The mutational analysis of mitochondrial DNA in maternal inheritance of polycystic ovarian syndrome.

Introduction: Polycystic Ovarian Syndrome (PCOS) is a globally prevalent condition that leads to infertility in women. While environmental factors contribute to PCOS, maternal genetics also play a significant role. Currently, there is no definitive test for identifying predisposition to PCOS. Hence, our objective is to discover novel maternal genetic risk factors for PCOS by investigating the genomes of patients from Pakistan. Methods: We utilized Next-Generation Sequencing (NGS) to sequence the complete mitochondrial DNA of three PCOS patients. Subsequently, we employed MitoTIP (Mitochondrial tRNA Informatics Predictor) and PON-mt-tRNA tools to identify variations in the mitochondrial DNA. Our analysis focused on the genes MT-RNR1, MT-RNR2, MT-ATP6, MT-TL2, and MT-CYTB, which displayed common variations in all three genomes. Additionally, we observed individual variations. The D-loop region exhibited the highest frequency of mutations, followed by the non-coding regions of RNR1 and RNR2 genes. Moreover, we detected frameshift mutations in the mitochondrially encoded NADH Dehydrogenase 2 (MT-ND2) and mitochondrially encoded NADH Dehydrogenase 5 (ND5) genes within individual genomes. Results: Our analysis unveiled six regions with common variations in the mitochondrial DNA of all three PCOS patients. Notably, the MT-RNR1, MT-RNR2, MT-ATP6, MT-TL2, and MT-CYTB genes exhibited these variations. Additionally, we identified individual variations in the mitochondrial DNA. The D-loop region displayed the highest mutation frequency, followed by the non-coding regions of RNR1 and RNR2 genes. Furthermore, frameshift mutations were detected in the MT-ND2 and ND5 genes within individual genomes. Conclusion: Through our study, we have identified variations in mitochondrial DNA that may be associated with the development of PCOS and have the potential to serve as predisposition tests. Our findings highlight the presence of novel mutations in the MT-RNR1, MT-RNR2, MT-ATP6, MT-TL2, and MT-CYTB genes, as well as frameshift mutations in the MT-ND2 and ND5 genes. Pathogenicity analysis indicated that most variants were likely to result in benign cysts. However, the frameshift mutations in the ND2 gene were associated with a high risk of complications and pathogenicity in PCOS. This is the first report identifying these mutations and their association with PCOS, contributing to our understanding of the genetic factors underlying the condition.

TP53 and its Regulatory Genes as Prognosis of Cutaneous Melanoma.

The present study was the first comprehensive investigation of genetic mutation and expression levels of the p53 signaling genes in cutaneous melanoma through various genetic databases providing large datasets. The mutational landscape of p53 and its signaling genes was higher than expected, with TP53 followed by CDKN2A being the most mutated gene in cutaneous melanoma. Furthermore, the expression analysis showed that TP53, MDM2, CDKN2A, and TP53BP1 were overexpressed, while MDM4 and CDKN2B were under-expressed in cutaneous melanoma. Overall, TCGA data revealed that among all the other p53 signaling proteins, CDKN2A was significantly higher in both sun and non-sun-exposed healthy tissues than in melanoma. Likewise, MDM4 and TP53BP1 expressions were markedly greater in non-sun-exposed healthy tissues compared to other groups. However, CDKN2B expression was higher in the sun-exposed healthy tissues than in other tissues. In addition, various genes were expressed significantly differently among males and females. In addition, CDKN2A was highly expressed in the SK-MEL-30 skin cancer cell line, whereas, Immune cell type expression analysis revealed that the MDM4 was highly expressed in naïve B-cells. Furthermore, all six genes were significantly overexpressed in extraordinarily overweight or obese tumor tissues compared to healthy tissues. MDM2 expression and tumor stage were closely related. There were differences in gene expression across patient age groups and positive nodal status. TP53 showed a positive correlation with B cells, MDM2 with CD8+T cells, macrophages and neutrophils, and MDM4 with neutrophils. CDKN2A/B had a non-significant correlation with all six types of immune cells. However, TP53BP1 was positively correlated with all five types of immune cells except B cells. Only TP53, MDM2, and CDKN2A had a role in cutaneous melanoma-specific tumor immunity. All TP53 and its regulating genes may be predictive for prognosis. The results of the present study need to be validated through future screening, in vivo, and in vitro studies.

Stress Induced Cortisol Release Depresses The Secretion of Testosterone in Patients With Type 2 Diabetes Mellitus.

Background: Both hormonal and genetic data reveal that the stress hormone cortisol and its regulating genes may affect the level of testosterone in humans. It is uncertain whether type 2 diabetes mellitus would manifest similarly. Furthermore, a genetic strategy to screen out the stress system genes that may contribute to testosterone decline in humans is less understood. Objectives: In this study, we aimed to elucidate the link between stress and testosterone levels, both hormonally and genetically. Method: This study comprised 37 individuals with type 2 diabetes mellitus and 50 healthy individuals. For the analysis of hormones and the targeted genes, we used the RIA system and bioinformatics expertise. Results: The patients had significantly elevated cortisol and lower testosterone readings, according to data from hormonal analyses. The bioinformatics approach reveals that SHBG was intracellularly suppressed by 2 defined stress system genes: FKB5 and CYP17. TCF4/TCF8, ATRX, and AR in skeletal muscle were inversely related to stress system genes. Furthermore, all testosterone regulated genes were positively linked with SHBG in the current study. A strong relationship between GNAS and PKA with CYP17 and FKBP5 reveals that the Gαs-cAMP/PKA signaling pathway may be one of the regulatory pathways through which the suppression of testosterone system genes happens. In conclusion, this study demonstrated that beyond stress, the key stress system genes might affect cortisol levels, which in turn affect testosterone figures via the Gαs-cAMP/PKA signaling pathway.

Molecular Characterization and Phylogenetic Analysis of Earthworm Species Collected from Different Soil Habitats of Poonch Division Azad Jammu and Kashmir Pakistan.

This study aims to analyze molecular characterization and phylogenetic analysis of earthworm species collected from different soil habitats of Poonch division Azad Kashmir Pakistan by using CO1 gene partial sequencing methodology. Samples gathered randomly from 18 study sites (127 localities) by digging and hand sorting methods were preserved in pure ethanol at -20°C. The modified CTAB (Cetyltrimethyl ammonium bromide) method extracted high quality DNA from region of representative earthworm's caudal region. This extracted DNA was used to amplify the 700 bp partial region of the cytochrome oxidase I (COI) gene with LCO1490 and HCO2198 universal primers. All of the obtained amplified gene sequences were aligned, edited and analyzed using MEGA X software to characterize different species of earthworms. Thirty-eight (38) Barcoding sequences belonging to 11 different strains of earthworms were successfully generated. Their phylogenetic analysis revealed that 7 Barcoding sequences gave maximum similarity with the available online database, while the rest of the 4 sequences gave lower similarity than the maximum threshold level. The collected DNA barcode sequences were also clustered together by the maximum likelihood method and the resultant phylogenetic tree revealed they belong to different family lineages. Moreover the identified earthworm species have a close evolutionary link with the earthworm fauna of south and central Asia instead of Europe, which might be due to similar climate of both regions.

Molecular characterization of plant growth-promoting vermi-bacteria associated with Eisenia fetida gastrointestinal tract.

Earthworms are highly productive invertebrates and play a vital role in organic farming and improving soil structure and function. The gastrointestinal tract of earthworms possessed agricultural important bacteria. So, the current research aimed was to examine, screen, and identify the plant growth promoting bacteria existing in the digestive tract of Eisenia fetida called plant growth promoting vermi-bacteria. The plant growth promoting traits such as siderophore, phytohormone, and hydrolytic enzymes production, and phosphate solubiliation were assessed. Eleven vermi-bacteria i.e. Bacillus mycoides, B. aryabhattai, B. megaterium, Staphylococcus hominis, B. subtilis, B. spizizenii, B. licheniformis, B. mojavensis, B. toyonensis, B. anthracis, B. cereus, B. thuringiensis, and B. paranthracis were isolated and identified based on microscopic studies, biochemical tests, ribotyping, and agricultural traits. All vermi-bacteria are Gram-positive rods except Staphylococcus hominis and produce different compounds such as siderophore, indole acetic acid, catalase, oxidase, proteases, amylases, and lipases. All vermi-bacteria also act as phosphate solubilizers. Therefore, all isolated vermi-bacteria could be used as potential microbial biofertilizers to enhance crops production in Pakistan.

Toxicological impact of Zinc Nano Particles on tilapia fish (Oreochromis mossambicus).

In this study we investigated the acute toxicity of Zinc Nano Particles (ZnO NPs) and bulk ZnO on tilapia fish (Oreochromis mossambicus). Oreochromis mossambicus was exposed to the different concentration of ZnO NPs, ZnO and mixed solution of both ZnO NPs and ZnO (20 ppb, 20 ppb, 20 ppb) respectively for 96 h. A very high impact was recorded in hematological parameters which shows significant increased (p < 0.05) in count of white WBCs and platelets in all the experimental groups compared tocontrol group. The count of RBCs, Hb, hematocrit and MCHC were significantly decreased. The remarkable changes which were recorded during this study were histopathological lesions in the gills of exposed fish including, disorganization of gill lamella, cartilaginous core disruption, lifting of epithelium, loss of secondary gill lamellae, blood congestion, fusion of secondary gills lamellae, shortening of secondary gills lamellae, atrophy and curling. Disassembly were seen in plasma membrane of liver along with blood congestion, pyknosis, necrosis, hyperplasia and formation of vacuoles. Intestinal alterations which were observed include shortening of villi, necrosis, detachment and fusion of villi and extreme goblet cells formation. It is concluded from the present study that high level of ZnO NPs, ZnO and mixed solution has a strong tendency to alter hematological parameters, histological architecture, therefore, the indiscriminate use of ZnO NPs and ZnO can subsidize in reducing the population of Oreochromis mossambicus in natural water bodies.

A cross-sectional survey of hard ticks and molecular characterization of Rhipicephalus microplus parasitizing domestic animals of Khyber Pakhtunkhwa, Pakistan.

BACKGROUND: In tropical and subtropical countries, tick infestation causes major public health problems and considerable financial losses to the livestock industry. This study was aimed to assess the species composition of richness and analyze the phylogeny of Rhipicephalus microplus in the District Bannu of Khyber Pakhtunkhwa, Pakistan. METHODS: Collected ticks were identified morphologically and DNA extracted from R. microplus was amplified and subjected to sequencing. RESULTS: A total of 3,600 animals were examined among them 1,494 animals were found to be infested with ticks, including 669 cows, 476 buffaloes, 163 goats, and 186 sheep (p = 0.001). Tick infestation was significantly high (43.58%) in animals of age group (<1 year) (p-value = 0.027). Female animals were more (44.05%) infested with ticks than males (34.43%) (p = 0.001). The intensity of infestation was significantly higher in summer (77.49%) (p = 0.001). A total of 5,557 ticks were collected comprising three genera and six species. R. microplus was predominantly prevalent (n = 1,474; 26.52%), followed by Rhipicephalus annulatus (n = 1,215; 21.86%), Hyalomma anatolicum (n = 1,139; 20.49%), Hyalomma marginatum (n = 1,086; 19.54%), and Rhipicephalus turanicus (n = 761; 13.69%), while the least common was Haemaphysalis aciculifer (n = 80; 1.43%) (p = 0.001). Morphologically identified R. microplus species were also analyzed genetically by using two genetic markers 16S ribosomal RNA (16S rRNA) and internal transcribed spacer 2 (ITS2) genes. The phylogenetic study revealed that R. microplus is genetically diversified and clustered in clade B with R. microplus species from China, India, and Pakistan. CONCLUSION: Ticks infestation was significantly correlated with various factors including age, sex, season, and animal type. R. microplus genetically resembled species reported from India and China. However, major knowledge gaps concerning various species of ticks exist and many areas are still unexplored in Pakistan. Therefore, it is necessary to explore the epidemiological and molecular aspects of various tick species in other regions of southern Khyber Pakhtunkhwa.

Albendazole and mebendazole in the treatment of ancylostomiasis in school children between the ages of 6-15 in Swat, Pakistan.

A cross-sectional study was conducted from March to September, 2018 on the efficacies of albendazole and mebendazole against ancylostomiasis in school children of district Swat, Pakistan. Faecal samples were collected from primary school children and preserved in 10% formalin. The samples were then sent to the Laboratory of Parasitology, in the University of Malakand for microscopic analysis. On the basis of drug availability, the Ancylostoma dueodenale infected students were divided into two groups. Group A was treated with Albendazole 400-450mg while group B was orally treated with Mebendazole 350-400mg. Eggs per gram were calculated before and after the treatment. From the total sample of 296, 192 (64.8%) children were found infected with Ancylostoma duodenale. Of the total number of infected children, this study found 87.8% (n=137/156)of them with light intensity of infection, 10.8%(n=17/156) with moderate and 1.2% (n=2/156) with heavy intensity of infection. Albendazole showed a high rate 75% of efficacy than mebendazole 71% (p<0.05). The present study concluded that albendazole and mebendazole are drugs of choice for the treatment of Ancylostomiasis.

Natural Polyphyllins (I, II, D, VI, VII) Reverses Cancer Through Apoptosis, Autophagy, Mitophagy, Inflammation, and Necroptosis.

Cancer is the second leading cause of mortality worldwide. Conventional therapies, including surgery, radiation, and chemotherapy, have limited success because of secondary resistance. Therefore, safe, non-resistant, less toxic, and convenient drugs are urgently required. Natural products (NPs), primarily sourced from medicinal plants, are ideal for cancer treatment because of their low toxicity and high success. NPs cure cancer by regulating different pathways, such as PI3K/AKT/mTOR, ER stress, JNK, Wnt, STAT3, MAPKs, NF-kB, MEK-ERK, inflammation, oxidative stress, apoptosis, autophagy, mitophagy, and necroptosis. Among the NPs, steroid saponins, including polyphyllins (I, II, D, VI, and VII), have potent pharmacological, analgesic, and anticancer activities for the induction of cytotoxicity. Recent research has demonstrated that polyphyllins (PPs) possess potent effects against different cancers through apoptosis, autophagy, inflammation, and necroptosis. This review summarizes the available studies on PPs against cancer to provide a basis for future research.

A comparative study on the effects of selected pesticides on hemato-biochemistry and tissue histology of freshwater fish Cirrhinus mrigala (Hamilton, 1822).

The aim of the present study was to investigate the comparative effects of pesticides Chlorfenapyr, Dimethoate and Acetamiprid on the health of Cirrhinus mrigala under long term exposure. Eighty C. mrigala were divided in four equal groups; one control and three treated groups. The blood was collected from both control and treated groups at intervals of 10th, 20th and 30th days for hemato-biochemistry and histopathological alterations. The result indicates significant difference (P < 0.05) in RBCs, Hb, PCV and MCHC whereas elevation in WBCs and Platelets counts were recorded. In 10th day sampling, MCV value of Dimethoate and Acetamiprid treatment had no difference in comparison with the control group, however it is significantly increased (P < 0.05) in rest of sampling. The MCH value of exposed fish showed significant increased (P < 0.05) after 20th and 30th days for Chlorfenapyr and after 30th days for Acetamiprid exposure while insignificantly increased for rest of sampling. It was also found that these pesticides significantly decrease (p < 0.05) the T3 and T4 levels while increase in the TSH, cortical, ALP, AST, ALT and LDH levels in the serum of the treated fishes in contrast to control group. Similarly, histopathological analysis of gills and liver showed significant alterations in all the treated groups. Toxicity trends of these pesticides was ranked as Chlorfenapyr > Acetamiprid > Dimethoate. It is concluded that indiscriminate use of such pesticides poses a noxious threat to non-target organisms, harm the ecosystems and jeopardizes human health.

Molecular mechanisms of anticancer activities of polyphyllin VII.

Cancer is the leading cause of mortality in the world. The major therapies for cancer treatment are chemotherapy, surgery, and radiation therapy. All these therapies expensive, toxic and show resistance. The plant-derived compounds are considered safe, cost-effective and target cancer through different pathways. In these pathways include oxidative stress, mitochondrial dependent and independent, STAT3, NF-kB, MAPKs, cell cycle, and autophagy pathways. One of the new plants derived compounds is Polyphyllin VII (PPVII), which target cancer through different molecular mechanisms. In literature, there is a review gap of studies on PPVII; therefore in the current review, we summarized the available studies on PPVII to provide a base for future research.

Development of DNA barcodes for selected Acacia species by using rbcL and matK DNA markers.

Acacia species are very important tree species in tropical and subtropical countries of the World for their economic and medicinal benefits. Precise identification of Acacia is very important to distinguish the invasive species from rare species however, it is difficult to differentiate Acacia species based on morphological charcters. In addition, precise identification is also important for wood charcterization in the forest industry as these species are declining due to illegal logging and deforestation. To overcome thsese limitations of morphological identification, DNA barcoding is being used as an efficient and quick approach for precise identification of tree species. In this study, we selected two chloroplast and plastid base DNA markers (rbcL and matK) for the identification of five selected tree species of Acacia (A. albida, A. ampliceps, A. catechu, A. coriacea and A. tortilis). The genomic DNA of the selected Acacia species was extracted, amplified through PCR using specific primers and subsequently sequenced through Sanger sequencing. In matK DNA marker the average AT nucleotide contents were higher (59.46%) and GC contents were lower (40.44%) as compared to the AT (55.40%) and GC content (44.54%) in rbcL marker. The means genetic distance K2P between the Acacia species was higher in matK (0.704%) as compared to rbcL (0.230%). All Acacia species could be identified based on unique SNPs profile. Based on SNP data profiles, DNA sequence based scannable QR codes were developed for accurate identification of Acacia species. The phylogenetic analysis based on both markers (rbcL and matK) showed that both A. coriacea and A. tortilis were closely related with each other and clustered in the same group while other two species A. albida and A. catechu were grouped together. The specie A. ampliceps remained ungrouped distantly, compared with other four species. These finding highlights the potential of DNA barcoding for efficient and reproducible identification of Acacia species.

The complete mitochondrial genome of blue pansy, Junonia orithya (Lepidoptera: Nymphalidae: Nymphalinae) from Pakistan.

Junonia orithya's complete mitochondrial genome (mitogenome) is determined to be 14,214 bp in length, including 37 typical mitochondrial genes and an AT-rich region. Its gene order and orientation are identical to those of other butterfly species. All PCGs are initiated by typical ATN codons, except for cox1 gene which is started by CGA codon. Nine genes use complete termination codon (TAA), whereas the COX1, COX2, NADH1 and NAH4 genes end with single T. Except for trnS1(AGN), all tRNA genes display typical secondary cloverleaf structures as those of other insects. The 331 bp long AT-rich region contains several features common to the other lepidopterans, such as the ATAGA motif followed by a 18 bp poly-T stretch, two microsatellite-like (TA) 9 elements, a 5 bp poly-A stretch immediately upstream of trNAM gene from Pakistan.

Phylogenetic association of Cyprinid (Schizothorax) inferred from complete mitochondrial genome.

Whole mitochondrial DNA (mtDNA) of S.esocinusand S. plagiostomus was found to be 16,591 and 16,564 bp respectively with 13 protein-coding genes, 2 rRNAs genes, 22 tRNA genes and 2 non-coding region. For valuing the phylogenetic relationship of a species on the basis of whole mitogenome are considered to be a great importance. In this study, we sequenced whole mitogenome of S.esocinusand S. plagiostomus and compared with the whole mitogenome sequences of members of other families (Balitoridae, Nemanchillidae and Cobatidae). The monophyly of the family Cyprinidae and a clade comprising Balitoridae and Nemanchillidae while Cobatidae with paraphyletic origin was strongly supported by the resultant phylogenies, recognized that Cyprinidae was closely related with the family Cobatidae than other ones. The results indicated that whole mitochondrial genome has a great importance in studying variation in genes and phylogenetic relationship in the subfamily Schizothoracinae. This data offering the molecular phylogenetic frame work of important Schizothorax species found in Swat.

Comparative phylogenetic analysis of Schizothorax plagiostomus and Schizothorax esocinus with other members of subfamilies of cyprinidae on the basis of complete mitochondrial genome and 12S, 16S ribosomal RNA from Northren areas of Pakistan.

We assessed the relationship of Schizothoracinae species with other subfamilies Alburninae, Xenocyprinae, Cultrinae and Squaliobarbinae of family Cyprinidae by creating the phylogenetic trees using complete mitogenome and 12S and 16S RNA. Our representative species show the great affiliation with other but separated from a group composed of Metzia mesembrinum, Metzia longinasus, Metzia lineata and Metzia formosae of subfamily Alburninae while other subfamilies formed distinct group. The members of subfamily Schizothoracinae shows separate line of evolution from subfamily Barbinae.

Comparative phylogeny of (Schizothorax esocinus) with reference to 12s and 16 sribosomal RNA from River Swat, Pakistan.

Schizothorax esocinus is one of the important cold water delicious fish belonging to family Cyprinidae and found in River Swat which is one of the main river flowing in Khyber Pakhtunkhwa, Pakistan.The complete mitochondrial genomes of the S. esocinus species was found to be 16,591bp. Phylogenetic analysis of S. esocinus based on 12 s ribosomal RNA and 16S rRNA confirmed that, the phylogenetic position of S. esocinus was slightly different, but clustered closed to S. plagiostomus, S. labiatus, S. richardsonii, S. nepalensis, and S. progastus predicting a close homology in genome and hence a monophyletic line of evolution also this specie showed close relationship and homologs to the Aspiorhynchus laticeps, Spinibarbus sinensis, Aspiorhynchus laticeps, and Percocypris respectively. This study provided phylogenetic relationship between Schizothoracinae fishes and with the other fishes of family cyprinidae even at the subfamily level.

Antileishmanial activity and cytotoxicity of ZnO-based nano-formulations.

INTRODUCTION: Nanoparticles (NPs) can be toxic due to their nano-range sizes. Zinc oxide (ZnO) has good biocompatibility and is commercially used in cosmetics. Moreover, ZnO NPs have potential biomedical uses, but their safety remains unclear. METHODS: A range of doped ZnO NPs was evaluated for antileishmanial activity and in vitro toxicity in brine shrimp and human macrophages, and N-doped ZnO NPs were evaluated for in vivo toxicity in male BALB/C mice. N-doped ZnO NPs were administered via two routes: intra-peritoneal injection and topically as a paste. The dosages were 10, 50, and 100 mg/kg/day for 14 days. RESULTS: Topical administration was safe at all dosages, but intra-peritoneal injection displayed toxicity at higher doses, namely, 50 and 100 mg/kg/day. The pathological results for the i.p. dose groups were mild to severe degenerative changes in parenchyma cells, increases in Kupffer cells, disappearance of hepatic plates, increases in cell size, ballooning, cytoplasmic changes, and nuclear pyknosis in the liver. Kidney histology was also altered in the i.p. administration group (dose 100 mg/kg/day), with inflammatory changes in the focal area. We associate pathological abnormalities with the presence of doped ZnO NPs at the diseased site, which was verified by PIXE analysis of the liver and kidney samples of the treated and untreated mice groups. CONCLUSION: The toxicity of the doped ZnO NPs can serve as an essential determinant for the effects of ZnO NPs on environmental toxicity and can be used for guidelines for safer use of ZnO-based nanomaterials in topical treatment of leishmaniasis and other biomedical applications.

Epidemiological and clinical correlates of oral squamous cell carcinoma in patients from north-west Pakistan.

OBJECTIVE: To investigate the association of oral squamous cell carcinoma with demographic variables and oral health indicators. . METHODS: The observational case-control study was conducted at the Institute of Radiotherapy and Nuclear Medicine, Peshawar, Pakistan, from November, 2015, to August, 2016.Pathologically confirmed patients with oral cancer represented the cases, while The controls were ethnically and linguistically-matched subjects without any oral pathology. Demographical, clinical and pathological data was taken down to assess variables, risk factors, and oral health indicators. Descriptive statistics and logistic regression modelling were employed for data analysis. RESULTS: Of the 551 subjects, 341(62%) were males, and 210(38%) were females. Of the total, 276(50.1%) were cases and 275(49.9%) were controls. The mean age of the cases was 55.0}13.4 years and that of the controls was 52.8}14.9 years (p=0.073). Poor oral hygiene, periodontal diseases, material of toothbrush, and no use of mouthwash were significant predictors of oral cancer (p<0.05 each). Smoking and using smokeless tobacco were also significant variables. CONCLUSIONS: Oral health indicators in combination with smoking conferred an increased risk of oral cancer.

Detection and Molecular Characterization of Staphylococci from Eggs of Household Chickens.

Eggs are a healthy and nutritious food source, but may be contaminated by bacteria. Previous studies have reported the presence of staphylococci in eggs of farmed chickens, but no study has evaluated the staphylococcal population of eggs from household chickens. In this study, staphylococci from eggs (n = 275) of household chickens collected from November 2016 to March 2017 from different villages of Khyber Pakhtunkhwa province, Pakistan, were characterized. Seven species of staphylococci were identified from 65 eggs, including the predominant species, Staphylococcus xylosus (49/275; 17.8%). S. xylosus isolates (n = 73) were tested for antimicrobial susceptibility, presence of resistance genes, genetic relatedness, and inhibitory activity against other bacteria. The majority of isolates were resistant to oxacillin (83.6%) and tetracycline (24.7%), but also exhibited resistance to daptomycin and linezolid (5.5% each). Of the 10 resistance genes tested, isolates were only positive for mecA (35.6%; 26/73), mecC/C1 (2.7%; 2/73), and tet(K) (14/73; 19%). Using pulsed-field gel electrophoresis (PFGE), nine clusters had identical PFGE patterns. Isolates produced inhibitory activity against a broad spectrum of bacteria; 20.5%, 19.2%, 17.8%, and 16.4% of S. xylosus were able to inhibit growth of Salmonella enterica serotype Typhi, methicillin-susceptible Staphylococcus aureus, Escherichia coli, and methicillin-resistant Staphylococcus aureus, respectively. This study demonstrated the presence of genetically related antimicrobial-resistant S. xylosus from eggs from household chickens. Like table eggs, eggs of household chickens also contain staphylococci that may be resistant to antimicrobials used to treat human infections. These data will allow comparison between staphylococci from eggs from different sources and may indicate the relative safety of eggs from household chickens. Further study of these egg types and their microbial composition is warranted.

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.