Investigation of NLR Genes Reveals Divergent Evolution on NLRome in Diploid and Polyploid Species in Genus Trifolium.

Crop wild relatives contain a greater variety of phenotypic and genotypic diversity compared to their domesticated counterparts. Trifolium crop species have limited genetic diversity to cope with biotic and abiotic stresses due to artificial selection for consumer preferences. Here, we investigated the distribution and evolution of nucleotide-binding site leucine-rich repeat receptor (NLR) genes in the genus of Trifolium with the objective to identify reference NLR genes. We identified 412, 350, 306, 389 and 241 NLR genes were identified from Trifolium. subterraneum, T. pratense, T. occidentale, subgenome-A of T. repens and subgenome-B of T. repens, respectively. Phylogenetic and clustering analysis reveals seven sub-groups in genus Trifolium. Specific subgroups such as G4-CNL, CCG10-CNL and TIR-CNL show distinct duplication patterns in specific species, which suggests subgroup duplications that are the hallmarks of their divergent evolution. Furthermore, our results strongly suggest the overall expansion of NLR repertoire in T. subterraneum is due to gene duplication events and birth of gene families after speciation. Moreover, the NLRome of the allopolyploid species T. repens has evolved asymmetrically, with the subgenome -A showing expansion, while the subgenome-B underwent contraction. These findings provide crucial background data for comprehending NLR evolution in the Fabaceae family and offer a more comprehensive analysis of NLR genes as disease resistance genes.

Organochlorines in the riverine ecosystem of Punjab province, Pakistan: contamination status, seasonal variation, source apportionment, and ecological risk assessment.

The aim of the present study was to determine the occurrence, spatio-temporal variations, source apportioning, and ecological risk assessment of selected PCBs and OCPs in surface water and sediments collected riverine environment of Punjab province, Pakistan. The concentration of ΣOCPs (water: 64-455 ng/L; sediments: 117-616 ng/g) and ΣPCBs (water: 2-132 ng/L; sediments: 3.27-200 ng/g) was found comparatively higher than the levels reported from other parts of the world. The higher concentrations of DDTs and HCHs were detected in both studied matrices, whereas among PCBs, CB-28, 49 and CB-37, 82 were dominant in water and sediments, respectively. The isomeric ratios including α-HCH/γ-HCH, (DDE + DDD) / DDTs, and α /ß-endosulfan reflected the recent use of lindane, technical DDT, and endosulfan in the study area. The WHO-TEQ values of DL-PCBs ranged from 3.6 × 10-6 to 0.115 ng/L and 8.7 × 10-6 to 0.157 ng/g in surface water and sediments in both seasons, respectively. The spatial variation analysis revealed that the sites in the industrial and agricultural zones were highly contaminated. The OCPs and PCBs fluxes to downstream areas were estimated to be 12.4 tons/year and 1.9 tons/year, respectively. The significant ecological risks were estimated to be posed by OCPs and PCBs, as their levels in 67% and 62% of surface water and sediment samples were exceeding the threshold limits, highlighting effects to ecological integrities.

Effect of aqueous garlic (Allium sativum) extract against di-(2-ethylhexyl) phthalate induced reproductive toxicity in male mice.

This study was designed to investigate testicular and male reproductive tract histopathologies and lipid profile against di-ethylhexyl phthalate (DEHP) exposure in mice and curative potentials of aqueous garlic (Allium sativum) extract. Four groups (n = 10) were named and treated as follow (a) control (C): (normal feed and drinking water + 0.2 ml corn oil); (b) aqueous garlic extract group (AGE): (500 mg/kg body weight of aqueous garlic extract); (c) DEHP group: (500 mg/kg body weight of DEHP, dissolved in corn oil; (d) AGE + DEHP group (500 mg/kg body weight garlic aqueous extract, and DEHP 500 mg/kg body weight dissolved in corn oil). The doses were given once daily through gavages for 28 days and on the 29th day, all the animals were euthanized through cervical dislocation and reproductive organs and blood samples were collected. The results showed that exposure to DEHP caused a significant effect on body weight, testicular weight, serum cholesterol, triglycerides, lipid profile, average cross-sectional area (ACSA) of the seminiferous tubule, ACSA of the lumen of seminiferous tubule, spermatogenic cells, Leydig's cells number, vas deferens diameter, lumen, muscular thickness, and epithelial cell height of vas deferens. This study revealed that exposure to DEHP can be injurious to male reproductive health and aqueous garlic extract can decrease the toxic effects of DEHP in male mice.

A preliminary assessment of microplastics in indoor dust of a developing country in South Asia.

Microplastics (MPs) pollution is an emerging global environmental concern. Considering the high fraction of time people spend indoors, the human population can be directly exposed to this contamination through indoor dust. This preliminary study evaluates MPs' abundance and human health risk assessment in the deposited indoor dust. A total of forty dust samples (n = 20) were collected from homes in two different cities (Pakistan) in steel mesh pouches using the vacuum cleaner. The identification and quantification of MPs were conducted with a stereo microscope, whereas the qualitative assessment was performed with Fourier transform infrared spectroscopy (FTIR). The US EPA parameters to calculate the human health risk assessment were used to determine MPs' risk per-day/month/year. Overall, microfibers were the dominant category, followed by microfilms, micro-fragments, and nurdles. The chemical categorization of MPs was revealed as polyester, polyethylene, copolymers of polypropylene, and polyurethane. In Lahore, an average abundance of 241.45 (items/m2) MPs were observed compared to Sahiwal, with 162.1 (items/m2). More than 90% of the identified MPs were microfibers, with higher detection frequency and abundance in Lahore than Sahiwal. The human health risk assessment revealed high exposure risk because of indoor MPs. Moreover, toddlers were more vulnerable as compared to adults at both low and high exposure risk scenarios. There is an imminent need to conduct in-depth risk assessment focusing on the respirable fraction of MPs.

Comparative mutational analysis of SARS-CoV-2 isolates from Pakistan and structural-functional implications using computational modelling and simulation approaches.

SARS-CoV-2, an RNA virus, has been prone to high mutations since its first emergence in Wuhan, China, and throughout its spread. Its genome has been sequenced continuously by many countries, including Pakistan, but the results vary. Understanding its genomic patterns and connecting them with phenotypic features will help in devising therapeutic strategies. Thus, in this study, we explored the mutation landscape of 250 Pakistani isolates of SARS-CoV-2 genomes to check the genome diversity and examine the impact of these mutations on protein stability and viral pathogenesis in comparison with a reference sequence (Wuhan NC 045512.2). Our results revealed that structural proteins mainly exhibit more mutations than others in the Pakistani isolates; in particular, the nucleocapsid protein is highly mutated. In comparison, the spike protein is the most mutated protein globally. Furthermore, nsp12 was found to be the most mutated NSP in the Pakistani isolates and worldwide. Regarding accessory proteins, ORF3A is the most mutated in the Pakistani isolates, whereas ORF8 is highly mutated in world isolates. These mutations decrease the structural stability of their proteins and alter different biological pathways. Molecular docking, the dissociation constant (KD), and MM/GBSA analysis showed that mutations in the S protein alter its binding with ACE2. The spike protein mutations D614G-S943T-V622F (-75.17 kcal/mol), D614G-Q677H (-75.78 kcal/mol), and N74K-D614G (-73.84 kcal/mol) exhibit stronger binding energy than the wild type (-66.34 kcal/mol), thus increasing infectivity. Furthermore, the simulation results strongly corroborated the predicted protein servers. Our analysis findings also showed that E, M, ORF6, ORF7A, ORF7B, and ORF10 are the most stable coding genes; they may be suitable targets for vaccine and drug development.

Evaluation of Anti-clotting and thrombolytic potential of the aqueous-methanolic extract of Jasminum sambac.

This study was done to investigate the anti-clotting and thrombolytic potential of ariel part of Jasminum sambac (L.). Healthy male rabbits were divided into five (5) groups with each group containing six (6) animals. Aqueous-methanolic extract of the plant was prepared and given at different doses of 200, 300 and 600mg/kg to three groups in comparison to negative and positive control groups. The aqueous-methanolic extract showed a dose-dependent increase in the activated partial thromboplastin (APTT) and prothrombin time (PT), bleeding time (BT) and clotting time (CT) (p<0.05). Warfarin 2mg/kg was used as the standard. The plant extract also showed significant (p<0.05) clot lysis in comparison with standard urokinase. Moreover, it also prolonged the ADP induced platelet adhesion at doses of 200, 300 and 600µg/mL dependently. HPLC analysis showed rutin, quercetin, salicylic acid and ascorbic acid as vital phytoconstituents in aqueous-methanolic extract. Anticoagulant and thrombolytic effect of Jasminum sambac justified its therapeutic utility in cardiovascular disorders and this may be due to the presence of salicylic acid, rutin and quercetin in the extract.

Therapeutic Role of Mango Peels in Management of Dyslipidemia and Oxidative Stress in Obese Females.

Obesity is a chronic metabolic and noncommunicable disease that affects 50% of world population. Reactive oxygen species and oxidative stress are interconnected with the obesity and several metabolic disorders, gaining the attention of scientific community to combat this problem naturally. Among various fruits, mango as a yellow fruit is rich in polyphenols, carotenoids, terpenes, and flavonoids that act as antioxidants to protect against free radicals produced in the body. The present study was performed to explore in vivo antioxidant potential of mango peels against dyslipidemia and oxidative stress in overweight subjects. The female volunteers (n = 31) between 25 and 45 years of age having a body mass index (BMI) of 25.0-29.9 (overweight) were included in this study, while participants with complications as diabetes, hypertension, cardiovascular, and liver diseases were excluded. The treatment group consumed 1 g mango peel powder for 84 days. The subjects were analyzed for biochemical analysis, antioxidant status, and anthropometric measurements at baseline and end of the study period. Further, at the end of study, the safety evaluation tests were also performed. The results showed that upon consumption of mango peel powder, low-density lipoproteins (LDL), cholesterol, triglyceride, urea, and creatinine levels were decreased and high-density lipoprotein (HDL) level was increased (P ≤ 0.05), while thiobarbituric acid reactive substances (TBARS) showed increased antioxidant status (P ≤ 0.05) which suggests that mango peels have a strong management potential against oxidative stress and dyslipidemia in obese subjects.

Pharmaco-Technical Evaluation of Statistically Formulated and Optimized Dual Drug-Loaded Silica Nanoparticles for Improved Antifungal Efficacy and Wound Healing.

The current research aimed at designing mesoporous silica nanoparticles (MSNs) for a controlled coadministration of salicylic acid (SA) and ketoconazole (KCZ) to effectively treat highly resistant fungal infections. The sol-gel method was used to formulate MSNs, which were further optimized using central composite rotatable design (CCRD) by investigating mathematical impact of independent formulation variables such as pH, stirring time, and stirring speed on dependent variables entrapment efficiency (EE) and drug release. The selected optimized MSNs and pure drugs were subjected to comparative in vitro/in vivo antifungal studies, skin irritation, cytotoxicity, and histopathological evaluations. The obtained negatively charged (-23.1), free flowing spherical, highly porous structured MSNs having a size distribution of 300-500 nm were suggestive of high storage stability and improved cell proliferation due to enhanced oxygen supply to cells. The physico-chemical evaluation of SA/KCZ-loaded MSNs performed through powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), and thermal gravimetric analysis (TGA) indicates absolute lack of any interaction between formulation components and successful encapsulation of both drugs in MSNs. The EESA, EEKCZ, SA release, and KCZ release varied significantly from 34 to 89%, 36 to 85%, 39 to 88%, and 43 to 90%, respectively, indicating the quadratic impact of formulation variables on obtained MSNs. For MSNs, the skin tolerability and cell viability percentage rate were also having an extraordinary advantage over suspension of pure drugs. The optimized SA/KCZ-loaded MSNs demonstrated comparatively enhanced in vitro/in vivo antifungal activities and rapid wound healing efficacy in histopathological evaluation without any skin irritation impact, suggesting the MSNs potential for the simultaneous codelivery of antifungal and keratolyic agents in sustained release fashion.

Polychlorinated biphenyls in indoor dust from urban dwellings of Lahore, Pakistan: Congener profile, toxicity equivalency, and human health implications.

This study is the pioneer assessment of the PCBs in indoor dust particles (from air conditioners) of an urbanized megacity from South Asian. The ∑35 PCB concentration ranged from 0.27 to 152.9 ng/g (mean: 24.84 ± 22.10 ng/g). The tri- and tetra-PCBs were dominant homologues, contributing 57.36% of the total PCB concentrations. The mean levels of Σ8 -dioxin-like (DL), Σ6 -indicator PCBs and WHO2005 -TEQ for DL-PCBs were 2.22 ± 2.55 ng/g, 9.49 ± 8.04 ng/g and 4.77 ± 4.89 pg/g, respectively. The multiple linear regression indicated a significant correlation of dusting frequency (p = 1.06 × 10-04) and age of the house (p = 1.02 × 10-06) with PCB concentrations in indoor environment. The spatial variation of PCB profile revealed relatively higher concentrations from sites near to illegal waste burning spots, electrical locomotive workshops, and grid stations. Human health risk assessment of PCBs for adults and toddlers through all three exposure routes (ie, inhalation, ingestion, and dermal contact) demonstrated that toddlers were vulnerable to high cancer risk (4.32 × 10-04 ), while adults were susceptible from low to moderate levels of risk (3.16 × 10-05 ). Therefore, comprehensive investigations for PCBs in the indoor settings, focusing particularly on the sensitive populations with relationship to the electronic devices, transformers, and illegal waste burning sites, are recommended.

Organochlorine pesticides (OCPs) in air-conditioner filter dust of indoor urban setting: Implication for health risk in a developing country.

This preliminary investigation highlights the occurrence of organochlorine pesticides (OCPs) in the indoor environment of a megacity, Lahore, Pakistan using the dust ensnared by air-conditioner filters. The Σ16 OCPs concentration ranged from 7.53 to 1272.87 ng/g with the highest percent contribution by ΣDDT (dichlorodiphenyltrichloroethane; 87.21%) and aldrin (6.58%). The spatial variation of OCPs profile revealed relatively higher concentration from homes near to agricultural and abandoned DDT manufacturing sites. Calculated isomer ratios revealed historic sources of hexachlorocyclohexanes (HCHs) and the fresh input of technical DDT and chlordane by the dwellers. The air conditioner dust was helpful to better understand the health risk in the indoor environment. So far a high lifetime cancer risk (10-3 ) was predicted for toddlers via accidental ingestion, inhalation, and dermal exposure. Similarly, the non-carcinogenic risk-based hazard quotient was found to be high for toddlers (6.94) and within the permissible limit (<1) for adults.

Screening of human health risk to infants associated with the polychlorinated biphenyl (PCB) levels in human milk from Punjab Province, Pakistan.

This study assessed the polychlorinated biphenyl (PCB) levels in human milk and its associated health risk to infants from rural and urban settings of five districts of Punjab Province, Pakistan. The ∑34PCB concentrations ranged from 30.9 to 68.3 ng g-1 on lipid weight (l.w.) basis. The ∑8DL-PCB concentrations were ranged from 0.29 to 1.35 ng g-1 l.w., (mean 6.2 ± 8.7 ng g-1 l.w.), with toxicity equivalent to polychlorinated dibenzodioxins (PCDDs) ranging from 8.58 × 10-6 to 0.05 ng TEQ g-1 l.w. The spatial trend of PCB levels in human milk revealed higher bioaccumulative levels for urban mothers as compared with rural counterparts. The estimated daily intake (EDI) values of DL-PCBs to infants through trans-mammary transfer were considerably higher than tolerable daily intake limits established by WHO (i.e., 1-4 pg TEQ kg-1 bw) and other globally recognized organizations. Similarly, the hazard quotient values for TEQ ∑8DL-PCBs (range 1.21 to 79.87) were far above the benchmark value of 1 at all the sampling sites, indicating the high levels of adverse health risks to infants in the region through breast milk consumption. The ∑34PCB levels were found to be negatively correlated with mother' age (r = -0.31; p = 0.02), parity (r = - 0.85; p = 0.001), and infant' birth weight (r = - 0.73; p = 0.01). The present study suggests undertaking comprehensive public health risk assessment studies and firm regulatory measures to safeguard human health risks.

Quantification of polychlorinated biphenyl contamination using human placenta as biomarker from Punjab Province, Pakistan.

The present study biomonitored the placental polychlorinated biphenyl (PCB) concentrations in women from Punjab Province, Pakistan, that provides the pioneer data for occurrence and body burden of PCBs in placental tissues from South Asian women. The ∑34PCB concentrations in placental tissues were ranged from 20.2 to 115.98 ng/g lipid weight (lip. wt.), with predominance of tetra-PCB (54.67%). The levels of ∑8DL-PCBs and ∑6-indicator PCBs were ranged from 2.03 to 27.64 ng/g (lip. wt.) and 1.71 to 30.8 ng/g (lip. wt.), respectively. The WHO-TEQ2005 values for DL-PCBs were ranged from 1.18 × 10-5 to 0.067 ng/g (lip. wt.), with highest value evaluated for CB-126. The estimated daily intake (EDI) for DL-PCBs was ranged from 9.27 × 10-8 to 5.25 × 10-4 pg WHO-TEQ/kg body weight (bw), which was within the tolerable daily intake (TDI) values established by international organizations. The spatial distribution patterns of Σ34PCB concentrations from study area have shown relative higher concentrations in samples from urban and industrial cities than rural areas, and industrial and urban releases along with e-waste handling were recognized as vital PCB sources in the environment. In order to ascertain the transplacental transfer of PCBs, the fetal growth parameters were correlated with the ∑34PCB concentrations in placental tissues. The relationship between ∑34PCB concentrations in placental tissues and infant's anthropometric measures through multiple linear regression showed a negative correlation of infant's body weight (R2 = 0.0728), crown to heel length (R2 = 0.068), head circumference (R2 = 0.0342), chest circumference (R2 = 0.0001), and mid arm circumference (R2 = 0.0096) that noticeably highlights the inhibited fetal anthropometric development associated with maternal PCB bioaccumulation. Hence, an immediate elimination of ongoing PCB addition in the studied area has been emphasized and further investigations are suggested to appropriately manage the public and neonatal health risks in the region.

Occurrence, ecological risk assessment, and spatio-temporal variation of polychlorinated biphenyls (PCBs) in water and sediments along River Ravi and its northern tributaries, Pakistan.

Ecological risk assessment, spatio-temporal variation, and source apportionment of polychlorinated biphenyls (PCBs) were studied in surface sediments and water from River Ravi and its three northern tributaries (Nullah Deg, Nullah Basantar, and Nullah Bein) in Pakistan. In total, 35 PCB congeners were analyzed along 27 sampling stations in pre-monsoon and post-monsoon seasons. The ∑35PCB concentration ranged from 1.06 to 95.76 ng/g (dw) in sediments and 1.94 to 11.66 ng/L in water samples, with hexa-CBs and tetra-CBs as most dominant homologs in sediments and water matrixes, respectively. The ∑8DL-PCB levels were 0.33-22.13 ng/g (dw) and 0.16-1.95 ng/L in sediments and water samples, respectively. The WHO-toxic equivalent values were ranged from 1.18 × 10-6 to 0.012 ng/L and 1.8 × 10-6 to 0.031 ng/g in water and sediments matrixes, respectively. The ecological risk assessment indicates considerable potential ecological risk during pre-monsoon season ([Formula: see text] = 95.17) and moderate potential ecological risk during post-monsoon season ([Formula: see text] = 49.11). The industrial and urban releases were recognized as key ongoing sources for high PCB levels in environment. Therefore, we recommend more freshwater ecological studies to be conducted in the study area and firm regulatory initiatives are required to be taken in debt to the Stockholm Convention, 2001 to cop up with PCB contamination on emergency basis.