Asif Nadeem

Characterization and Comparison of the Leukocyte Transcriptomes of Three Cattle Breeds

In this study, mRNA-Seq was used to characterize and compare the leukocyte transcriptomes from two taurine breeds (Holstein and Jersey), and one indicine breed (Cholistani). At the genomic level, we identified breed-specific base changes in protein coding regions. Among 7,793,425 coding bases, only 165 differed between Holstein and Jersey, and 3,383 (0.04%) differed between Holstein and Cholistani, 817 (25%) of which resulted in amino acid changes in 627 genes. At the transcriptional level, we assembled transcripts and estimated their abundances including those from more than 3,000 unannotated intergeneic regions. Differential gene expression analysis showed a high similarity between Holstein and Jersey, and a much greater difference between the taurine breeds and the indicine breed. We identified gene ontology pathways that were systematically altered, including the electron transport chain and immune response pathways that may contribute to different levels of heat tolerance and disease resistance in taurine and indicine breeds. At the post-transcriptional level, sequencing mRNA allowed us to identify a number of genes undergoing differential alternative splicing among different breeds. This study provided a high-resolution survey of the variation between bovine transcriptomes at different levels and may provide important biological insights into the phenotypic differentiation among cattle breeds.

Frequencies of PrP genotypes and their implication for breeding against scrapie susceptibility in nine Pakistani sheep breeds

Prion protein (PrP) gene of 308 sheep was genotyped to investigate polymorphisms at scrapie-associated codons 136, 154 and 171 to assess the resistance of nine different Pakistani sheep breeds to natural/typical scrapie. As a result six genotypes were established on the basis of polymorphic codons 154 and 171. The most scrapie-susceptible codon 136 (A/V) was monomorphic (A) in all breeds. Wild-type genotype ARQ/ARQ was detected with maximum prevalence ranging from 63.2% in crossbred Pak-karakul to 100% in native Buchi, Kachi and Thalli breeds. The most frequent of typical scrapie-associated genotypes was ARQ/ARR as indicated by five of nine breeds. The coding region of PrP gene of 49 animals from the total sampled was also sequenced to ascertain additional polymorphisms. Polymorphism was found in 13 animals of the six breeds in codons 101(Q/R), 112(M/T), 146(N/S) and 189(Q/L) and ten genotypes were established on the basis of these polymorphic codons. Only Hissardale possessed five of the ten genotypes. The most frequent genotype was M112ARQ/T112ARQ detected in Hissardale, Pak-karakul and Awassi, whereas genotypes ARQr231/ARQr231 and ARQR231/ARQr231 (established on the basis of silent polymorphism agg/cgg-R/R) were detected in all breeds. Some animals consisted of three polymorphisms at different PrP codons that are not common in European breeds. An infrequent double heterozygosity (c/c a/g g/t) for codon 171 resulting in a genotype R/H was also detected in three animals each one from Kajli, Hissardale and Pak-karakul. This study concludes that all native sheep breeds are poor in scrapie-resistant PrP genotypes and could contract scrapie if exposed to prions.

Prion protein gene polymorphisms in four goat breeds of Pakistan

Four different goat breeds (Pak-Angora, Dera Din Panah, Naachi and Teddy) of Pakistan were selected to investigate polymorphism in the prion protein gene (PrP gene) responsible for scrapie disease resistance in goats. Initially, genotyping of 187 animals of these four breeds by restriction fragment length polymorphism (RFLP) was done to see the genotype for codon 136 and 154. All the animals were monomorphic with a genotype of AARR except one animal of Teddy breed having the genotype of AARH. Sequencing of PrP gene of twenty animals representing these four goat breeds revealed two genotypes PPSSSS and PPSSPS with haplotypes PSS and PSP of PrP gene at the codon numbers 42, 138, and 240. All four breeds showed both wild type monomorphic sequence and mutant polymorphic sequences of these codons. The mutants of 42 and 138 codons translate the same amino acids as with the wild type sequences, while the mutant of codon 240 is responsible for a different amino acid translation i.e., serine to proline. In short, this study provides preliminary information about alleles and genotypes of PrP gene in four goat breeds of Pakistan.

Expression and identification of folate-sensitive fragile sites in British Suffolk sheep (Ovis aries)

An investigation to understand the dynamics and biological significance of fragile site expression, and identification of 5-fluorodeoxyuridine (FUdR) induced chromosomal gaps/breaks, were carried out in an experimental flock of 45 Suffolk sheep. The statistical comparison revealed, highly significant variation in the frequency of chromosomal fragile site expression between control and FUdR cultures. Mean (± S.D.) values for cells with gaps and breaks, or aberrant cell count (AC), and the number of aberrations (NoA) per animal were 2.02 ± 0.34, 2.42 ± 0.48, 13.26 ± 0.85 and 21.87 ± 1.88 (P < 0.01) in control and FUdR cultures, respectively. The comparison of age revealed nonsignificant variation between control and FUdR cultures. The G-band analysis of fragile site data revealed gaps in 29 autosomal and two X-chromosomal bands in the control cultures, whereas FUdR treated cultures scored 78 unstable bands in autosomes of which 56 were significantly fragile. X-chromosomes expressed breaks and gaps in six G-negative bands and five of them (Xq13, Xq15, Xq17, Xq24 and Xq26) were significantly fragile. The distribution comparison of autosomal fragile sites between sex groups did not reveal any significant variation. Female X-chromosomes were significantly more fragile than the male X-chromosomes. The distribution comparison for age groups (lambs versus adults) revealed significantly higher number of fragile bands in adults. Comparison of published data on reciprocal translocations in sheep with the fragile-site data obtained in this study indicated that the break sites of both phenomena were correlated. Similarities were also found between fragile sites and breakpoints of evolutionary significance in family Bovidae.

The contribution of donkeys to human health

Donkeys have been serving mankind for 5000 years [1]. The phrase ‘beasts of burden’ describes their utility as pack animals and [2] in many parts of the world they play a significant economic and social role in the transport of water, building materials, relief supplies, animal feed and other critical supplies [2,3]. Donkey cart ambulances are an increasing trend in Africa where they are used for transporting sick people to hospital. Donkeys are particularly suited to this role because they are easy to keep and do not show fatigue [2,4]. It is estimated that there are about 90 million donkeys worldwide and they are especially widespread in Central and South American and parts of Europe. China has the largest population with about 11 million donkeys [5]. In recent years, donkey-related research is increasing with the goal of using this species to further improve human health and provide greater societal benefit. Milk is an important source of nutrients including protein, carbohydrate and fats. For thousands of years, cows have provided milk for human consumption in societies around the world. In developed societies of the northern hemisphere, the fat in cow’s milk is increasingly being viewed as a risk factor for human disease because of its relatively high percentage of saturated fatty acids. These saturated fatty acids are associated with chronic cardiovascular diseases that can have profound negative effects on human health. Recent research demonstrates that the wide range of fatty acids present in donkey’s milk may provide positive health effects with human consumption [6], leading to a growing interest in donkey’s milk as a source for human nutrition [7]. Donkey milk is low in fat with high polyunsaturated fatty acids (PUFAs) and resultant advantageous atherogenic and thrombogenic indices. Eicosapentanoic acid and arachidonic acid are present in very small amounts. The high PUFA content and other beneficial nutrients in donkey milk suggest that it may be useful for human nutrition especially for infants and the elderly [8]. Dietary PUFAs also play a pivotal role in maintaining energy balance and minimising body fat deposition by upregulating mitochondrial uncoupling [7]. A comparison of donkey and cow milk for energy balance, lipid metabolism, antioxidant/detoxifying effects and anti-inflammatory effects shows enhanced mitochondrial activity and increased expression of mitochondrial markers after consumption of raw donkey milk by rats as compared with consumption of raw cow milk [9]. In addition to nutritional benefits, consumption of donkey milk has been associated with improved immune responses. Consumption of donkey milk increases interleukins and tumour necrosis factor as compared with levels seen with consumption of goat milk. These findings suggest that donkey milk might be investigated as a beneficial dietary component for immune compromised individuals as well as the sick and elderly [10]. Donkey milk contains other factors that may be beneficial for human health. It has been suggested as an alternative to human and cow milk for individuals with allergic diseases, inflammatory disorders and atopy [10] Donkey milk has been recommended for feeding to infants who are allergic to cow milk [6]. In an experimental study, some infants were provided with cow milk with the addition of soy protein with poor results. When donkey milk with added triglycerides was provided to infants, the milk was well-tolerated by all patients. Patients receiving donkey milk had no recognisable clinical reactions and gained more weight [11]. The low prevalence of intramammary infections in donkeys suggests that donkey milk might be a safe food. However, donkeys are a potential source of Brucella infection, both for people living in close contact with donkeys [12] and through ingestion of unpasteurised milk. Toxoplasma gondii has also been identified with PCR in milk from infected, asymptomatic donkeys but further studies are need to assess the risk of T. gondii transmission through milk [13,14]. Donkey meat occasionally enters the human food chain via adulteration [15]. However, donkey meat has potential as a beneficial alternative to traditional red meats because of its high nutritional profile and components [16]. Donkeys represent a potential threat to human health via meat infected with T. gondii [17] and neospora [18]. The study by Gharsa et al., included in EVJ’s online collection on Working Equids, also highlights the importance of donkeys as a potential source of bacterial species carrying virulence factors, which may have important public health implications [19]. Donkeys have potential as a source of tissues and molecules that can be of use in human medicine: human cardiac disorders are an important cause of morbidity and mortality and heart valve disorders may be treated by prosthetic valve replacement. Donkey pericardium is a potential alternative bioprosthetic heart valve material because it is thin, has minimal calcification values and possesses high tensile strength [20]. Donkeys produce a variety of peptides that may be beneficial to man. Donkeys are now commonly used for the commercial production of antiantibodies. Peptide HP-6, a peptide derived from donkey serum albumin, may: promote the proliferation of cells related to the haematopoietic system; enhance mouse haemopoiesis function; and increase resistance to chemotherapeutic injury [21]. This study in rats showed promising effects in vivo and in vitro and additional human studies may be warranted [21]. LH pentapeptide, derived from donkey serum, inhibited the growth of transplanted tumour cells and stopped their infiltration into other organs [22]. Donkeys are also used in some parts of the world at high altitudes for the production of antivenom drugs. Horses, commonly used for this purpose in other parts of the globe, are not well-adapted to high altitudes. Future research may be appropriate to determine whether this type of antivenom production may be commercially appropriate [23] In addition to their potential contributions to the advancement of human health, donkeys may also provide mental and moral support to human individuals. The human–animal bond that develops with donkeys can help individuals with motivation and development of psychoaffective and psychocognitive processes. Rehabilitation sessions with donkeys can help in identification of individual strengths so that motivation may be strengthened [24]. In summary, while most research relating to donkey health published within equine veterinary medicine and included in this online collection focuses on medical disorders of the donkey [25–27], it is important not to overlook the benefits that donkeys may provide for human health while being cognisant of the potential hazards to man that donkeys may pose.

Genetic variability at seven codons of the prion protein gene in nine Pakistani sheep breeds

) in the central nervous system (CNS).The different genotypes of prion protein gene have beenshown to make animals variably susceptible to this disease.We determined the genotypes of 284 sheep from six native(Buchi, Kachi, Kajli, Lohi, Sipli and Thalli), two crossbred(Hissardale and Pak-Karakul) and one imported (Awassi)breeds of sheep in the Punjab province of Pakistan at sevencodons of the prion protein (

Genetic and genomic dissection of Prolactin revealed potential association with milk production traits in riverine buffalo

Milk yield and quality has been a major selection criterion for genetic improvement in livestock species. Role of Prolactin gene in determining milk quality in terms of protein profile, lactose, lipids and other imperative macromolecules is very important. In this context, genetic profiling of Prolactin gene in riverine buffalo of Pakistan was performed and potential genetic markers were identified illustrating worth of this gene in marker-assisted selection of superior dairy buffaloes. Series of wet and dry lab experimentation was performed starting with genomic DNA isolation from true to breed representatives of indigenous river buffalo (Nili-Ravi). After amplification of coding regions of Prolactin gene, products were eluted and sequenced by Sanger’s chain termination method and aligned to get variations in genomic region. A total of 15 novel variations were identified and analyzed statistically for their significance at population level, haplotypes were constructed, and association was estimated. Phylogenetic analysis was performed to evaluate the rate of evolution for Prolactin gene in various mammalian species. Lastly, biological networking for this molecule was predicted to get the bigger pictorial of its functional machinery. Pathway analysis was performed to find its physiological mode of action in milk synthesis. This is a first report toward complete genetic screening of Prolactin gene in Pakistani buffaloes. Results of this study not only provide an insight for potential role of Prolactin gene in milk-producing abilities of buffalo but also suggest new directions for exploration of more genes that may have promising role to enhance future milk production capabilities of river buffalo breeds of Asian region through marker-assisted selection.

Human Hair Analysis among Four Different Castes Having Potential Application in Forensic Investigation

Hair is the most frequently found trace evidence in the crime cases. It assists in establishing a triangular relation between a crime scene, a victim and a criminal. Morphology of hair can be helpful in forensic investigations to distinguish hair from one race to another. In this context for the purpose of analysis, cuticle inner margin (distinct or not distinct), cuticle thickness and ovoid bodies (present or absent) were considered. Four different castes (Awan, Butt, Gujjar and Rajput) were selected to analyze the structural similarity and differences from each other. Compound microscope was used for purpose of observation. There was no significant gender based difference among all castes. But significant differences were found among individuals of different castes. The purpose of this study was to develop a data base by analyzing the cuticle thickness, inner cuticle margin and ovoid bodies in human head hair in different races of Pakistan to narrow down forensic analysis in criminal scene investigation.

Identification of a novel mutation (p.Ile198Thr) in gene TYR in a Pakistani family with nonsyndromic oculocutaneous albinism

The TYR gene (MIM #6069333) is located at position 11q14.3 on the human chromosome, and encodes tyrosinase, which is expressed in melanocytes and controls the biosynthesis of melanin. Most TYR mutations eliminate the activity of tyrosinase, preventing melanocytes from producing any melanin throughout life. People with this form of albinism have white hair, light‐coloured eyes and very pale skin. Some mutations in TYR reduce but do not completely eliminate tyrosinase activity, and allow some melanin to be produced. We report a Pakistani family with four members affected by oculocutaneous albinism (OCA). Blood samples were collected from all affected individuals, normal siblings and their parents. Genomic DNA was extracted, and sequence analysis of all the coding exons and adjacent intronic sequences of TYR was performed, which identified a novel missense substitution (p.Ile198Thr). Sequencing of TYR in 90 unrelated healthy individuals showed no sequence variant at this location. Our study expands the mutational spectrum of OCA1.

A novel selection signature in stearoyl-coenzyme A desaturase (SCD) gene for enhanced milk fat content in Bubalus bubalis

Modern molecular interventions are dynamic gears for breeding animals with superior genetic make-up. These scientific efforts lead us toward sustainable dairy herds with improved milk production in terms of yield and quality. Many of candidate genes have been dissected at molecular level, and suitable genetic markers have been identified in cattle, but this work has not been validated in buffaloes so far. Stearoyl-coenzyme A desaturase (SCD) has been a potential candidate gene for fat content of milk. Genomic analysis of SCD revealed a total of six variations that were identified through DNA sequencing of animals with lower and higher butter fat %age. After statistical analysis, genotype AB of p.K158I could be associated (P value <0.0001) with higher milk fat %age (10.5 ± 0.5464). This SNP was validated on larger data set by cleaved amplified polymorphic sequences (CAPS) by using DdeI. To scrutinize the functional consequences of p.K158I, 3D protein structure of SCD was predicted by homology modeling and this variation was found located in the vicinity of functional domain and a part of transmembrane helix of this membrane integrated protein. This is a first report toward genetic screening of SCD gene at molecular level in buffalo. This report illustrates the implication of SCD gene and in particular p.K158I variation, in imparting its effect on milk fat %age, which can be targeted in selection of superior dairy buffaloes.