is a very important mammal from the bovine family renowned for providing milk, meat and hide around the globe. {beta}-casein is one of the principal proteins found in cows milk encoded by the CSN2 gene with 12-13 reported genetic variants present on Chr.6 in the cows genome. The current study investigated A1/A2 associated variant g.6:85451298C>A (rs43703011) in exon 7th according to the GenBank ID: M55158.1 position 8101C>A (c.245C>A), which is responsible to change the 67th amino acid of the {beta}-casein polypeptide from proline (CCT, A2) to histidine (CAT, A1). The milk containing A2 protein variant is good while A1 milk has some negative effects on human health due to the production of {beta}-casmomorphin-7. Genetic screening of the subject variant was conducted using ARMS-PCR on 48 native and exotic cows from Pakistan. Our initial results showed that 37.5% of the population is homozygous wild-type (C/C), 56.25% is heterozygous (C/A) and the remaining 6.25% is homozygous mutant-type (A/A). Additionally, the Chi-square association test was applied using the PLINK data analysis toolset which presented a significant p-value of 2.811x10-3 and OR of 3.8 with an alternative allele frequency of 0.2 and 0.5 in native and exotic populations respectively. Hence, there is significant variability in A1/A2 genotype which may be addressed by adopting selective breeding programs in Pakistan.

genetics↗

bioRxiv2022

Genetic Association of LDHA Variant g.2582481G>A with Racing Performance of Pakistani Pigeons

Pigeons are being used as a means of communication back in history due to their efficient flight stamina, navigational (homing) ability and endurance to harsh weather conditions. Due to these distinctive characteristics, pigeon racing and rearing has become a popular sport and hobby throughout the world, predominantly in Pakistan. The genetic tendency of LDHA gene in pigeons should be considered for understanding racing ability. The present study aimed to examine the genetic association of LDHA variant g.2582481G>A with racing performance in Pakistani pigeons. Our results demonstrated that 55% of the pigeons population was homozygous wild-type (G/G), 37.5% was heterozygous (G/A) and the remaining 7.5% was homozygous-mutant (A/A) at this locus. Furthermore, alternative allele frequencies within the best and low-performing cohorts of pigeons were 0.45 and 0.075 respectively. Furthermore, the Chi-Square association test was applied using PLINK data analysis toolset which showed a significant p-value of 1.381x10-4 with odds ratio (OR) of 10.09. Consequently, Pakistani pigeon population suggests a significant association of g.2582481G>A with racing performance similar to the other pigeon population of the world.

genetics↗

bioRxiv2021

Differential Gene Expression Pipeline for Whole Transcriptome RNA-Seq Data using Personal Computer

Advances in the next generation sequencing (NGS) technologies, their cost effectiveness and well-developed pipelines using computational tools/softwares has allowed researchers to reveal ground-breaking discoveries in multi-omics data analysis. However, there is still uncertainty due to massive upsurge in parallel tools and difficulty in choosing best practiced pipeline for expression profiling of RNA sequenced (RNA-seq) data. Here, we detail the optimized pipeline that works at a fast pace with enhanced accuracy on personal computer rather than using cloud or high-performance computing clusters (HPC). The steps include quality check, base filtration, quasi-mapping, quantification of samples, estimation and counting of transcript/gene expression abundances, identification and clustering of differentially expressed features and visualization of the data. The tools FastQC, Trimmomatic, Salmon and some other scripts in Trinity toolkit were applied on two paired-end datasets. An extension of this pipeline may also be formulated in future for the gene ontology enrichment analysis and functional annotation of the differential expression matrix to make this data biologically more significant.

bioinformatics↗