Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.

Predisposing deleterious variants in the cancer-associated human kinases in the global populations.

Human kinases play essential and diverse roles in the cellular activities of maintaining homeostasis and growth. Genetic mutations in the genes encoding the kinases (or phosphotransferases) have been linked with various types of cancers. In this study, we cataloged mutations in 500 kinases genes in >65,000 individuals of global populations from the Human Genetic Diversity Project (HGDP) and ExAC databases, and assessed their potentially deleterious impact by using the in silico tools SIFT, Polyphen2, and CADD. The analysis highlighted 35 deleterious non-synonymous SNVs in the ExAC and 5 SNVs in the HGDP project. Notably, a higher number of deleterious mutations was observed in the Non-Finnish Europeans (26 SNVs), followed by the Africans (14 SNVs), East Asians (13 SNVs), and South Asians (12 SNVs). The gene set enrichment analysis highlighted NTRK1 and FGFR3 being most significantly enriched among the kinases. The gene expression analysis revealed over-expression of NTRK1 in liver cancer, whereas, FGFR3 was found over-expressed in lung, breast, and liver cancers compared to their expression in the respective normal tissues. Also, 13 potential drugs were identified that target the NTRK1 protein, whereas 6 potential drugs for the FGFR3 target were identified. Taken together, the study provides a framework for exploring the predisposing germline mutations in kinases to suggest the underlying pathogenic mechanisms in cancers. The potential drugs are also suggested for personalized cancer management.

Exploration of newly synthesized amantadine-thiourea conjugates for their DNA binding, anti-elastase, and anti-glioma potentials.

Three newly synthesized amantadine thiourea conjugates namely MS-1 N-(((3 s,5 s,7 s)-adamantan-1-yl)carbamothioyl)benzamide, MS-2 N-(((3 s,5 s,7 s)-adamantan-1-yl)carbamothioyl)-4-methylbenzamide and MS-3 N-((3 s,5 s,7 s)-adamantan-1-ylcarbamothioyl)-4-chlorobenzamide were investigated for their structures, bindings (DNA/ elastase), and for their impact on healthy and cancerous cells. Theoretical (DFT/docking) and experimental {UV-visible (UV-), fluorescence (Flu-), and cyclic voltammetry (CV)} studies indicated binding interactions of each conjugate with DNA and elastase enzyme. Theoretically and experimentally calculated binding parameters for conjugate - DNA interaction revealed MS-3 - DNA to have most significant binding with comparatively greater values of binding parameters {(Kb/M-1: docking, 3.8 × 105; UV-, 5.95 × 103; Flu-,1.55 × 105; CV, 1.52 × 104), (∆G/ kJmol-1: docking, -32.09; UV-, -22.40; Flu-,-30.81; CV, -24.82)}. The docked structures, greater bindings site size values (n), and the trend in DNA viscosity changes in the presence of each conjugate concentration confirmed a mixed binding mode of interaction among them. Conjugate - elastase binding by docking agreed with the experimental anti-elastase findings. Cytotoxicity studies of each tested conjugate demonstrated greater cytotoxicity for cancerous (MG-U87) cells in comparison to control, while for the normal (HEK-293) cells the cytotoxicity was found comparatively low. Overall exploration suggested that MS-3 is the most effective candidate for DNA binding, anti-elastase, and for anti-glioma activities.

Noval soliton solution, sensitivity and stability analysis to the fractional gKdV-ZK equation.

This work examines the fractional generalized Korteweg-de-Vries-Zakharov-Kuznetsov equation (gKdV-ZKe) by utilizing three well-known analytical methods, the modified [Formula: see text]-expansion method, [Formula: see text]-expansion method and the Kudryashov method. The gKdV-ZK equation is a nonlinear model describing the influence of magnetic field on weak ion-acoustic waves in plasma made up of cool and hot electrons. The kink, singular, anti-kink, periodic, and bright soliton solutions are observed. The effect of the fractional parameter on wave shapes have been analyzed by displaying various graphs for fractional-order values of [Formula: see text]. In addition, we utilize the Hamiltonian property to observe the stability of the attained solution and Galilean transformation for sensitivity analysis. The suggested methods can also be utilized to evaluate the nonlinear models that are being developed in a variety of scientific and technological fields, such as plasma physics. Findings show the effectiveness simplicity, and generalizability of the chosen computational approach, even when applied to complex models.

Investigating vertical transmission and maternofoetal outcomes in pregnant women with COVID-19: a case-control study from Pakistan.

Objectives: To estimate the frequency of severe acute respiratory syndrome coronavirus-2 among pregnant women, the impact in terms of obstetrical and clinical outcomes and vertical transmission to the neonates. METHODS: The prospective, case-control study was conducted at Zainab Panjwani Memorial Hospital, Karachi, from March to December 2021, and comprised pregnant women regardless of gestational age who exhibited symptoms or had a suspicion of exposure to any confirmed coronavirus disease-2019 individual. They were screened for severe acute respiratory syndrome coronavirus-2 infection using polymerase chain reaction or serology. Those who tested negative were designated as control group A, while who had a positive serology result along with a negative polymerase chain reaction were taken as recovered case group B1, and those who tested positive for polymerase chain reaction were called the positive case group B2. Groups B1 and B2 were followed up till delivery. The clinical presentation of coronavirus disease-2019 infection in pregnancy and its obstetrical and neonatal outcomes was assessed. Products of conception were tested for the detection of the severe acute respiratory syndrome coronavirus-2 genome. The viral genome from group B2 cases was sequenced to confirm vertical transmission. Data was analysed using GraphPad Prism V8. RESULTS: Of the 139 pregnant women, 74(53.2%) were in group A with mean age 25.87±6.90 years, 49(35.3%) were in group B1 with mean age 25.53±7.02 years, and 16(11.5%) were in group B2 with mean age 27.12±5.03 years. The gestational age at which termination of pregnancy occurred was 38.3±1.26 weeks in group B1 and 38.3±1.85 weeks for group B2. There were 96 neonates across the 3 groups. Of the 11(11.45%) neonates in group B2, 1(9.09%) had postnatal transmission of severe acute respiratory syndrome coronavirus-2 and this mother-neonate case was taken as the Indexed case. The severe acute respiratory syndrome coronavirus-2 genome isolated from the neonate showed similar mutations as the viral strain infecting the mother. Conclusion: The risk of vertical transmission was found to be low. The severe acute respiratory syndrome coronavirus-2 genome was the same for both the mother and the neonate.

Intelligent prediction modeling for flexural capacity of FRP-strengthened reinforced concrete beams using machine learning algorithms.

Fiber-reinforced polymers (FRP) are widely utilized to improve the efficiency and durability of concrete structures, either through external bonding or internal reinforcement. However, the response of FRP-strengthened reinforced concrete (RC) members, both in field applications and experimental settings, often deviates from the estimation based on existing code provisions. This discrepancy can be attributed to the limitations of code provisions in fully capturing the nature of FRP-strengthened RC members. Accordingly, machine learning methods, including gene expression programming (GEP) and multi-expression programming (MEP), were utilized in this study to predict the flexural capacity of the FRP-strengthened RC beam. To develop data-driven estimation models, an extensive collection of experimental data on FRP-strengthened RC beams was compiled from the experimental studies. For the assessment of the accuracy of developed models, various statistical indicators were utilized. The machine learning (ML) based models were compared with empirical and conventional linear regression models to substantiate their superiority, providing evidence of enhanced performance. The GEP model demonstrated outstanding predictive performance with a correlation coefficient (R) of 0.98 for both the training and validation phases, accompanied by minimal mean absolute errors (MAE) of 4.08 and 5.39, respectively. In contrast, the MEP model achieved a slightly lower accuracy, with an R of 0.96 in both the training and validation phases. Moreover, the ML-based models exhibited notably superior performances compared to the empirical models. Hence, the ML-based models presented in this study demonstrated promising prospects for practical implementation in engineering applications. Moreover, the SHapley Additive exPlanation (SHAP) method was used to interpret the feature's importance and influence on the flexural capacity. It was observed that beam width, section effective depth, and the tensile longitudinal bars reinforcement ratio significantly contribute to the prediction of the flexural capacity of the FRP-strengthened reinforced concrete beam.

Heat stress and stallion fertility.

The threat posed by increased surface temperatures worldwide has attracted the attention of researchers to the reaction of animals to heat stress. Spermatogenesis in animals such as stallions is a temperature-dependent process, ideally occurring at temperatures slightly below the core body temperature. Thus, proper thermoregulation is essential, especially because stallion spermatogenesis and the resulting spermatozoa are negatively affected by increased testicular temperature. Consequently, the failure of thermoregulation resulting in heat stress may diminish sperm quality and increase the likelihood of stallion infertility. In this review, we emphasize upon the impact of heat stress on spermatogenesis and the somatic and germ cells and describe the subsequent testicular alterations. In addition, we explore the functions and molecular responses of heat shock proteins, including HSP60, HSP70, HSP90, and HSP105, in heat-induced stress conditions. Finally, we discuss the use of various therapies to alleviate heat stress-induced reproductive harm by modulating distinct signaling pathways.

Systemic resistance induced in tomato plants by Beauveria bassiana-derived proteins against tomato yellow leaf curl virus and aphid Myzus persicae.

BACKGROUND: Tomato (Solanum lycopersicum L.) is an economically important vegetable crop around the globe. Tomato yellow leaf curling (TYLC) is the most devastating viral disease posing a serious threat to tomato production throughout the tropical and subtropical world. Induction of microbe-mediated systemic resistance in plants has been of great interest in recent years as a novel microbiological tool in disease and insect pest management. This in-vitro study aimed to determine the effectiveness of different strains (BB252, BB72 and ARSEF-2860) of a hypocreal fungus Beauveria bassiana against TYLCV disease and aphid Myzus persicae. Potted tomato plants exogenously treated with conidial and filtrate suspensions of B. bassiana strains and of their partially purified or purified proteins were exposed to TYLCV inoculum and aphid M. persicae. RESULTS: Results showed a significant suppression of TYLCV disease severity index by the exogenous application of conidial, filtrate and protein treatments of all B. bassiana strains and this response was directly proportional to the treatment concentration. Similarly, mean fecundity rate of M. persicae was also significantly reduced by the highest concentration of ARSEF-2860-derived elicitor protein PeBb1, followed by the highest concentrations of BB252- and BB72-derived partially purified proteins. Moreover, these B. bassiana-derived proteins also caused a significant upregulation of most of the plant immune marker genes associated with plant defense. CONCLUSION: Overall, the study findings suggest that these B. bassiana strains and their partially purified or purified elicitor proteins could be effective biological tools for the management of TYLCV and aphid infestation on tomato plants. © 2023 Society of Chemical Industry.

Construction of diverse water wave structures for coupled nonlinear fractional Drinfel'd-Sokolov-Wilson model with Beta derivative and its modulus instability.

This paper aims to analyze the coupled nonlinear fractional Drinfel'd-Sokolov-Wilson (FDSW) model with beta derivative. The nonlinear FDSW equation plays an important role in describing dispersive water wave structures in mathematical physics and engineering, which is used to describe nonlinear surface gravity waves propagating over horizontal sea bed. We have applied the travelling wave transformation that converts the FDSW model to nonlinear ordinary differential equations. After that, we applied the generalized rational exponential function method (GERFM). Diverse types of soliton solution structures in the form of singular bright, periodic, dark, bell-shaped and trigonometric functions are attained via the proposed method. By selecting a suitable parametric value, the 3D, 2D and contour plots for some solutions are also displayed to visualize their nature in a better way. The modulation instability for the model is also discussed. The results show that the presented method is simple and powerful to get a novel soliton solution for nonlinear PDEs.

Zucchini Yellow Mosaic Virus (ZYMV) as a Serious Biotic Stress to Cucurbits: Prevalence, Diversity, and Its Implications for Crop Sustainability.

Zucchini yellow mosaic virus (ZYMV) is a severe threat to cucurbit crops worldwide, including Pakistan. This study was pursued to evaluate the prevalence, geographic distribution, and molecular diversity of ZYMV isolates infecting cucurbits in Pakistan's Pothwar region. Almost all the plant viruses act as a biotic stress on the host plants, which results in a yield loss. These viruses cause losses in single-infection or in mixed-infection cucurbit crops, and we have found a number of mixed-infected samples belonging to the Curubitaceae family. Serological detection of the tested potyviruses in the collected cucurbit samples revealed that ZYMV was the most prevalent virus, with a disease incidence (DI) at 35.2%, followed by Papaya ringspot virus (PRSV) with an incidence of 2.2%, and Watermelon mosaic virus (WMV) having an incidence as little as 0.5% in 2016. In the year 2017, a relatively higher disease incidence of 39.7%, 2.4%, and 0.3% for ZYMV, WMV, and PRSV, respectively, was recorded. ZYMV was the most prevalent virus with the highest incidence in Attock, Rawalpindi, and Islamabad, while PRSV was observed to be the highest in Islamabad and Jhelum. WMV infection was observed only in Rawalpindi and Chakwal. Newly detected Pakistani ZYMV isolates shared 95.8-97.0% nucleotide identities among themselves and 77.1-97.8% with other isolates retrieved from GenBank. Phylogenetic relationships obtained using different ZYMV isolates retrieved from GenBank and validated by in silico restriction analysis revealed that four Pakistani isolates clustered with other ZYMV isolates in group IIb with Chinese, Italian, Polish, and French isolates, while another isolate (MK848239) formed a separate minor clade within IIb. The isolate MK8482490, reported to infect bitter gourd in Pakistan, shared a minor clade with a Chinese isolate (KX884570). Recombination analysis revealed that the recently found ZYMV isolate (MK848239) is most likely a recombinant of Pakistani (MK848237) and Italian (MK956829) isolates, with a recombinant breakpoint between 266 and 814 nucleotide positions. Local isolate comparison and recombination detection may aid in the development of a breeding program that identifies resistant sources against recombinant isolates because the ZYMV is prevalent in a few cucurbit species grown in the surveyed areas and causes heavy losses and economic damage to the agricultural community.

Unlocking the secrets of soil microbes: How decades-long contamination and heavy metals accumulation from sewage water and industrial effluents shape soil biological health.

Heavy metals contamination is posing severe threat to the soil health and environmental sustainability. Application of industrial and sewage waste as irrigation and growing urbanization and agricultural industry is the main reason for heavy metals pollution. Therefore, the present study was planned to assess the influence of different irrigation sources such as industrial effluents, sewage wastewater, tube well water, and canal water on the soil physio-chemical, soil biological, and enzymatic characteristics. Results showed that sewage waste and industrial effluents affect the soil pH, organic matter, total organic carbon, and cation exchange capacity. The highest total nickel (383.71 mg kg-1), lead (312.46 mg kg-1), cadmium (147.75 mg kg-1), and chromium (163.64 mg kg-1) were recorded with industrial effluents application. Whereas, industrial effluent greatly reduced the soil microbial biomass carbon (SMB-C), soil microbial biomass nitrogen (SMB-N), soil microbial biomass phosphorus (SMB-P), and soil microbial biomass sulphur (SMB-S) in the winter season at sowing time. Industrial effluent and sewage waste inhibited the soil enzymes activities. For instance, the minimum activity of amidase, urease, alkaline-phosphatase, ß-glucosidase, arylsulphatase and dehydrogenase activity was noted with HMs contamination. The higher levels of metals accumulation was observed in vegetables grown in soil contaminated with untreated waste water and industrial effluent in comparison to soil irrigated with canal and tube well water. The mean increase in soil microbial parameters and enzyme activities was also observed in response to the change in season from winter to spring due to increase in soil mean temperature. The SMB-C, SMB-N, SMB-P and SMB-S showed significant positive correlation with soil enzymes (amidase, urease, alkaline-phosphatase, ß-glucosidase, arylsulphatase and dehydrogenase). The heavy metals accumulation in soil is toxic to microorganisms and inhibits enzyme functions critical for nutrient cycling and organic matter decomposition and can disrupt the delicate balance of soil ecosystem and may lead to long-term damage of soil biological health.

A generalized procedure for joint monitoring and probabilistic quantification of extreme climate events at regional level.

Droughts and heat waves are currently recognized as two of the most serious threats associated with climate changes. Drought is characterized by prolonged dry periods, low precipitation, and high temperature, while heat wave refers to an extended period of exceptionally high temperature, surpassing the region's average for that time of year. There is a close relationship between droughts and heat waves, as both are often caused by similar weather patterns and can exacerbate each other's impacts. Therefore, it is crucial to monitor and quantify both droughts and heat waves jointly at a regional level in order to develop sustainable policies and effectively manage water resources. This article develops a new index, the standardized composite index for climate extremes (SCICE), for joint monitoring and probabilistic quantification of extreme climate events at regional level. The procedure of SCICE is mainly based on the joint standardization of standardized precipitation index (SPI) and standardized temperature index (STI). In the application of SCICE, results reveal that the long-term probabilities of the joint occurrence of dry and hot events are significantly greater than those of wet and cold events. Furthermore, the outcomes of the comparative assessment support the validity of using SCICE as a compact statistical approach in regional drought analysis. In summation, the study demonstrates the capability of SCICE to effectively characterize and assess the joint monitoring of drought and heat waves at a regional level, providing a comprehensive approach to understanding the joint impact of climate extremes.

Effects of insulin-like growth factor-1 on the proliferation and apoptosis of stallion testicular cells under normal and heat stress culture conditions.

This study investigated the effect of heat stress on stallion testicular cells (TCs) and the effect of insulin-like growth factor (IGF)-1 on TC viability, proliferation, and apoptosis, including different stages of germ cells. TCs were divided into control or treatment groups with 0.01, 0.1, 1, 10, and 100 ng/mL of recombinant human IGF-1 (rhIGF-1) for 24 h at 34 °C and 37 °C. The population and viability were measured before and after treatment. The effects of rhIGF-1 on TC viability, proliferation, and apoptosis were determined using RT-qPCR. Proliferating cell nuclear antigen (PCNA) and marker of proliferation Ki-67 (MKI-67) were used as proliferation markers. Myeloid leukemia-1 (MCL-1) was used as an antiapoptotic marker. BCL2 antagonist/killer-1 (BAK-1) was used as a proapoptotic marker. The relative abundance of mRNA transcript of undifferentiated cell transcription factor 1 (UTF-1), protein gene product 9.5 (PGP9.5), and deleted in azoospermia-like (DAZL), was measured for spermatogenesis progression. TCs treated with 1 ng/mL rhIGF-1 at 34 °C exhibited the highest viability. Significant upregulation of the relative abundance of mRNA transcript of PCNA, MKI-67, and MCL-1 was observed in treated TCs compared with untreated TCs; however, BAK-1 was significantly downregulated in treated TCs. Germ cells treated with 1 ng/mL rhIGF-1 exhibited the highest relative abundance of mRNA transcript of UTF-1 and DAZL, whereas TCs exposed to 0.1 ng/mL showed the highest PGP9.5 level. These data confirm that heat stress in stallions decreases TC viability. These findings may help identify a basal IGF-1 level for TC proliferation and apoptosis during heat stress-induced testicular degeneration in stallions.

Fabrication of Temperature- and Humidity-Independent Silver Nanoparticle's Carbon Composite-Based Strain Sensor Through Additive Manufacturing Process.

A highly sensitive low-cost strain sensor was fabricated in this research study based on microdispensing direct write (MDDW) technique. MDDW is an additive manufacturing approach that involves direct deposition of functional material to the substrate. The devices were printed directly onto a polymeric substrate by optimizing the fabrication parameters. A composite of silver and carbon was used as active sensor material where both materials in the composite have opposite resistance temperature coefficients. The ratio of materials in the composite was selected so that the effect of temperature on the resistance of overall composite was canceled out. This resulted in achieving temperature compensation or inherent independence of the strain sensor resistance on temperature without requiring any additional sensors and components. The sensor was further encapsulated by electrospray deposition, which is also an additive manufacturing approach, to eliminate the effect of humidity as well. Electrical and morphological characterizations were performed to investigate the output response of the sensors and their physical and structural properties. An analog signal conditioning circuit was developed for seamless interfacing of the sensor with any electronic system. The sensor had an excellent gauge factor of 45 and a strain sensitivity of 45 Ω/µÉ› that is higher than most of the conventional strain sensors. The sensor's response showed excellent temperature and humidity compensation reducing the relative effect of temperature on the resistance by ∼99.5% and humidity by ∼99.8%.

ALKBH5 inhibitors as a potential treatment strategy in heart failure-inferences from gene expression profiling.

Heart Failure (HF) is a complex clinical syndrome in which the heart is unable to provide enough blood flow to meet metabolic needs and lacks efficient venous return. HF is a major risk factor for morbidity and mortality with cardiovascular diseases globally. Despite enormous research, the molecular markers relevant to disease prognosis and management remain not well understood. Here, we analyzed the whole transcriptomes of 18 failing hearts and 15 non-failing hearts (predominantly of Caucasian origin), by applying the standard in silico tools. The analyses revealed novel gene-markers including ALKBH5 of mRNA demethylation and KMT2E of histone modification processes, significantly over-expressed in the HF compared with the non-failing hearts (FDR < 0.05). To validate the over-expression of ALKBH5, we determined the global m6A level in hypoxic H9c2 cells using a dot blot assay. The global m6A level was found markedly lower in the hypoxic H9c2 cells than in the control cells. Additionally, the expression of ALKBH5 in the H9c2 cells was quantified by the qPCR and found to be 1.18 times higher at 12 h (p < 0.05), and 1.67 times higher at 24 h of hypoxia (p < 0.01) compared with the control cells, indicating a likely role of ALKBH5 in the failing cardiac cells. Furthermore, we identified several compounds through the virtual screening of 11,272 drug-like molecules of the ZINC15 database to inhibit the ALKBH5 in a molecular docking process. Collectively, the study revealed novel markers potentially involved in the pathophysiology of HF and suggested plausible therapeutic molecules for the management of the disease.

Influence of natural and non-natural diets on the fitness and rearing of Pectinophora gossypiella Saunders.

In order to develop integrated management approaches for Pectinophora gossypiella, basic studies are crucial. The two-sex life table is the most important tool for describing the fitness and population parameters of both sexes (male and female) of an insect, while the traditional life table only explains the female sex of an insect. However, no study has reported on the biology of P. gossypiella using two-sex life table tools. Therefore, this study explains the rearing dynamics of P. gossypiella on a cotton seed-based artificial diet and a natural diet (mature cotton bolls). According to the results, the oviposition period of P. gossypiella was recorded to be longer on the artificial diet (9.07 ± 0.24) compared to the natural diet (7.40 ± 0.11). The total fecundity of P. gossypiella was greater on the artificial diet (125.94 ± 3.06) in comparison to the natural diet (60.37 ± 1.10). The population parameters, including intrinsic rate of increase, finite rate of increase, gross reproductive rate, and net reproductive rate of P. gossypiella were highest on the artificial diet in comparison to the natural diet. This study concluded that the cotton seed-based artificial diet was most suitable for the rearing of P. gossypiella. In the future, P. gossypiella may be studied in depth in light of the findings in this study.

Study of drug resistance-associated genetic mutations, and phylo-genetic analysis of HCV in the Province of Sindh, Pakistan.

Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. The aim of the current study was to identify such mutations in the NS3, and NS5B genes in DAAs treatment-naïve Pakistani chronic HCV 3a patients. Peripheral blood samples were collected from 233 chronic HCV 3a patients at different tertiary care hospitals in Karachi, Pakistan, between August 2020 to September 2021. PCR-amplified target regions of the NS3/NS5B gene were subjected to Sanger sequencing to identify resistance-associated mutations. Phylogenetic analysis of the identified amino acid sequences was performed using HCV3a sequences of the global population in the virus pathogen resource (VIPR) database. Sequence analysis identified five amino acid mutations, Leu36Pro, Gln41His, Gln80Lys/Arg, Ala156Tyr, and Gln168Arg in the NS3 region, and two mutations Leu159Phe and Cys316Arg in the NS5B region. Phylogenetic analysis revealed a high genetic diversity in the studied isolates. Overall, the prevalence of resistance-associated substitutions was almost similar to other geographic regions worldwide. This data could be helpful in selecting the most effective treatment regimen for HCV chronically infected people in Pakistan.