Exploring Trichoderma Species in Industrial Wastewater: Morphological and Molecular Insights from Isolates.

The genus Trichoderma holds economic significance due to its widespread distribution and diverse applications, including biological control, enzyme production, and various biotechnological uses. The accurate identification of Trichoderma species is crucial given their close association with human activities. Despite previous efforts in classification, a comprehensive analysis combining morphological and molecular approaches is necessary. This study focuses on the isolation of four Trichoderma species from industrial wastewater in Pakistan, expanding on the known diversity in the region; isolation involved collecting samples from industrial wastewater effluents at specific sites in Punjab, Pakistan. Trichoderma strains were cultured and purified on solid media, with subsequent biomass production for bisorptional activity. Morphological characterization included colony features and microscopic examinations. DNA extraction, polymerase chain reaction (PCR), and sequencing of the internal transcribed spacer (ITS) region were conducted for molecular analysis. Phylogenetic analysis was performed using the Maximum Likelihood Algorithm. The study identified three Trichoderma species, viz. T. citrinoviride, T. erinaceum, and T. longibrachiatum. Each species was characterized morphologically and supported by molecular-phylogenetic analysis. Illustrations of microscopic features and a phylogenetic tree based on the ITS-nrDNA region were recorded. T. citrinoviride and T. longibrachiatum, isolated from steel mill and tanneries wastewater, respectively, were differentiated based on morphological characteristics such as phialides and conidia. The combination of morphological and molecular techniques enhances the accuracy of species identification. The study highlights the significance of Trichoderma in industrial wastewater environments and underscores the need for continued research in this area. Future research should focus on exploring the ecological roles and potential applications of the newly identified Trichoderma species. Additionally, further investigations into the biotechnological potential of these species, including enzyme production and bioremediation capabilities, would contribute to their practical applications.

Enhanced Citric Acid Production through Aspergillus niger: Insights from Fermentation Studies Using Sugarcane Molasses.

The production of citric acid, a vital agricultural commodity utilized across various industries such as food, beverages, pharmaceuticals, agriculture, detergents, and cosmetics, predominantly relies on microbial fermentation, with Aspergillus niger accounting for approximately 90% of global production. In this study, we aimed to optimize the key factors influencing citric acid production, with a focus on strains, fermentation techniques, and carbon sources, particularly sugarcane molasses. A. niger, sourced from the Botany department/Biotechnology laboratories at Govt. College of Science, Lahore, was employed for citric acid production. The process involved inoculum preparation through spore collection from 3 to 5 days of cultured PDA slants. The fermentation medium, comprising cane molasses with a 15% sugar concentration, was meticulously prepared and optimized for various factors, including magnesium sulfate, potassium ferrocyanide, time of addition of potassium ferrocyanide, ammonium oxalate, and calcium chloride. Our optimization results shed light on the significant impact of different factors on citric acid production. For instance, the addition of 0.4 g/L magnesium sulfate led to a maximum yield of 75%, while 2 g/L potassium ferrocyanide, added at 24 h, achieved a yield of 78%. Remarkably, ammonium oxalate, at a concentration of 10 g/L, resulted in a notable 77% yield. Conversely, the addition of calcium chloride exhibited negligible effects on citric acid production, with the control group yielding more at 78%. Our study underscores the potential for optimizing factors to enhance citric acid production by A. niger in submerged fermentation. These findings highlight the pivotal role of magnesium sulfate, potassium ferrocyanide, and ammonium oxalate in augmenting citric acid yields while emphasizing the minimal impact of calcium chloride. Ultimately, these insights contribute to advancing our understanding of microbial citric acid biosynthesis, providing valuable implications for industrial applications and future research endeavors.

Optimizing germination: comparative assessment of various growth media on dragon fruit germination and early growth.

Dragon fruit (Selenicereus undatus), known for its captivating appearance and remarkable nutritional profile, has garnered considerable attention in recent years. Despite its popularity, there's a dearth of research on optimal conditions for seed germination and early growth stages such as seedling shoot length, which are crucial for optimal crop yield. This study aims to bridge this gap by evaluating various growing media's performance on dragon fruit germination and early growth stages. Dragon fruit seeds were obtained from local markets in Pakistan and evaluated in five different growing media: cocopeat, peat moss, sand, vermiculite, and compost. Germination parameters were observed for 45 days, including seed germination percentage, mean germination time, and mean daily germination percentage, among others while early growth was monitored for 240 days. Statistical analysis was conducted using ANOVA and Tukey's HSD test. Significant differences were found among the growing media regarding germination percentage, mean germination time, and mean daily germination. Vermiculite exhibited the highest germination rate (93.33%), while compost showed the least (70%). Peat moss and sand media facilitated rapid germination, while compost showed slower rates. Stem length was significantly influenced by the growth media, with compost supporting the longest stems. Vermiculite emerged as the most effective medium for dragon fruit seed germination, while compost showed slower but steady growth. These findings provide valuable insights for optimizing dragon fruit cultivation, aiding commercial growers and enthusiasts in achieving higher yields and quality. Further research could explore additional factors influencing dragon fruit growth and development.

A novel test and treat program for hepatitis C virus infection utilizing HCV core antigen testing, among police and general population, Islamabad, Pakistan, 2022.

Hepatitis C virus core antigen (HCVcAg) testing can simplify and decrease costs of HCV infection confirmation compared to molecular testing (nucleic acid testing). We piloted HCVcAg testing for the confirmation of active infection. The study was conducted during June through December 2022 among the police and the general population of Islamabad, Pakistan age 18 years and older. Initial screening for HCV antibody was conducted using a rapid diagnostic test (RDT) for all consenting participants. Those who tested positive had venous blood samples tested for HCVcAg, platelets and aspartate aminotransferase (AST). Persons with HCVcAg values ≥3 fmol/L were defined as viremic, and they were offered treatment with direct acting antiviral (DAA) medications, sofosbuvir and daclatasvir. Aspartate aminotransferase to platelet ratio index (APRI) was calculated for each HCV infected person, and those with an APRI score <1.5 received treatment for 12 weeks, while those with APRI ≥ to 1.5 received 24 weeks of treatment. A total of 15,628 persons were screened for anti-HCV using RDT and 643 (4.1%) tested positive. HCVcAg values of ≥3 fmol/L was found in 399/643 (62.1%), and all were offered and accepted treatment. Of those treated, 273/399 (68.4%) returned for a follow-up SVR and HCVcAg was not detected in 261/273, a 95.6% cure rate. The pilot study demonstrated the effectiveness of reaching and treating an urban population using RDT for screening and HCVcAg for confirmation of infection and test of cure.

Ethnomedicinal Plants and Herbal Preparations Used by Rural Communities in Tehsil Hajira (Poonch District of Azad Kashmir, Pakistan).

The present study emphasizes the importance of documenting ethnomedicinal plants and herbal practices of the local rural communities of Tehsil Hajira (Pakistan). The aim was to document, explore and quantify the traditional ethnomedicinal knowledge. Ethnobotanical data were collected using semi-structured questionnaires and analyzed using various quantitative indices. The results showed that 144 medicinal plant species from 70 families and 128 genera play an important role in herbal preparations. The most common type of preparation was powder (19.0%), followed by paste (16.7%), aqueous extract (15.7%), decoction (14.7%) and juice (11.0%). Fragaria nubicola (0.94) and Viola canescens (0.93) had the highest relative frequency of mention (RFC), while Berberis lycium (1.22) and Fragaria nubicola (1.18) had the highest use value (UV). Geranium wallichianum (85.5), Ligustrum lucidum (83) and Indigofera heterantha (71.5) were the most important species in the study area with the highest relative importance (RI) value. The diseases treated were categorized into 17 classes, with diseases of the digestive system and liver having the highest Informant Consensus Factor (ICF) value, followed by diseases of the oropharynx and musculoskeletal system. Important plants mentioned for the treatment of various diseases of the gastrointestinal tract are Zanthoxylum alatum, Berberis lycium, Mentha longifolia, Punica granatum, Rubus ellipticus and Viola canescens. New applications of rarely documented plants from this area are: Oxalis corniculata paste of the whole plant to treat vitiligo, Carthamus tinctorius flowers to treat chicken pox, Dioscorea deltoidea tuber powder to treat productive cough, Inula cappa root decoction to treat miscarriage, Habenaria digitata tuber juice for the treatment of fever, Viola canescens leaves and flowers for the treatment of sore throat and Achillea millefolium root and leaf juice for the treatment of pneumonia. These plants may contain interesting biochemical compounds and should be subjected to further pharmacological studies to develop new drugs. Traditional medicinal knowledge in the area under study is mainly limited to the elderly, traditional healers and midwives. Therefore, resource conservation strategies and future pharmacological studies are strongly recommended.

Revealing the dynamics of saffron growth: Optimizing corm size and planting depth for increased yield synergies.

Saffron, the "golden spice" derived from Crocus sativus L., is renowned for its richness in secondary metabolites such as crocin and safranal, contributing to its unique properties. Facing challenges like decreasing global production, optimizing cultivation techniques becomes imperative for enhanced yields. Although the impact of factors like planting density, planting depth, spacing, and corm size on saffron growth has been studied, the interaction between corm size and planting depth remains underexplored. This study systematically investigates the interactive effects of corm size and planting depth on saffron growth and yield, providing evidence-based guidelines for optimizing cultivation. A factorial experiment, employing a completely randomized design, was conducted to assess the influence of corm size (05-10g, 10.1-15g, 15.1-20g) and planting depth (10cm, 15cm, 20cm) on saffron yield. Uniform-sized corms were obtained, and a suitable soil mixture was prepared for cultivation. Morphological and agronomic parameters were measured, and statistical analyses were performed using ANOVA and Tukey's HSD test. The study revealed that planting depth significantly affected saffron emergence. The corms sown under 15cm depth showed 100% emergence regardless of corm size (either 05-10g, 10.1-15g, 15.1-20g) followed by 10cm depth corms. Corm dry weight exhibited a complex interaction, where larger corms benefited from deeper planting, while intermediate-sized corms thrived at shallower depths. Similar patterns were observed in shoot fresh weight and dry weight. Specifically, the largest corm size (t3, 15.1-20g) produced the greatest fresh-weight biomass at the deepest planting depth of 20cm (T3), while intermediate-sized corms (t2, 10.1-15g) were superior at the shallowest 10cm depth (T1). The total plant biomass demonstrated that larger corms excelled in deeper planting, while intermediate-sized corms were optimal at moderate depths. This research highlights the intricate interplay between corm size and planting depth in influencing saffron growth. Larger corms generally promote higher biomass, but the interaction with planting depth is crucial. Understanding these dynamics can aid farmers in tailoring cultivation practices for optimal saffron yields. The study emphasizes the need for a coordinated approach to corm selection and depth placement, providing valuable insights for sustainable saffron production and economic growth.

Mitigation of cadmium-induced stress in maize via synergistic application of biochar and gibberellic acid to enhance morpho-physiological and biochemical traits.

Cadmium (Cd), being a heavy metal, tends to accumulate in soils primarily through industrial activities, agricultural practices, and atmospheric deposition. Maize, being a staple crop for many regions, is particularly vulnerable to Cd contamination, leading to compromised growth, reduced yields, and potential health risks for consumers. Biochar (BC), a carbon-rich material derived from the pyrolysis of organic matter has been shown to improve soil structure, nutrient retention and microbial activity. The choice of biochar as an ameliorative agent stems from its well-documented capacity to enhance soil quality and mitigate heavy metal stress. The study aims to contribute to the understanding of the efficacy of biochar in combination with GA3, a plant growth regulator known for its role in promoting various physiological processes, in mitigating the adverse effects of Cd stress. The detailed investigation into morpho-physiological attributes and biochemical responses under controlled laboratory conditions provides valuable insights into the potential benefits of these interventions. The experimental design consisted of three replicates in a complete randomized design (CRD), wherein soil, each containing 10 kg was subjected to varying concentrations of cadmium (0, 8 and 16 mg/kg) and biochar (0.75% w/w base). Twelve different treatment combinations were applied, involving the cultivation of 36 maize plants in soil contaminated with Cd (T1: Control (No Cd stress; T2: Mild Cd stress (8 mg Cd/kg soil); T3: Severe Cd stress (16 mg Cd/kg soil); T4: 10 ppm GA3 (No Cd stress); T5: 10 ppm GA3 + Mild Cd stress; T6: 10 ppm GA3 + Severe Cd stress; T7: 0.75% Biochar (No Cd stress); T8: 0.75% Biochar + Mild Cd stress; T9: 0.75% Biochar + Severe Cd stress; T10: 10 ppm GA3 + 0.75% Biochar (No Cd stress); T11: 10 ppm GA3 + 0.75% Biochar + Mild Cd stress; T12: 10 ppm GA3 + 0.75% Biochar + Severe Cd stress). The combined application of GA3 and BC significantly enhanced multiple parameters including germination (27.83%), root length (59.53%), shoot length (20.49%), leaf protein (121.53%), root protein (99.93%), shoot protein (33.65%), leaf phenolics (47.90%), root phenolics (25.82%), shoot phenolics (25.85%), leaf chlorophyll a (57.03%), leaf chlorophyll b (23.19%), total chlorophyll (43.77%), leaf malondialdehyde (125.07%), root malondialdehyde (78.03%) and shoot malondialdehyde (131.16%) across various Cd levels compared to the control group. The synergistic effect of GA3 and BC manifested in optimal leaf protein and malondialdehyde levels indicating induced tolerance and mitigation of Cd detrimental impact on plant growth. The enriched soils showed resistance to heavy metal toxicity emphasizing the potential of BC and GA3 as viable strategy for enhancing maize growth. The application of biochar and gibberellic acid emerges as an effective means to mitigate cadmium-induced stress in maize, presenting a promising avenue for sustainable agricultural practices.

Effect of salinity on growth and biochemical responses of brinjal varieties: implications for salt tolerance and antioxidant mechanisms.

Salinity poses significant challenges to agricultural productivity, impacting crops' growth, morphology and biochemical parameters. A pot experiment of three months was conducted between February to April 2023 in the Department of Botany, The Islamia University of Bahawalpur. Four brinjal (eggplant) varieties: ICS-BR-1351, HBR-313-D, HBR-314-E, and HBR-334-D were selected and assessed for the effects of salinity on various growth and biochemical attributes. The experiment was completely randomized in design with three replicates each. This study revealed that increased salinity significantly reduced the shoot length, root length, and leaf number across all varieties, with maximum adverse effects observed at a 300mM NaCl concentration. Among the tested varieties, ICS-BR-1351 demonstrated superior performance in most growth parameters, suggesting potential salt tolerance. Biochemically, salinity decreased chlorophyll content across all varieties, with the sharpest decline observed at the highest salt concentration. V4 (HBR-334-D) showed a 57% decrease in chlorophyll followed by V3 (HBR-314-E) at 56%, V2 (HBR-313-D) at 54%, and V1 (ICS-BR-1351) at 33% decrease at maximum salt levels as compared to control. Conversely, carotenoid content increased up to -42.11% in V3 followed by V2 at -81.48%, V4 at -94.11%, and - 233% in V1 at 300mM NaCl stress as compared to respective controls. V3 (HBR-314-E) has the maximum value for carotenoids while V1 has the lowest value for carotenoids as compared to the other three brinjal varieties. In addition to pigments, the study indicated a salinity-induced decrease in total proteins and total soluble sugar, whereas total amino acids and flavonoids increased. Total proteins showed a decrease in V2 (49.46%) followed by V3 (36.44%), V4 (53.42%), and V1 (53.79%) at maximum salt concentration as compared to plants treated with tap water only. Whereas, total soluble sugars showed a decrease of 52.07% in V3, 41.53% in V2, 19.49% in V1, and 18.99% in V4 at the highest salt level. While discussing total amino acid, plants showed a -9.64% increase in V1 as compared to V4 (-31.10%), V2 (-36.62%), and V3 (-22.61%) with high salt levels in comparison with controls. Plant flavonoid content increased in V3 (-15.61%), V2 (-19.03%), V4 (-18.27%) and V1 (-27.85%) at 300mM salt concentration. Notably, salinity elevated the content of anthocyanin, lycopene, malondialdehyde (MDA), and hydrogen peroxide (H2O2) across all varieties. Antioxidant enzymes like peroxidase, catalase, and superoxide dismutase also increased under salt stress, suggesting an adaptive response to combat oxidative damage. However, V3 (HBR-314-E) has shown an increase in anthocyanin at -80.00%, lycopene at -24.81%, MDA at -168.04%, hydrogen peroxide at -24.22%, POD at -10.71%, CAT as-36.63 and SOD as -99.14% at 300mM NaCl stress as compared to control and other varieties. The enhanced accumulation of antioxidants and other protective compounds suggests an adaptive mechanism in brinjal to combat salt-induced oxidative stress. The salt tolerance of different brinjal varieties was assessed by principal component analysis (PCA), and the order of salt tolerance was V1 (ICS-BR-1351) > V4 (HBR-334-D), > V2 (HBR-313-D) > V3 (HBR-314-E). Among the varieties studied, ICS-BR-1351 demonstrated resilience against saline conditions, potentially offering a promising candidate for saline-prone agricultural areas.

Exploring the benefits of wild plants in dietary nutrition: investigating perspectives, choices, health impacts and sustainable practices.

BACKGROUND: This ethnobotanical study in Dunyapur, District Lodhran, Pakistan, focuses on traditional medicinal knowledge, exploring 41 plants across 28 families. The research involves 496 informants from diverse backgrounds, including farmers, herbalists, housewives, teachers, and shopkeepers. The prevalence of herbs (68%) aligns with their accessibility and rapid regrowth, shaping the local medicinal landscape. The study investigates socio-demographic features, emphasizing the importance of considering the community's diverse perspectives. METHODS: The research employs quantitative ethnobotanical data analysis, introducing various indices like PPV, FUV, FIV, RFC, UV, and RI. The analysis of plant growth habits underscores the dominance of herbs, and the method of preparation evaluation identifies decoction as the most common (23%). Leaves (27%) are the most utilized plant part, and Resedaceae stands out with the highest FUV (0.38). FIV highlights the ecological and cultural significance of Poaceae, Boraginaceae, Fabaceae, and Solanaceae. RESULTS: The RFC values range from 0.016 to 0.032, with Cucumis melo having the highest value (0.032), indicating its frequent citation and cultural significance. The study reveals specific plants like Melia azedarach, Peganum harmala and Salvadora oleoides with high PR values for skin issues, reflecting their widespread acceptance and effectiveness. Oligomeris linifolia emerges with the highest UV (0.38), emphasizing its greater significance in local traditional practices. Leptadenia pyrotechnica records the highest RI (9.85), underlining its exceptional importance in the community's traditional pharmacopeia. CONCLUSION: The findings offer a holistic understanding of ethnobotanical knowledge in Dunyapur, emphasizing the role of local contexts and ecological factors in shaping traditional plant uses. The study contributes valuable insights into the diverse practices within the community, laying the foundation for sustainable integration of traditional knowledge into broader healthcare frameworks.

Antibacterial, antioxidant, and anticancer potential of green fabricated silver nanoparticles made from Viburnum grandiflorum leaf extract.

BACKGROUND: Recently, researchers are focusing on creating new tools to combat the antibiotic resistant bacteria and malignancy issues, which pose significant threats to humanity. Biosynthesized silver nanoparticles (AgNPs) are thought to be a potential solution to these issues. The biosynthesis method, known for its environmentally friendly and cost-effective characteristics, can produce small-sized AgNPs with antimicrobial and anticancer properties. In this study, AgNPs were bio-fabricated from the distilled water and methanolic extracts of Viburnum grandiflorum leaves. Physio-chemical characterization of the bio-fabricated AgNPs was conducted using UV-visible spectroscopy, scanning electron microscopy, energy dispersive X-ray, and X-ray diffraction analysis. RESULTS: AgNPs produced from the methanol extract were smaller in size (12.28 nm) compared to those from the aqueous extract (17.77 nm). The bioengineered AgNPs exhibited a circular shape with a crystalline nature. These biosynthesized AgNPs demonstrated excellent bactericidal activity against both gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria. Highest antibacterial activity was observed with the methanol extract against P. aeruginosa (14.66 ± 0.74 mm). AgNPs from the methanol extract also displayed the highest antioxidant activity, with an IC50 value of 188.00 ± 2.67 µg/mL against 2,2-diphenyl-1-picrylhydrazyl (DPPH). Furthermore, AgNPs exhibited notable cytotoxic activity against Rhabdomyosarcoma cell line (RD cell) of human muscle cancer cell. The IC50 values calculated from the MTT assay were 26.28 ± 1.58 and 21.49 ± 1.44 µg/mL for AgNPs synthesized from aqueous and methanol extracts, respectively. CONCLUSION: The methanol extract of V. grandiflorum leaves demonstrates significant potential for synthesizing AgNPs with effective antibacterial, antioxidant, and anticancer actions, making them applicable in various biomedical applications.

Boosting Solanum tuberosum resistance to Alternaria solani through green synthesized ferric oxide (Fe2O3) nanoparticles.

Potato (Solanum tuberosum) is the third crucial global crop facing threats from Alternaria solani, a necrotrophic fungal pathogen causing early blight disease. Beyond crop impact, it leads to substantial production reduction and economic losses worldwide. This study introduces a green synthesis method for producing Ferric Oxide nanoparticles (FNPs) using dried Guava (Psidium guajava) leaves. Guava leaf extract acts as a reducing agent, with iron (III) chloride hexahydrate (FeCl3·6H2O) as the oxidizing agent. This study employed various characterization techniques for Ferric Oxide nanoparticles (FNPs). Fourier Transform Infrared Spectroscopy (FTIR) revealed peaks at 877 cm-1, 1180 cm-1, 1630 cm-1, 1833 cm-1, 2344 cm-1, and 3614 cm-1, associated with Maghemite vibrations, polyphenol compounds, and amino acids. UV-Vis spectroscopy exhibited a characteristic absorbance peak at 252 nm for FNPs. Scanning Electron Microscope (SEM) images illustrated particle sizes of 29-41 nm, and Energy Dispersive Spectroscopy (EDS) indicated elemental composition. X-ray diffraction (XRD) confirmed crystalline FNPs with peaks at 26.78, 30.64, 36.06, 38.21, 43.64, 53.52, 57.42, 63.14 and 78.32. Disease resistance assays demonstrated FNPs' effectiveness against A. solani, reducing disease incidence and severity. In the leaf detach assay, concentrations of 15, 10 and 5 mg/L showed a dose-dependent reduction in disease severity and incidence. The Greenhouse Assay confirmed FNPs' concentration-dependent effect on disease incidence and severity. The study also explored FNPs' potential as biocontrol agents showing no adverse effects on overall plant development. Additionally, the study highlighted the agronomic potential of FNPs in enhancing plant growth and development emphasizing their role as micronutrients in biofortification. The findings suggest the promising application of FNPs in plant protection and biofortification strategies.

Context-dependent accuracy of the cobas plasma separation card for HCV RNA viral load measurement.

Collection and preservation of plasma are challenging in remote or under-resourced settings. The cobas® Plasma Separation Card (PSC) is an alternative specimen type for blood-borne pathogen nucleic acid quantitation. We assessed PSC as a specimen type for HCV RNA quantitation in Pakistan. Plasma from venous blood and PSC from finger prick blood were prepared at two sites: Site 1 (in Lahore, n = 199) consisted of laboratory-based outpatient clinics. Specimens were prepared in the same facility and stored frozen. Site 2 was a catchment area within a resource-limited, semi-urban locality of Islamabad with limited access to healthcare services (n = 151). Community public health outreach staff collected blood and prepared the PSC in the participants' homes. Specimens were transported to the central hepatitis laboratory in Lahore to be stored frozen until tested. HCV RNA testing was performed using the cobas HCV RNA test in a central laboratory. Concordance with respect to RNA detectability was high at Site 1 (97.4%), but lower at Site 2 (82.4%). At Site 1, HCV viral load in plasma and PSC were well correlated across the linear range with a 0.21 log10 IU/mL mean bias toward higher concentrations in PSC. At Site 2, HCV viral load in plasma and PSC were poorly correlated. There was a 0.11 log10 IU/mL mean bias toward higher concentrations in PSC. PSC performance can be excellent in underserved settings where refrigerated transport of traditional specimens is difficult. In very challenging field settings, extra support must be provided to ensure correct specimen collection and handling.

HepFREEPak: protocol for a multi-centre, prospective observational study examining efficacy and impact of current therapies for the treatment of hepatitis C in Pakistan and reporting resistance to antiviral drugs: study protocol.

BACKGROUND: Pakistan has one of the highest burdens of Hepatitis C virus (HCV) infection globally. To achieve the World Health Organization's goals for HCV elimination, there is a need for substantial scale-up in testing, treatment, and a reduction in new infections. Data on the population impact of scaling up treatment is not available in Pakistan, nor is there reliable data on the incidence of infection/reinfection. This project will fill this gap by providing important empirical data on the incidence of infection (primary and reinfection) in Pakistan. Then, by using this data in epidemic models, the study will determine whether response rates achieved with affordable therapies (sofosbuvir plus daclatasvir) will be sufficient to eliminate HCV in Pakistan. METHODS: This prospective multi-centre cohort study will screen 25,000 individuals for HCV antibody (Ab) and RNA (if Ab-positive) at various centers in Pakistan- Karachi (Sindh) and Punjab, providing estimates of the disease prevalence. HCV positive patients will be treated with sofosbuvir and daclatasvir for 12-weeks, (extended to 24-weeks in those with cirrhosis) and the proportion responding to this first-line treatment estimated. Patients who test HCV Ab negative will be recalled 12 months later to test for new HCV infections, providing estimates of the incidence rate. Patients diagnosed with HCV (~ 4,000) will be treated and tested for Sustained Virological Response (SVR). Questionnaires to assess risk factors, productivity, health care usage and quality of life will be completed at both the initial screening and at 12-month follow-up, allowing mathematical modelling and economic analysis to assess the current treatment strategies. Viral resistance will be analysed and patients who have successfully completed treatment will be retested 12 months later to estimate the rate of re-infection. CONCLUSION: The HepFREEPak study will provide evidence on the efficacy of available and widely used treatment options in Pakistan. It will also provide data on the incidence rate of primary infections and re-infections. Data on incidence risk factors will allow us to model and incorporate heterogeneity of risk and how that affects screening and treatment strategies. These data will identify any gaps in current test-and-treat programs to achieve HCV elimination in Pakistan. STUDY REGISTRATION: This study was registered on clinicaltrials.gov (NCT04943588) on June 29, 2021.

Synergistic effect of biochar-based compounds from vegetable wastes and gibberellic acid on wheat growth under salinity stress.

Soil salinization is a prevalent form of land degradation particularly in water-deficient regions threatening agricultural sustainability. Present desalinization methods demand excessive water use. Biochar has been recognized as a potential remedy for saline soils and Gibberellic acids (GA3) are known to mediate various biochemical processes aiding in stress mitigation. This study was undertaken at The Islamia University of Bahawalpur during winter 2022-23 to explore the combined effect of biochar and GA3 on wheat (Triticum aestivum L.) in saline conditions. Employing a fully randomized design wheat seeds in 24 pots were subjected to two salinity levels with three replications across eight treatments: T1 to T8 ranging from controls with different soil electrical conductivities (ECs) to treatments involving combinations of GA3, biochar and varying soil ECs. These treatments included T1 (control with soil EC of 2.43dS/m), T2 (salinity stress with soil EC of 5.11dS/m), T3 (10 ppm GA3 with soil EC of 2.43dS/m), T4 (10 ppm GA3 with soil EC of 5.11dS/m), T5 (0.75% Biochar with soil EC of 2.43dS/m), T6 (0.75% Biochar with soil EC of 5.11dS/m), T7 (10 ppm GA3 combined with 0.75% biochar at soil EC of 2.43dS/m) and T8 (10 ppm GA3 plus 0.75% biochar at soil EC of 5.11dS/m). The results indicated that the combined applications of GA3 and biochar significantly enhanced plant growth in saline conditions viz. germination rate by 73%, shoot length of 15.54 cm, root length of 4.96 cm, plant height of 16.89 cm, shoot fresh weight 43.18 g, shoot dry weight 11.57 g, root fresh weight 24.26 g, root dry weight 9.31 g, plant water content 60.77%, photosynthetic rate 18.58(CO2 m-2 s-1) carotenoid 3.03 g, chlorophyll a 1.01 g, chlorophyll b 0.69 g, total chlorophyll contents by 1.9 g as compared to the control. The findings suggest that the combined application of these agents offers a sustainable and effective strategy for cultivating wheat in saline soils. The synergy between biochar and GA3 presents a promising avenue for sustainable wheat cultivation in saline conditions. This combined approach not only improves plant growth but also offers an innovative, water-efficient solution for enhancing agricultural productivity in saline-affected regions.

Alleviation of cadmium and drought stress in wheat by improving growth and chlorophyll contents amended with GA3 enriched deashed biochar.

Drought and cadmium (Cd) stress are both major issues that significantly affect the growth and development of wheat plants. Both drought stress and Cd toxicity disrupt physiological processes i.e., nutrient uptake, cell expansion, and enzymatic reactions resulting in poor crop growth. To overcome these issues, the use of activated carbon and gibberellic acid (GA3) are considered valuable amendments. However, the current study aimed to add value using GA3-enriched biochar (GA3-BC). That's why, a lab experiment was conducted on wheat to assess the effectiveness of GA3-BC against Cd and drought stress. For GA3 enrichment in biochar, 10 µg GA3/g biochar was mixed. There were 3 levels of GA3-BC i.e., 0, 0.6 (GA3-BC1), and 0.9% (GA3-BC). All levels were applied in 3 replicates under no stress (0Cd + no drought), drought stress (DS), and 6 mg Cd/ kg soil (6Cd). Results showed that GA3-BC2 caused a significant improvement in shoot length (44.99%), root length (99.73%), seedling length (60.13%) and shoot fresh weight (63.59%) over control at 6Cd + drought stress. A significant improvement in chlorophyll a, chlorophyll b, and total chlorophyll while a decrease in electrolyte leakage and regulation of antioxidants i.e., lipid peroxidation, SOD, CAT, APx, GR, GPx, GST, and DPHH also signified the effectiveness of GA3-BC2 compared to control at 6Cd + drought stress. In conclusion, GA3-BC2 is an efficacious amendment for simultaneously alleviating drought and Cd stress in wheat. More investigations are recommended at the field level on different cereal crops cultivated in different soil textures to declare GA3-BC2 as the best treatment for mitigation of drought stress and Cd toxicity.

Exploration of the Wild Edible Plants Used for Basic Health Care by Local People of Bahawalpur and Adjacent Regions, Pakistan.

The current study aimed to explore the traditional knowledge and practices of indigenous communities in Bahawalpur and adjacent regions, Pakistan, to treat a variety of diseases with a particular focus on the usage of ethnomedicinal wild plants. The objective of the study was to collect and evaluate local and indigenous knowledge regarding the quantity, variety, use and management of resources by the people. Data were gathered using Rapid Appraisal Approach (RAA), which involved in-person interviews. The data were analyzed using descriptive statistics methods as well as common ethnobotanical analytical techniques viz. use value (UV), relative frequency of citation (RFC), informant consensus factor (ICF), fidelity level (FL), relative importance (RI), frequency index (FI), family use value (FUV), family importance value (FIV), popular therapeutic use value (POPUT), plant part value (PPV), preference ranking (PR), cultural significance index (CSI), rank order priority (ROP) and Jaccard index (JI). A total of 158 plant species from 49 families were identified. The perennial herbs made up 72% of the documented species of therapeutic plants. There were 21 different species in each of the two major plant families i.e., Fabaceae and Poaceae. The maximum ICF were calculated for gonorrhea and paralysis (1.0) and maximum POPUT was calculated for skin disorders (0.088) and cough (0.077), respectively. The maximum UV was found for Rumex crispus (0.57) and RI for Leucaena leucocephala (4.38). Heliotropium crispum had the highest FL value (83%) and is used to treat kidney diseases. Alhagi maurorum had the greatest RFC (0.009) and FI (0.93). Arecaceae (0.45) and Fabaceae (13.39%) had the greatest FUV while Poaceae and Fabaceae had the highest FIV (13.29 for each). Leaves were the most frequently used plant part (35%). Alhagi maurorum (4.0) and Rumex crispus (32.57) had the highest CSI and ROP, respectively. The maximum JI (28.31) was calculated for Cholistan desert. These results showed the local population's reliance on herbal remedies to treat prevalent illnesses. To demonstrate the viability of species, it's crucial to emphasize both the use and conservation of these species. Adopting innovative applications, enhancing their value, and prioritizing the protection of multipurpose wild plants in inhabited environments is vital.

Barriers and Strategies for Hepatitis B and C Elimination in Pakistan.

BACKGROUND: Pakistan has a high hepatitis burden for both hepatitis C virus (HCV) and hepatitis B virus (HBV). To achieve World Health Organization (WHO) 2030 targets for hepatitis elimination, there is a need to constitute progress in the country, find the barriers and strategies for HCV elimination, and take actions to address the gaps. METHODS: We collected data from (1) WHO estimates in 2020, (2) midterm review questionnaire of the WHO regional action plan, and (3) WHO estimates on immunization. We analyzed these data to inform (1) the burden defined as prevalence and mortality and (2) response in 3 thematic areas: governance, policy, and finance; strategic information; and service delivery. RESULTS: The prevalence of hepatitis B in the general population is 1.6% with 12 000 deaths/year. The prevalence of hepatitis C in the general population is 7.5% with 19 000 deaths and 545 000 new cases (incidence)/year. The selected indicators to monitor progress on viral hepatitis in Pakistan were governance and financing, policies and guidelines, and strategic information. The overall governance indicators are good with a focal point, a national hepatitis strategy, an operational plan, strategy for price reduction, and involvement of civil society but the costed action plan and the advocacy strategy are missing. The indicators on policies and guidelines are also adequately addressed. The hepatitis B and C testing and treatment guidelines are available, there is a policy to screen all blood donations, and there is an injection safety policy, but the policy for timely hepatitis B vaccine birth dose and hepatitis B vaccination for the vulnerable is missing. Both indicators regarding strategic information, that is measures of key hepatitis indicators and regular data review, are missing. The status of 5 key interventions in Pakistan show that the hepatitis B vaccination coverage is 74% and only 3% of newborn children are given the hepatitis B vaccine birth dose. Only 22% of HCV cases have been diagnosed and 2% have received treatment. Treatment response is 96%. Same-day testing and treatment of hepatitis C reduced the overall dropout rate and improved the cascade of care. Decentralization and task shifting are important tools to improve service delivery and reach communities. Finances to implement hepatitis elimination is a major barrier. CONCLUSIONS: Pakistan has the highest hepatitis disease burden. With the current pace, hepatitis elimination appears impossible. Introduction of the birth dose of hepatitis B vaccine and improving access and affordability of testing can improve the testing and treatment numbers. Finances need to be mobilized from within the country and outside to support disease elimination.

Investigation of phytotherapeutic potential of herbal mixtures and their effects on salbutamol induced cardiotoxicity and hyperlipidemia in rabbits.

BACKGROUND: Cardiovascular diseases (CVDs) are the major cause of deaths all over the world. The high level of blood cholesterol and oxidative stress are major risk factors for heart diseases. The phytotherapeutics have attracted attention as potential agents for preventing and treating oxidative stress associated diseases. The objective of present study was to evaluate the synergetic cardio-protective and antilipidemic potential of medicinal plants viz. Coriandrum sativum, Piper nigrum and Cactus grandiflorus. Cardio-protective and anti-lipidemic potential of herbal mixture was evaluated against salbutamol induced cardiotoxicity in rabbits. For this purpose, rabbits were divided into six groups as normal control, salbutamol control, curative and standard drug curative. RESULTS: Salbutamol significantly (p < 0.05) increased the level of serum cardiac biomarkers (ALT, CK-MB, AST and LDH) and lipids (LDL, triglycerides, cholesterol) in rabbits. The prior and post administration of herbal mixture significantly (p < 0.05) lowered the elevated level of serum cardiac biomarkers and lipids equal to normal control. Gross pathological examination revealed that heart of salbutamol control animals became hardened, congested and were enlarged than preventive and curative groups. The phytotherapeutic analysis of medicinal plants revealed the presence of phenols, tannins, alkaloids and steroids. CONCLUSION: The results showed that this herbal mixture has strong cardio-protective and anti-lipidemic potential.

Citrus sinensis Peel Oil Extraction and Evaluation as an Antibacterial and Antifungal Agent.

Throughout the tropical and subtropical climates, the genus Citrus can be found. The current study was conducted to extract the Citrus sinensis peel oil and evaluate its antibacterial, antifungal and antiparasitic potential. Petroleum ether was used to extract the C. sinensis peel oil through a Soxhlet apparatus. The antimicrobial and antifungal potential was determined via agar well diffusion method and minimum inhibitory concentrations (MIC) were calculated (test bacterial strains: Staphylococcus aureus, Escherichia coli and Streptococcus agalactiae; test fungal strains: Aspergillus flavus, Aspergillus niger, Altrnaria alternata). Antiparasitic activity against Leishmaniatropica was determined following standard protocol using amphotericin-B as positive and Dimethyl Sulfoxide (DMSO) as a negative control and the percentage inhibition was calculated. The oil extracted was brownish yellow with a tangy smell, water-insoluble, density (0.778 g/cm3) and specific gravity (0.843 g/cm). In antibacterial activity, the diameter of the zone of inhibition was maximum against E. coli (14 mm) and minimum for S. agalactiae (10 mm). While in antifungal activity diameter of the zone of inhibition was maximum against A. flavus (12.5 mm) and minimum for A. alternata (8.6 mm). S. agalactiae exhibited the minimum MIC value (6 mg/mL) and in fungal strains A. alternata exhibited the minimum value (2 mm). Citrus sinensis peel oil displayed antileishmanial efficiency of 60% at 50 µg/mL concentration after 48 h of incubation. C. sinensis peel oil demonstrated antimicrobial capabilities, implying that it could be used as a natural preservative in food or as an effective treatment against a variety of pathogenic organisms. Industries should extract oil from the waste of citrus fruits which will be beneficial from an economic point of view.

Cancer Registry - What is the Need to be Attentive and Why Now?

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