First report on prevalence, molecular characterization and phylogenetic study of Toxoplasma gondii infecting sheep of the Malakand Division of Pakistan.

BACKGROUND: Toxoplasma gondii is an apicomplexan protozoan parasite that infects one-third of the population of the world, including humans, animals, birds, and other vertebrates. The present investigation is the first molecular attempt in the Malakand Division of Pakistan to determine the epidemiology and phylogenetic study of Toxoplasma gondii infecting small ruminants. METHODOLOGY: A total of (N = 450) blood samples of sheep were randomly collected during the study period (December 2020 to November 2021), and DNA detection was done using PCR by amplifying ITS-1 genes. SPSS.20 and MEGA-11 software were used for statistical significance and phylogenetic analysis. RESULTS: The overall prevalence of T. gondii infection among sheep was 14.44 % (65/450). A high infection rate was found in more than five-year-olds at 18.33 % (11/60). Sequencing and BLAST analysis of PCR-positive samples confirmed the presence of T. gondii. Randomly, three isolates were sequenced and submitted to GenBank under accession numbers (PP028089-PP028091), respectively. The BLAST analysis of the obtained sequences based on the ITS-1 gene showed 99 % similarities with reported genotypes found in goats of Malakand, Pakistan (PP028089) and dogs of Brazil (MF766454). The study concludes that T. gondii is notably prevalent among the sheep population in the region, emphasizing the significant role of risk factors in disease transmission across animals and potentially to humans. Further research, zoonotic potential analysis, and targeted control measures are warranted to address and manage this parasitic infection effectively.

Altitudinal distribution and seasonal migration of asiatic black bear (Ursus thibetanus) in kaghan and Siran Valleys, Pakistan.

The Asiatic black bear (Ursus thibetanus) is considered one of the largest carnivores inhabiting different territories including coniferous and broad-leaved forests, extending from sea level to the elevation of 4300 m. The Kaghan and Siran Valleys in Pakistan's District Mansehra were the location of the current study. To evaluate the altitudinal distribution and seasonal movement of Asiatic black bears in the region, we used three approaches (sign distribution survey, questionnaire-based survey, and scat collecting). A total of 1858 bear signs were observed during the field survey, the highest number of signs was dig marks (1213) followed by plant uprooting. Dig marks (MER: 7.574) were observed repeatedly in each potential site of both valleys followed by plant uprooting (MER: 1.594). Similarly, the altitudinal distribution of black bears was determined through the frequency of signs concerning elevation (m). Elevation of the study area ranged from 1300 to 3500 m and the most abundant population was recorded at the elevation of 2501-3000 m followed by 2001-2500 m. About 52% of local communities encountered black bears during the summer season in the field followed by Spring (23%.8) and winter (15.3%). Manshi Reserver forest (24.5%) from Kaghan and Panjul Reserve forest (31.6) from Siran Valley are the potential summer migratory spots. Eleven scat samples were collected with a 0.103 encounter rate (ER). Most of the scats (about 70%) were observed within the maize crop field in the summer season at low elevation, while just 30% of the scat was observed in the winter season in the forest.

Comparative valuation of the chlorpyrifos, acetamiprid, and lambda-cyhalothrin toxicity and their hematological and histopathological consequences in pigeons.

Globally agrochemicals are widely used in the agricultural sectors, posing potential eco-toxicological risks and disrupting various lifeforms including birds. Thus, the current work was conducted to compare the acute toxic impacts of pesticides (e.g., chlorpyrifos, acetamiprid, and lambda-cyhalothrin) on the pigeon's health. In total 50 adult pigeons were purchased from a local market where these pigeons were fed on pollution-free food. Post adaptation period (15 days), the pigeons were arbitrarily separated into five distinct groups after having been identified in this manner by chance (each group containing 10 pigeons). Control group (group 1) was not treated with any pesticide while the remaining groups (groups 2, 3, and 4) were treated with 0.25-mg/kg body weight of chlorpyrifos, acetamiprid, lambda-cyhalothrin, and a mixture of all three pesticides (group 5), respectively. After 36 days of exposure, the groups that had been exposed to the pesticide showed a significant (p < 0.05) increase in both the total number of platelets and the number of white blood cells (WBCs), in comparison to the control group. On the other hand, the groups that were exposed to the insecticides had significantly lower levels of red blood cells (RBCs), hemoglobin (Hb), and packed cell volume (PCV) (p < 0.05). The value of mean corpuscular volume (MCV) was significantly (p < 0.05) reduced in acetamiprid-exposed group, while a significant increase was observed in other pesticide-exposed groups. Obvious histopathological changes were observed in the tissues of control group and no such changes were reported by control group. Necrosis, pyknosis, lymphocyte infiltration, congestion of blood, dissolution of plasma membrane, and vacuolation were observed in the livers of pesticide-treated pigeons. The intestinal study showed the formation of goblet cells, villi rupturing, degeneration of serosa, necrosis, and pyknosis in treated groups. Renal alterations, dilation of renal tubules, reduction of glomerulus tissue, and edema were observed. This study manifests that the uncontrolled use of pesticides impairs ecosystems and poses a substantial health risk to wildlife and ultimately to human.

Mathematical modelling of atrial and ventricular pressure-volume dynamics and their change with heart rate.

This paper presents mathematical models that can simulate the cardiovascular system of a healthy sheep under normal resting conditions in which the heart rate changes significantly. The models include several new modelling features that are introduced progressively. The contraction of the cardiac chambers is modelled using a time-dependent muscle force with constant elasticity instead of time dependent elasticity. A new hypothesis about the mechanical contraction of the atria generates realistic pressure volume (PV) loops. The inter-ventricular interaction is modelled as well. Additionally, hysteresis is incorporated in the aortic valve to produce an end-systolic reverse (negative) flow. Most of the model parameter values are based on previous literature data while time periods of delay, atrial and ventricular contraction are derived using experimental data from 14 sheep. We provide new relationships between contraction time and delay as a function of heart period. The effects of different aspects of our modelling on the mean cardiac output, stroke volume, ejection time, ejection fraction and PV loops are studied. Model outputs are compared with published experimental results where possible, and are within a wide range of physiological observations.

Immunoinformatics design of B and T-cell epitope-based SARS-CoV-2 peptide vaccination.

SARS-COV-2 is a virulent respiratory virus, first identified in China (Wuhan) at the end of 2019. Scientists and researchers are trying to find any possible solution to this deadly viral disease. Different drug source agents have been identified, including western medicine, natural products, and traditional Chinese medicine. They have the potential to counteract COVID-19. This virus immediately affects the liver and causes a decrease in oxygen levels. In this study, multiple vacciome approaches were employed for designing a multi-epitope subunit vaccine for battling against SARS-COV-2. Vaccine designing, immunogenicity, allergenic, and physico-chemical assessment were performed by using the vacciome approach. The vaccine design is likely to be antigenic and produce potent interactions with ACE2 and NSP3 receptors. The developed vaccine has also been given to in-silico cloning models and immune response predictions. A total number of 12 CTL and 12 HTL antigenic epitopes were predicted from three selected covid-19 virulent proteins (spike protein, nucleocapsid protein, and membrane proteins, respectively) based on C-terminal cleavage and MHC binding scores. These predicted epitopes were amalgamated by AYY and GPGPG linkers, and a ß-defensins adjuvant was inserted into the N-terminus of this vaccine. This analysis shows that the recommended vaccine can produce immune responses against SARS-COV-2. Designing and developing of the mentioned vaccine will require further experimental validation.

Morphological characteristics of native chicken of village Chhajjian, Haripur Pakistan.

The present study was conducted to describe the variations in morphological characteristics of different selected populations of indigenous chickens. Five populations of chickens in different (localities) of Chhajjian, KP, Pakistan, were studied based on qualitative traits recorded for a total of 100 chickens. Each of the study populations contains multiple variants of plumage colors and other physical features. The average flock size was observed to be 38. Predominant plumage color was grayish and other mixtures along with different percentages in different localities. Pea comb was the dominant comb type in all localities. Most of the chickens were yellow skinned. Males in all populations were heavier and taller than the females. This recorded variation in morphological traits will help in the conservation of these chickens.

Dynamics and conformational propensities of staphylococcal CntA.

Enzymes use transition metals as co-factors for catalytic roles in biological processes. Notably, manganese, iron, cobalt, nickel, copper and zinc are abundantly used. Staphylococcus aureus, a commensal bacterium asymptomatically, lies on the human body causing variety of infections. S. aureus is equipped by advanced virulence-regulatory circuits of metal acquisition like Cnt that acquires metals at infection sites by utilizing a nicotianamine-like metallophore staphylopine. Despite significant growth in structural studies, how CntA of Cnt system transmits conformational signal upon staphylopine recognition remains elusive. Here, we analyzed the structural changes adopted by CntA during close-to-open transition by computational approaches. CntA uses a bi-domain architectural form of domain II which performed 37° rigid body rotation and 1.1 Å translation assisted by inter-domain hinge cluster residues. Important clustered communities were found regulating the conformational changes in CntA where communities 4 and 5 are found crucial. Besides open and close states, the fluctuating regions sampled two additional intermediate states which were considered close or open previously. CntA prefers fluctuating the non-conserved regions rather than conserved where domain II turned out to be rigid and maintains a stable fold. Overall, the CntA system is a potential target for structural biologist to hamper such conformational behaviors at family level.Communicated by Ramaswamy H. Sarma.

Microbiological quality control of some non-sterile preparations commonly used in Pakistan.

The quality of medicines in some developing countries including Pakistan is not very satisfactory regarding safety and efficacy. In addition to sterile preparations, the microbial contamination of non sterile preparations should also be monitored according to USP microbial limits for these preparations. This study was designed to check the microbial quality of some commonly used non-sterile preparations available in Pakistan. Total 133 samples containing national and multinational brands of different dosage forms were collected from retail setups of Sargodha, Khushab and Chakwal cities. The total aerobic bacterial count and fungal contamination was tested by pour plate method. The absence of objectionable microorganisms was confirmed by using selective media, biochemical testing and microscopy. Bioburden of these preparations was also tested after a storage period of six months. The bio burden varied among all the selected non-sterile preparations whether of local or multinational brands. The highest load was observed in syrups, among which syrup number 1 showed maximum aerobic count (8.4×106). Lowest count was observed in tablets, among which tablet preparation number 1 contained 1.5×103aerobic bacteria. Creams and capsules produced no recovered bacteria. The fungal contaminants were also observed in all dosage forms except tablets. The isolated organisms included Gramnegative bacteria which contained objectionable ones such as Salmonella, Shigella, Pseudomonas and E.coli and some airborne moulds including Aspergillus spp., Penicillium spp., Fusarium spp. and Acremonium spp. Several measures such as GMPs, monitoring programs and SOPs should be followed by the pharmaceutical companies to reduce the microbial contamination level in the non sterile preparations. The regulatory agencies have to implement strict analysis strategy to check the microbial quality of the medicines before their release for sale in the market.

Bioaccumulation of Heavy Metals in Water, Sediments, and Tissues and Their Histopathological Effects on Anodonta cygnea (Linea, 1876) in Kabul River, Khyber Pakhtunkhwa, Pakistan.

The present investigation aimed to assess the concentrations of selected heavy metals in water and sediments and their bioaccumulation in tissues of freshwater mussels and their histopathological effects on the digestive gland, gills, and gonads of Anodonta cygnea. Water, sediments, and freshwater mussel samples were collected at four sites, that is, reference and polluted sites, along the Kabul River, Khyber Pakhtunkhwa. The polluted sites were receiving effluents from the industrial, agricultural, municipal, and domestic sources. The order of metals in the water was Zn > Pb > Ni > Cu > Mn > Fe > Cr > Cd, in sediments the order was Fe > Zn > Cr > Ni > Mn > Pb > Cu > Cd, and in the soft tissues the order was Fe > Zn > Mn > Pb > Cu > Cr > Ni > Cd. Histopathological alterations observed in polluted sites of Kabul River were inflammation, hydropic vacuolation, and lipofuscin pigments (in digestive gland), gill lamellar fusion, dilated hemolymphatic sinus, clumping, and generation of cilia and hemocytic infiltration (in gills), and atresia, necrosis, granulocytoma, hemocytic infiltration, and lipofuscin pigments (in gonads). The histopathological alterations in the organs of Anodonta cygnea can be considered as reliable biomarkers in biomonitoring of heavy metal pollution in aquatic ecosystems.

Interspecific studies of circadian genes period and timeless in Drosophila.

The level of rescue of clock function in genetically arrhythmic Drosophila melanogaster hosts using interspecific clock gene transformation was used to study the putative intermolecular coevolution between interacting clock proteins. Among them PER and TIM are the two important negative regulators of the circadian clock feedback loop. We transformed either the D. pseudoobscura per or tim transgenes into the corresponding arrhythmic D. melanogaster mutant (per01 or tim01) and observed >50% rhythmicity but the period of activity rhythm was either longer (D. pseudoobscura-per) or shorter than 24 h (D. pseudoobscura-tim) compared to controls. By introducing both transgenes simultaneously into double mutants, we observed that the period of the activity rhythm was rescued by the pair of hemizygous transgenes (~24 h). These flies also showed a more optimal level of temperature compensation for the period. Under LD 12:12 these flies have a D. pseudoobscura like activity profile with the absence of morning anticipation as well as a very prominent earlier evening peak of activity rhythm. These observation are consistent with the view that TIM and PER form a heterospecific coevolved module at least for the circadian period of activity rhythms. However the strength of rhythmicity was reduced by having both transgenes present, so while evidence for a coevolution between PER and TIM is observed for some characters it is not for others.

Identification and characterization of mobile genetic elements LINEs from Brassica genome.

Among transposable elements (TEs), the LTR retrotransposons are abundant followed by non-LTR retrotransposons in plant genomes, the lateral being represented by LINEs and SINEs. Computational and molecular approaches were used for the characterization of Brassica LINEs, their diversity and phylogenetic relationships. Four autonomous and four non-autonomous LINE families were identified and characterized from Brassica. Most of the autonomous LINEs displayed two open reading frames, ORF1 and ORF2, where ORF1 is a gag protein domain, while ORF2 encodes endonuclease (EN) and a reverse transcriptase (RT). Three of four families encoded an additional RNase H (RH) domain in pol gene common to 'R' and 'I' type of LINEs. The PCR analyses based on LINEs RT fragments indicate their high diversity and widespread occurrence in tested 40 Brassica cultivars. Database searches revealed the homology in LINE sequences in closely related genera Arabidopsis indicating their origin from common ancestors predating their separation. The alignment of 58 LINEs RT sequences from Brassica, Arabidopsis and other plants depicted 4 conserved domains (domain II-V) showing similarity to previously detected domains. Based on RT alignment of Brassica and 3 known LINEs from monocots, Brassicaceae LINEs clustered in separate clade, further resolving 4 Brassica-Arabidopsis specific families in 2 sub-clades. High similarities were observed in RT sequences in the members of same family, while low homology was detected in members across the families. The investigation led to the characterization of Brassica specific LINE families and their diversity across Brassica species and their cultivars.

The landscape and structural diversity of LTR retrotransposons in Musa genome.

Long terminal repeat retrotransposons represent a major component of plant genomes and act as drivers of genome evolution and diversity. Musa is an important fruit crop and also used as a starchy vegetable in many countries. BAC sequence analysis by dot plot was employed to investigate the LTR retrotransposons from Musa genomes. Fifty intact LTR retrotransposons from selected Musa BACs were identified by dot plot analysis and further BLASTN searches retrieved 153 intact copies, 61 truncated, and a great number of partial copies/remnants from GenBank database. LARD-like elements were also identified with several copies dispersed among the Musa genotypes. The predominant elements were the LTR retrotransposons Copia and Gypsy, while Caulimoviridae (pararetrovirus) were rare in the Musa genome. PCR amplification of reverse transcriptase (RT) sequences revealed their abundance in almost all tested Musa accessions and their ancient nature before the divergence of Musa species. The phylogenetic analysis based on RT sequences of Musa and other retrotransposons clustered them into Gypsy, Caulimoviridae, and Copia lineages. Most of the Musa-related elements clustered in their respective groups, while some grouped with other elements indicating homologous sequences. The present work will be helpful to understand the LTR retrotransposons landscape, giving a complete picture of the nature of the elements, their structural features, annotation, and evolutionary dynamics in the Musa genome.

Evolutionary genomics of miniature inverted-repeat transposable elements (MITEs) in Brassica.

Miniature inverted-repeat transposable elements (MITEs) are truncated derivatives of autonomous DNA transposons, and are dispersed abundantly in most eukaryotic genomes. We aimed to characterize various MITEs families in Brassica in terms of their presence, sequence characteristics and evolutionary activity. Dot plot analyses involving comparison of homoeologous bacterial artificial chromosome (BAC) sequences allowed identification of 15 novel families of mobile MITEs. Of which, 5 were Stowaway-like with TA Target Site Duplications (TSDs), 4 Tourist-like with TAA/TTA TSDs, 5 Mutator-like with 9-10 bp TSDs and 1 novel MITE (BoXMITE1) flanked by 3 bp TSDs. Our data suggested that there are about 30,000 MITE-related sequences in Brassica rapa and B. oleracea genomes. In situ hybridization showed one abundant family was dispersed in the A-genome, while another was located near 45S rDNA sites. PCR analysis using primers flanking sequences of MITE elements detected MITE insertion polymorphisms between and within the three Brassica (AA, BB, CC) genomes, with many insertions being specific to single genomes and others showing evidence of more recent evolutionary insertions. Our BAC sequence comparison strategy enables identification of evolutionarily active MITEs with no prior knowledge of MITE sequences. The details of MITE families reported in Brassica enable their identification, characterization and annotation. Insertion polymorphisms of MITEs and their transposition activity indicated important mechanism of genome evolution and diversification. MITE families derived from known Mariner, Harbinger and Mutator DNA transposons were discovered, as well as some novel structures. The identification of Brassica MITEs will have broad applications in Brassica genomics, breeding, hybridization and phylogeny through their use as DNA markers.

Ethno veterinary uses of medicinal plants of district Karak, Pakistan.

ETHNOPHARMACOLOGICAL RELEVANCE: In the study area, the traditional knowledge regarding the uses of local wild medicinal plants for treating diseases of domestic animals and birds is totally in the custody of elders of the existing community. The young ones are not much aware about such important practices. AIM OF THE STUDY: The main aim of the study was to document and to release this knowledge from the custody of elders and share with the community. MATERIALS AND METHODS: Total 115 people between 20 and 80 years of age were interviewed and information was collected through semi-structured questionnaires. The data obtained were quantitatively analyzed using the use value (UV) formula. The collected specimens were pressed, dried, preserved, mounted on Herbarium sheets, identified properly and were submitted in the Herbarium, Department of Botany, Hazara University, Mansehra, Pakistan. RESULTS: With the co-ordination and cooperation of the local people, 46 plant species of 42 genera belonging to 31 families were collected, 3 were monocotyledons while 43 plant species belonged to dicotyledonae class. Considering taxonomic characteristics, it was confirmed that 12 trees, 10 shrubs and 22 herbs were commonly used by the local people in ethno veterinary practices. Two plants like Cistanche tubulosa and Cuscuta reflexa from family Orobanchaceae and family Cuscutaceae respectively lack chlorophyll and are parasites on host plants like Doedonia, Ziziphus, Calligonum and Calotropis. The powder of both plants showed great ethno veterinary value. The parts of 46 plant species commonly used for ethno veterinary practices were whole plants (32.60%), leaves (26.08%), fruits (17.39%), stems (13.04%) and roots (10.86%). Medicinal plants were administered through various routes i.e. oral (78.26%), skin (17.21%) and smoke (4.34%). CONCLUSION: The traditional knowledge of local plants of ethno veterinary values is mainly possessed by elders and transmitted from generation to generation with chances of elimination of such traditional knowledge due to less awareness. The present study was designed to document this ethno veterinary related knowledge and to share it with community members for use in future.

Population dynamics of mosquitoes and malaria vector incrimination in district Charsadda, Khyber Pakhtunkhwa (KP) Pakistan.

This study aimed to obtain updated information on mosquito diversity and malaria vector incrimination in Charsadda Khyber Pakhtunkhwa to help in devising effective control and preventive measures in the area. Monthly survey of indoor mosquitoes for one year was carried out in three villages, Dhaki Totaqi and Mathra. Female Anopheline were used to detect Circumsporozoites protein (CSP) using Enzyme Linked Immunosorbent Assay. Among 17 mosquito species, Culex quinquefasciatus, Anopheles splendidus, Anopheles stephensi, Anopheles fluviatilus, Anopheles culicifacies and Culex tritaeniorhynchus were predominant. Dhaki village had the highest mosquito species diversity (1.015) and similar species richness (0.7) and evenness (0.5) with village Mathra. Slide positivity rate (SPR) shows that the rate of malaria transmission increases with mosquito population. Four anopheline species i.e. A. stephensi, A. fluviatilis, A. splendidus, and A. culicifacies were CSP positive. The CSP rate was 0.8%, where two specimens of A. splendidus and one of A. fluviatilus were positive for Plasmodium falciparum, Plasmodium vivax was represented by two variants 210 and 247. Among the 20 CSP positive specimens, variant 210 was found in 12 (one belonging to A. culicifacies, two each of A. stephensi and A. fluviatilus, seven specimens of A. splendidus) and 247 in 8 specimens (two of A. stephensi, three each of A. fluviatilus and A. splendidus). The number of infected mosquitoes collected from animal sheds was higher (15) though non-significant (P>0.05) than that from bedrooms (8).

Molecular evolution of a pervasive natural amino-acid substitution in Drosophila cryptochrome.

Genetic variations in circadian clock genes may serve as molecular adaptations, allowing populations to adapt to local environments. Here, we carried out a survey of genetic variation in Drosophila cryptochrome (cry), the fly's dedicated circadian photoreceptor. An initial screen of 10 European cry alleles revealed substantial variation, including seven non-synonymous changes. The SNP frequency spectra and the excessive linkage disequilibrium in this locus suggested that this variation is maintained by natural selection. We focused on a non-conservative SNP involving a leucine-histidine replacement (L232H) and found that this polymorphism is common, with both alleles at intermediate frequencies across 27 populations surveyed in Europe, irrespective of latitude. Remarkably, we were able to reproduce this natural observation in the laboratory using replicate population cages where the minor allele frequency was initially set to 10%. Within 20 generations, the two allelic variants converged to approximately equal frequencies. Further experiments using congenic strains, showed that this SNP has a phenotypic impact, with variants showing significantly different eclosion profiles. At the long term, these phase differences in eclosion may contribute to genetic differentiation among individuals, and shape the evolution of wild populations.