Characterization of a thermostable protease from Bacillus subtilis BSP strain.

This study used conservative one variable-at-a-time study and statistical surface response methods to increase the yields of an extracellular thermostable protease secreted by a newly identified thermophilic Bacillus subtilis BSP strain. Using conventional optimization techniques, physical parameters in submerged fermentation were adjusted at the shake flask level to reach 184 U/mL. These physicochemical parameters were further optimized by statistical surface response methodology using Box Behnken design, and the protease yield increased to 295 U/mL. The protease was purified and characterized biochemically. Both Ca2+ and Fe2+ increased the activity of the 36 kDa protease enzyme. Based on its strong inhibition by ethylenediaminetetracetate (EDTA), the enzyme was confirmed to be a metalloprotease. The protease was also resistant to various organic solvents (benzene, ethanol, methanol), surfactants (Triton X-100), sodium dodecyl sulfate (SDS), Tween 20, Tween-80 and oxidants hydrogen per oxide (H2O2). Characteristics, such as tolerance to high SDS and H2O2 concentrations, indicate that this protease has potential applications in the pharmaceutical and detergent industries.

Influence of maize genotypes and harvest stages on in-silo fermentation quality and nutritional value of corn silage during hot summer condition of the tropics.

The aim of the experiment was to evaluate the potential of promising summer maize genotypes and optimal stage of harvesting these genotypes for ensiling in terms of dry matter (DM), starch, and crude protein (CP) yields, silage fermentation quality, nutrients profile, total digestible nutrients, metabolizable energy (ME) content, Cornell Net Carbohydrate and Protein System (CNCPS) carbohydrate (CHO) subfractions composition, in vitro DM digestibility (DMD) and in situ starch degradation characteristics. Six maize genotypes were chosen for the study: DK9108 from Monsanto, P30Y87, P3939 from Pioneer, QPM-300 (quality protein maize) and W94 from the International Maize and Wheat Improvement Center (CIMMYT), and a local cultivar, Afgoii, from the Cereal Research Institute (Persabaq, KP). A total of 72 plots (8 m × 10 m) were blocked in three replicate fields, and within each field, each genotype was sown in four replicate plots according to a randomized complete block design. For the data analysis, the Proc-Mixed procedure of Statistical Analysis System with repeated measure analysis of variance was used. The DM yield was strongly influenced (P < 0.001) by maize genotypes, varying from 12.6 to 17.0 tons/ha. Except for total CHO and ammonia nitrogen (NH3-N), the contents of all measured chemical components varied (P < 0.001) among the genotypes. Further comparison revealed that, genotype P3939 had a higher (P < 0.05) content of CP (7.27 vs. 6.92%), starch (36.7 vs. 27.9%), DMD (65.4 vs. 60.0%), ME (2.51 vs. 2.30 Mcal/kg) and lactic acid (5.32 vs. 4.83%) and lowest content of NDF (37.3 vs. 43.1%), pH (3.7 vs. 4.10) compared to the local cultivar (Afgoii). Advancement of post-flowering maturity from 25 to 35% DM (23 to 41 days after flowering (DAF)) increased (P < 0.05) the DM yield (10.4 to 17.8 tons/ha), starch content (29.1 to 35.0%), DMD (65.3 to 67.3%) and ME (2.34 to 2.47 Mcal/kg), and decreased (P < 0.001) the contents of CP (7.42-6.73%), NDF (48.8-38.5%), pH (4.10 to 3.60), NH3-N (8.93-7.80%N) and effective degradability of starch (95.4 to 89.4). Results showed that for higher yields and silage nutritional and fermentation quality, maize crops should be harvested at whole crop DM content of 30-35% (34 to 41 DAF). It was further concluded that genotype P3939 is the most suitable summer maize genotype for silage production in terms of yields and silage nutritional and fermentation quality under the hot environmental conditions of the tropics.

Removal of Cr(VI) from Wastewater Using Acrylonitrile Grafted Cellulose Extracted from Sugarcane Bagasse.

A highly efficient low-cost adsorbent was prepared using raw and chemically modified cellulose isolated from sugarcane bagasse for decontamination of Cr(VI) from wastewater. First, cellulose pulp was isolated from sugarcane bagasse by subjecting it to acid hydrolysis, alkaline hydrolysis and bleaching with sodium chlorate (NaClO3). Then, the bleached cellulose pulp was chemically modified with acrylonitrile monomer in the presence Fenton's reagent (Fe+2/H2O2) to carry out grafting of acrylonitrile onto cellulose by atom transfer radical polymerization. The developed adsorbent (acrylonitrile grafted cellulose) was analyzed by X-ray diffraction analysis (XRD), scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FT-IR). Both raw cellulose and acrylonitrile grafted cellulose were used for chromium removal from wastewater. The effects of metal ion concentration, pH, adsorbent dose and time were studied, and their values were optimized. The optimum conditions for the adsorption of Cr(VI) onto raw and chemically modified cellulose were: metal ion concentration: 50 ppm, adsorbent dose: 1 g, pH: 6, and time: 60 min. The maximum efficiencies of 73% and 94% and adsorption capacities of 125.95 mg/g and 267.93 mg/g were achieved for raw and acrylonitrile grafted cellulose, respectively. High removal efficiency was achieved, owing to high surface area of 79.92 m2/g and functional active binding cites on grafted cellulose. Isotherm and kinetics studies show that the experimental data were fully fitted by the Freundlich isotherm model and pseudo first-order model. The adsorbent (acrylonitrile grafted cellulose) was regenerated using three different types of regenerating reagents and reused thirty times, and there was negligible decrease (19%) in removal efficiency after using it for 30 times. Hence, it is anticipated that acrylonitrile could be utilized as potential candidate material for commercial scale Cr(VI) removal from wastewater.

Apolipoprotein E Gene Variation in Pakistani Subjects with Type 2 Diabetes with and without Cardiovascular Complications.

Background: Apolipoprotein E (APOE) gene polymorphism has been implicated in the pathogenesis of various metabolic disorders, including type 2 diabetes mellitus (T2DM). Type 2 diabetes mellitus (T2DM) is a major public health concern worldwide, including in Pakistan. Cardiovascular problems linked with T2DM have a significant impact on individuals and society. The goal of this study is to investigate the relationship between Apolipoprotein E (ApoE) genotypes, dyslipidemia, and cardiovascular complications such as ischemic heart disease (IHD) and stroke. Methods: This study was carried out on 260 subjects divided into controls and diabetics. The diabetics were further divided into four subgroups such as D1: diabetics without cardiovascular issues, D2: diabetics with heart disease, D3: diabetics with stroke, and D4: diabetics with both heart disease and stroke. Anthropometric parameters (age, BMI) and risk factors (smoking, diabetes duration, hypertension) were assessed in all groups. Serum levels of TC, TG, LDL, HDL, VLDL, creatinine, BSF, and HbA1c were also measured. Apolipoprotein E gene polymorphism was determined using PCR-RFLP. Results: Hypertension, BMI, and dyslipidemia are defined as elevated levels of total cholesterol, triglycerides, LDL, and VLDL, and decreased levels of HDL. Uncontrolled hyperglycemia (elevated fasting blood sugar and glycated hemoglobin) in T2DM was linked to vascular complications such as IHD and stroke. Hypertension was prevalent in 79.3% of the population. Stage 2 hypertension was more prevalent in all age groups. It was also noted that common genotypes in the Pakistani population are 3/3, 4/4, 2/3, and 3/4. The frequency of genotypes 3/4 and 2/3 is highest in diabetics with stroke. Genotype 3/3 is present frequently in diabetics with IHD/stroke and patients with both these complications. However, genotype 4/4 is most frequently found in diabetics with IHD. Conclusions: It is concluded that BMI, hypertension, hyperglycemia, atherosclerosis, and dyslipidemia are linked with cardiovascular complications of type 2 diabetes. Apolipoprotein E gene polymorphism is associated with cardiovascular disease in patients with diabetes by affecting the lipid profile.

Detection of high-risk human papillomavirus infected cervical biopsies samples by immunohistochemical expression of the p16 tumor marker.

Cervical cancer is the fourth most common type of cancer in women worldwide. It is widely accepted that the main cause of cervical cancer, especially in underdeveloped countries like Pakistan, is the infection caused by the human papillomavirus (HPV). The current screening and diagnostic methods face several challenges in accurately detecting the various types of lesions caused by HPV. Therefore, the present study was conducted to assess the effectiveness of p16 immunohistochemistry (IHC) analysis as a diagnostic method in samples of cervical biopsies. One hundred cervical biopsy samples were obtained from female patients across various age groups (> 20- ≤ 30, > 31- ≤ 40, > 41- ≤ 50, > 51- ≤ 60 years). These samples were subsequently prepared for subsequent examination. All samples were analyzed using automated tissue processing followed by Hematoxylin and Eosin (H & E) staining, and p16 IHC tumour marker staining. The H & E slides showed changes in normal cervical tissues, while four cervical abnormalities were identified statistically significant using p16 marker including chronic cervicitis, nabothian cyst formation, cervical intraepithelial neoplasia, and cervical cancers (P value 0.014). Furthermore, among females of different age groups (> 31- ≤ 40, > 41- ≤ 50, > 51- ≤ 60 years) were found statistically significant suffering from cervical cancer (P value 0.04), HPV with cervical cancer (P value 0.01), HPV with cervical intraepithelial neoplasia (P value 0.01). Based on the available data, it can be inferred that the incorporation of the p16 tumor marker may be a valuable method for detecting high-risk HPV in cervical biopsies samples.

Anti-ulcerative colitis effects of chemically characterized extracts from Calliandra haematocephala in acetic acid-induced ulcerative colitis.

Background: Ulcerative colitis is a chronic immune-mediated inflammatory bowel disease that involves inflammation and ulcers of the colon and rectum. To date, no definite cure for this disease is available. Objective: The objective of the current study was to assess the effect of Calliandra haematocephala on inflammatory mediators and oxidative stress markers for the exploration of its anti-ulcerative colitis activity in rat models of acetic acid-induced ulcerative colitis. Methods: Methanolic and n-hexane extracts of areal parts of the plant were prepared by cold extraction method. Phytochemical analysis of both extracts was performed by qualitative analysis, quantitative methods, and high-performance liquid chromatography (HPLC). Prednisone at 2 mg/kg dose and plant extracts at 250, 500, and 750 mg/kg doses were given to Wistar rats for 11 days, which were given acetic acid on 8th day through the trans-rectal route for the induction of ulcerative colitis. A comparison of treatment groups was done with a normal control group and a colitis control group. To evaluate the anti-ulcerative colitis activity of Calliandra haematocephala, different parameters such as colon macroscopic damage, ulcer index, oxidative stress markers, histopathological examination, and mRNA expression of pro and anti-inflammatory mediators were evaluated. mRNA expression analysis was carried out by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Results: The phytochemical evaluation revealed polyphenols, flavonoids, tannins, alkaloids, and sterols in both extracts of the plant. Results of the present study exhibited that both extracts attenuated the large bowel inflammation and prevented colon ulceration at all tested doses. Macroscopic damage and ulcer scoreswere significantly decreased by both extracts. Malondialdehyde (MDA) levels and nitrite/nitrate concentrations in colon tissues were returned to normal levels while superoxide dismutase (SOD) activity was significantly improved by all doses. Histopathological examination exhibited that both extracts prevented the inflammatory changes, cellular infiltration, and colon thickening. Gene expression analysis by RT-qPCR revealed the downregulation of pro-inflammatory markers such as tumor necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) whereas the anti-inflammatory cytokines including Interleukin-4 (IL-4) and Interleukin-10 (IL-10) were found to be upregulated in treated rats. Conclusion: It was concluded based on study outcomes that methanolic and n-hexane extracts of Calliandra haematocephala exhibited anti-ulcerative colitis activity through modulation of antioxidant defense mechanisms and the immune system. In this context, C. haematocephala can be considered as a potential therapeutic approach for cure of ulcerative colitis after bioassay-directed isolation of bioactive phytochemicals and clinical evaluation.

Molecular characterization of plasma virome of hepatocellular carcinoma (HCC) patients.

Hepatocellular carcinoma (HCC) stands as the most common cancer type, arising from various causes, and responsible for a substantial number of cancer-related fatalities. Recent advancements in viral metagenomics have empowered scientists to delve into the intricate diversity of the virosphere, viral evolution, interactions between viruses and their hosts, and the identification of viral causes behind disease outbreaks, the development of specific symptoms, and their potential role in altering the host's physiology. The present study had the objective of "Molecular Characterization of HBV, HCV, anelloviruses, CMV, SENV-D, SENV-H, HEV, and HPV viruses among individuals suffering from HCC." A total of 381 HCC patients contributed 10 cc of blood each for this study. The research encompassed the assessment of tumor markers, followed by molecular characterization of HBV, HCV, Anelloviruses (TTV, TTMV, and TTMDV), SENV-H and SENV-D viruses, HEV, CMV, and HPV, as well as histopathological examinations. The outcomes of this study revealed that majority of the HCC patients 72.4% (276/381) were male as compared to females. HCV infection, at 76.4% (291 out of 381), exhibited a significant association (p < 0.05) with HCC. Most patients displayed singular lesions in the liver, with Child Pugh Score Type B being the predominant finding in 45.2% of cases. Plasma virome analysis indicated the prevalence of TTMDV (75%), followed by TTMV (70%) and TTV (42.1%) among anelloviruses in HCC patients. Similarly, SENV-H (52%) was followed by SENV-D (20%), with co-infections at 15%. The presence of CMV and HEV among the HCC patients was recorded 5% each however 3.5% of the patients showed the presence of HPV. In conclusion, this study underscores that HCC patients serve as reservoirs for various pathogenic and non-pathogenic viruses, potentially contributing to the development, progression, and severity of the disease.

Utilization of microalgae for agricultural runoff remediation and sustainable biofuel production through an integrated biorefinery approach.

Generally wastewater such agricultural runoff is considered a nuisance; however, it could be harnessed as a potential source of nutrients like nitrates and phosphates in integrated biorefinery context. In the current study, microalgae Chlorella sp. S5 was used for bioremediation of agricultural runoff and the leftover algal biomass was used as a potential source for production of biofuels in an integrated biorefinery context. The microalgae Chlorella sp. S5 was cultivated on Blue Green (BG 11) medium and a comprehensive optimization of different parameters including phosphates, nitrates, and pH was carried out to acquire maximum algal biomass enriched with high lipids content. Dry biomass was quantified using the solvent extraction technique, while the identification of nitrates and phosphates in agricultural runoff was carried out using commercial kits. The algal extracted lipids (oils) were employed in enzymatic trans-esterification for biodiesel production using whole-cell biomass of Bacillus subtilis Q4 MZ841642. The resultant fatty acid methyl esters (FAMEs) were analyzed using Fourier transform infrared (FTIR) spectroscopy and gas chromatography coupled with mass spectrometry (GC-MS). Subsequently, both the intact algal biomass and its lipid-depleted algal biomass were used for biogas production within a batch anaerobic digestion setup. Interestingly, Chlorella sp. S5 demonstrated a substantial reduction of 95% in nitrate and 91% in phosphate from agricultural runoff. The biodiesel derived from algal biomass exhibited a noteworthy total FAME content of 98.2%, meeting the quality standards set by American Society for Testing and Materials (ASTM) and European union (EU) standards. Furthermore, the biomethane yields obtained from whole biomass and lipid-depleted biomass were 330.34 NmL/g VSadded and 364.34 NmL/g VSadded, respectively. In conclusion, the findings underscore the potent utility of Chlorella sp. S5 as a multi-faceted resource, proficiently employed in a sequential cascade for treating agricultural runoff, producing biodiesel, and generating biogas within the integrated biorefinery concept.

Whole cell of pure Clostridium butyricum CBT-1 from anaerobic bioreactor effectively hydrolyzes agro-food waste into biohydrogen.

Recycling organic waste and converting them into renewable energy are a promising route for environment protection and effective biochemical reactions suitable for industrial hydrogen synthesis. This study targeted to isolate a pure anaerobic culture with potential to hydrolyze different biomass and production of biohydrogen. For this, a sample of full-scale anaerobic digester, fed with a multicomponent solid, was inoculated on Reinforced Clostridial Medium (RCM) in strict anaerobic conditions. An anaerobic Clostridium butyricum CBT-1 strain was isolated, identified from morphological and 16S rRNA sequence. The CBT-1 culture expressed amylase, cellulase and peroxidases activities. The strain exhibited visual decolorization of both Azure B and crystal violet dyes. In batch fermentation experiment, the CBT-1 produced highest of 3.06, 2.67 and 2.46 mol/mol H2 yield from glucose, starch and cellulose respectively, whereas, the CBT-1 showed low 0.43 mol H2/mol of substrate from untreated rice straw due to lignin in compact structure and comparatively high H2 yield of 1.91 and 2.01 mol H2/mol of substrate rice straw hydrolysate and kitchen food waste (KFWS) respectively. The cumulative volumetric yield of H2 was 358.15, 300.8 and 294.5NmL/gSub from glucose, starch and cellulose respectively. Similarly, the cumulative H2 volume was 76.7, 184.4, 237.2 NmL/gVS from untreated rice straw, rice straw hydrolysate and kitchen food waste. This study emphasizes the prospects to find similar robust anaerobic culture for hydrolyzing complex biomass. Such strains could be used as standard co-inoculum for biohydrogen obtaining and as the biocatalyst for commercial scale applications.

Eco-friendly textile desizing with indigenously produced amylase from Bacillus cereus AS2.

Starch is added to the fabric surface to secure weaving process. During finishing these sized particles are removed from the fabric and prepared it for printing and dyeing. Chemicals de-sizing agents damage fabric surfaces and reduce the quality of the product. An alternative to these conventional desizing agents is the use of biological molecules i.e. enzymes. The current study compares traditional de-sizing to bio-based de-sizing methods, as well as the optimization of fabric desizing settings using crude amylase. Amylase-producing Bacillus cereus AS2 was isolated from indigenous soil samples. The maximal fermentative de-sizing capability was discovered at 72 h, with no fabric surface degradation. Chemical desizing showed that the fabric lost all sizing agents to TEGEWA scale 9 within 1 h in presence of 5N HCl. Optimal studies for desizing showed that 1000 IU/ml of amylase resulted in maximum de-sizing within 15 h at 60 °C and 0.5% Triton-X. Water absorbance and weight loss, both parameters were used to check the desizing efficacy and it was found that de-sizing to same scale was occurred in the case of enzyme as well as commercially desized fabric. Enzyme desized cloth was found to be free of any starch particles in SEM micrographs, identical to industrially de-sized fabric, ensuring bioprocess efficacy.

Multiple strategies for the development of multienzyme complex for one-pot reactions.

The main intention in the modern era is to make life and activities on earth more comfortable by adding necessary products through biological machinery. Millions of tons of biological raw materials and lignocellulosic biomass are wasted by burning each year without providing benefits to living organisms. Instead of being the cause of disturbing the natural environment by increasing global warming and pollutants worldwide, now, it is the need of the hour to develop an advanced strategy to utilize these biological raw materials to produce renewable energy resources to meet the energy crisis. The review presents the idea of multiple enzymes in one step to hydrolyze complex biomaterials into useful products. The paper discusses how multiple enzymes are arranged in a cascade for complete hydrolysis of raw material in one-pot to prevent multistep, time consuming, and expensive methods. Furthermore, there was the immobilization of multiple enzymes in a cascade system with in vitro and in vivo conditions for reusability of enzymes. The role of genetic engineering, metabolic engineering, and random mutation techniques is described for the development of multiple enzyme cascades. Techniques that are involved in the improvement of native strain to recombinant strain for the enhancement of hydrolytic capacity were used. The preparative steps, before enzymatic hydrolysis like acid, and base treatment methods are more effective for improving the hydrolysis of biomass by multiple enzymes in a one-pot system. Finally, the applications of one-pot multienzyme complexes in biofuel production from lignocellulosic biomass, biosensor production, medicine, food industry, and the conversion of biopolymers into useful products are described.

Age-induced aortic modifications are accompanied by alterations in the antioxidant defense system in female rats.

Introduction: Aging leads to significant structural and functional changes in blood vessels, which disrupt their normal function and impact cardiovascular health. Current research is actively exploring the NRF2 antioxidative pathway, recognizing its role in protecting cells by preserving their antioxidant defenses against damage. However, there has been limited exploration into the role of the NRF2 pathway in vascular aging. The primary objective of this study was to determine whether age-related changes in the aorta are associated with variations in the baseline levels of antioxidant enzymes, with a particular emphasis on how the NRF2 pathway operates in the aortic wall. Methods: A group of healthy aging female SD rats was compared with their younger counterparts. Various assessments were conducted, including measuring blood pressure, analyzing serum lipid profiles, examining aortic tissue, and assessing the expression of antioxidant enzymes. Results: The results revealed significant differences in both blood pressure and serum lipid levels between the aged and younger rats. The examination of the aorta in older rats showed structural alterations, increased apoptosis, and the accumulation of fatty deposits. In the older rats, levels of SOD-1 (superoxide dismutase) and GSS (glutathione synthetase) were lower, whereas NRF2, KEAP-1 (Kelch-like ECH-associated protein 1), and HO-1 (Heme oxygenase 1) were higher. Discussion: This study advances our understanding of how aging affects the antioxidant system in blood vessels, particularly in relation to the regulation of the NRF2/HO-1 pathway in the aorta. These findings suggest that targeting the NRF2/HO-1 pathway could present anovel therapeutic approach for addressing age-related vascular issues.

Relative expression levels of growth hormone gene and growth rate in Indian major carp species.

The phenomenon of growth is a leading factor for aquaculture success. The uneven growth of major Indian carps (Labeo rohita, Catla catla, and Cirrhinus mrigala) is a serious issue in fish culture from an economic point of view. The growth hormone (GH) gene is crucial for selection in commercially cultivated fish species for better growth and production. Indian major carp (L. rohita, C. catla, and C. mrigala) are commonly cultured in Pakistan. The GH expression was examined using qPCR to understand growth in fish species better. Muscle tissue samples (n=480) from 160 individuals of the same age were collected from three species (L. rohita, C. catla, and C. mrigala). Individuals were divided into two groups (high-weight and low-weight groups), cultured under normal conditions. The housekeeping gene ß-actin validated GH expression in fast and slow-growing fishes from the same species. Results showed that GH expression varies across species and fish specimens that overweight their counterpart feature have higher GH expression. A selection for overweight fish in the aquaculture breeding systems is preferable as those fish could inherit their genomics to the future cohort, enhancing production, and commercial profit for farmers. Comprehensive research about different growth genes and the environmental aspects that influence fish growth is mandatory. No work has been reported regarding the growth gene analysis of fish from Pakistan. This report was Pakistan's first and baseline study regarding growth analysis of main culturable fish species at the molecular level.

Screening of environment water for the presence of blaNDM-1 gene containing microorganisms.

The acquisition of blaNDM-1 gene by Gram negative bacteria has emerged as critical threat to human health as these organisms display high resistance to available antibiotics. The presence of this gene has been observed in both clinical as well as environmental settings. The present study was carried out to evaluate the prevalence of blaNDM-1 containing microorganisms in water sources. A total of 241 water samples were collected from different sites in Islamabad, Rawalpindi and areas of Khyber Pakhtunkhwa. Meropenem resistant organisms were isolated and PCR was used for the detection of the blaNDM-1 gene among the resistant isolates of all the water samples, 43 meropenem resistant bacteria were isolated among which 12 isolates were found to carry blaNDM-1 gene. The blaNDM-1 isolates were resistant to most of the antibiotics but sensitive to colistin and polymyxin B. The presence of blaNDM-1 gene harboring organisms in aquatic environment is concerning and poses public health threat.