Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme.

This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 µg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 µg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 µg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 µg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Molecular Analysis and Risk Factors Associated With Theileria equi Infection in Domestic Donkeys and Mules of Punjab, Pakistan.

Theileriosis is an important disease of economic significance which badly affects the equine husbandry of developing countries. The present study was planned to investigate the molecular prevalence of theileriosis, associated risk factors, and alterations in hematological parameters of donkeys and mules from district Mianwali, Punjab, Pakistan. Blood samples from 150 equids (n = 75 donkeys; n = 75 mules) were examined microscopically, and the genomic DNA from each sample was processed for the amplification of the 18S rRNA gene of Theileria. The polymerase chain reaction confirmed isolates were purified followed by sequencing. The data regarding the analysis of risk factors were collected in a predesigned questionnaire and statistically analyzed by logistic regression analysis. An overall prevalence of 17.33% was noted in this study. Donkeys showed more prevalence followed by mules being 20.0% and 14.7%, respectively. The study isolates showed high resemblance (99%) with isolates from the United States of America, Spain, Brazil, Israel, Cuba, France, South Africa, Korea, Turkey, Tunisia, India, E. Caribbean, and Nigeria. The potential risk factors found to be significantly associated (P < .05) with disease dynamics were tick infestation on study animals, previous tick history, and house hygiene. A significant (P < .05) decrease in the number of platelets, erythrocytes, hemoglobin level, and packed cell volume was observed in donkeys and mules suffering from theileriosis compared with the healthy ones. The study is the first report regarding the molecular characterization of theileriosis in donkeys and mules in Pakistan. The findings will be effectual in designing effective control strategies for this disease in Punjab, Pakistan.

Targeting STAT-3 signaling pathway in cancer for development of novel drugs: Advancements and challenges.

Signal transducers and activators of transcription 3 (STAT-3) is a transcription factor that regulates the gene expression of several target genes. These factors are activated by the binding of cytokines and growth factors with STAT-3 specific receptors on cell membrane. Few years ago, STAT-3 was considered an acute phase response element having several cellular functions such as inflammation, cell survival, invasion, metastasis and proliferation, genetic alteration, and angiogenesis. STAT-3 is activated by several types of inflammatory cytokines, carcinogens, viruses, growth factors, and oncogenes. Thus, the STAT3 pathway is a potential target for cancer therapeutics. Abnormal STAT-3 activity in tumor development and cellular transformation can be targeted by several genomic and pharmacological methodologies. An extensive review of the literature has been conducted to emphasize the role of STAT-3 as a unique cancer drug target. This review article discusses in detail the wide range of STAT-3 inhibitors that show antitumor effects both in vitro and in vivo. Thus, targeting constitutive STAT-3 signaling is a remarkable therapeutic methodology for tumor progression. Finally, current limitations, trials and future perspectives of STAT-3 inhibitors are also critically discussed.

Chitosan oligosaccharide (COS): An overview.

The frequently studied polysaccharide, chitosan oligosaccharide/chitooligosaccharide (COS) is the major degradation product of chitosan/chitin via chemical hydrolysis or enzymatic degradation involving deacetylation and depolymerization processes. Innumerable studies have revealed in the recent decade that COS has various promising biomedical implications in the past analysis, current developments and potential applications in a biomedical, pharmaceutical and agricultural sector. Innovations into COS derivatization has broadened its application in cosmeceutical and nutraceutical productions as well as in water treatment and environmental safety. In relation to its parent biomaterials and other available polysaccharides, COS has low molecular weight (Mw), higher degree of deacetylation (DD), higher degree of polymerization (DP), less viscous and complete water solubility, which endowed it with significant biological properties like antimicrobial, antioxidant, anti-inflammatory and antihypertensive, as well as drug/DNA delivery ability. In addition, it is also revealed to exhibit antidiabetic, anti-obesity, anti-HIV-1, anti-Alzheimer's disease, hypocholesterolemic, calcium absorption and hemostatic effects. Furthermore, COS is shown to have higher cellular transduction and completely absorbable via intestinal epithelium due to its cationic sphere exposed on the more exposed shorter N-glucosamine (N-Glc) units. This paper narrates the recent developments in COS biomedical applications while paying considerable attention to its physicochemical properties and its chemical composition. Its pharmacokinetic aspects are also briefly discussed while highlighting potential overdose or lethal dosing. In addition, due to its multiple NGlc unit composition and vulnerability to degradation, its safety is given significant attention. Finally, a suggestion is made for extensive study on COS anti-HIV effects with well-refined batches.

Effect of timing of artificial insemination in relation to onset of standing estrus on pregnancy per AI in Nili-Ravi buffalo.

The aim of the present study was to determine the optimum time of artificial insemination after the beginning of standing estrus in buffalo. Nili-Ravi buffalo (n = 109) during breeding season were exposed to teaser bull at 12 hours interval to determine the standing heat (0 h). Buffalo were randomly allocated to different time groups and a single artificial insemination was performed either at 0 h (n = 30), 12 h (n = 27), 24 h (n = 28) or 36 h (n = 24). In a subset of buffalo (n = 38) ultrasonography was performed, twice daily from 0 h (onset of standing heat) to determine the time of ovulation. Pregnancy diagnosis was performed 35-40 days after AI. Results revealed that mean time of ovulation from onset of standing heat was 34.7 ± 0.96 h (range 30 to 42 h). Higher (P < 0.05) pregnancy per AI were achieved in buffalo when inseminated at 24 h (15/28; 53%) compared to 0 h (8/30; 26%) and 36 h (3/24; 13%). Pregnancy per AI, was in-between, in buffalo, inseminated at 12 h (10/27; 37%) and did not differ (P > 0.05) with those bred either at 24 h or 0 h. The odds ratio further confirmed that the occurrence of pregnancy per AI was two times higher in buffalo inseminated at 24 h as compared to those at 12 h. It is concluded that optimal pregnancy per AI can be achieved when buffalo are bred artificially 24 h after the onset of standing heat.

Effect of timing of artificial insemination in relation to onset of standing estrus on pregnancy per AI in Nili-Ravi buffalo

The aim of the present study was to determine the optimum time of artificial insemination after the beginning of standing estrus in buffalo. Nili-Ravi buffalo (n = 109) during breeding season were exposed to teaser bull at 12 hours interval to determine the standing heat (0 h). Buffalo were randomly allocated to different time groups and a single artificial insemination was performed either at 0 h (n = 30), 12 h (n = 27), 24 h (n = 28) or 36 h (n = 24). In a subset of buffalo (n = 38) ultrasonography was performed, twice daily from 0 h (onset of standing heat) to determine the time of ovulation. Pregnancy diagnosis was performed 35-40 days after AI. Results revealed that mean time of ovulation from onset of standing heat was 34.7 ± 0.96 h (range 30 to 42 h). Higher (P 0.05) with those bred either at 24 h or 0 h. The odds ratio further confirmed that the occurrence of pregnancy per AI was two times higher in buffalo inseminated at 24 h as compared to those at 12 h. It is concluded that optimal pregnancy per AI can be achieved when buffalo are bred artificially 24 h after the onset of standing heat.

Effect of timing of artificial insemination in relation to onset of standing estrus on pregnancy per AI in Nili-Ravi buffalo

The aim of the present study was to determine the optimum time of artificial insemination after the beginning of standing estrus in buffalo. Nili-Ravi buffalo (n = 109) during breeding season were exposed to teaser bull at 12 hours interval to determine the standing heat (0 h). Buffalo were randomly allocated to different time groups and a single artificial insemination was performed either at 0 h (n = 30), 12 h (n = 27), 24 h (n = 28) or 36 h (n = 24). In a subset of buffalo (n = 38) ultrasonography was performed, twice daily from 0 h (onset of standing heat) to determine the time of ovulation. Pregnancy diagnosis was performed 35-40 days after AI. Results revealed that mean time of ovulation from onset of standing heat was 34.7 ± 0.96 h (range 30 to 42 h). Higher (P < 0.05) pregnancy per AI were achieved in buffalo when inseminated at 24 h (15/28; 53%) compared to 0 h (8/30; 26%) and 36 h (3/24; 13%). Pregnancy per AI, was in-between, in buffalo, inseminated at 12 h (10/27; 37%) and did not differ (P > 0.05) with those bred either at 24 h or 0 h. The odds ratio further confirmed that the occurrence of pregnancy per AI was two times higher in buffalo inseminated at 24 h as compared to those at 12 h. It is concluded that optimal pregnancy per AI can be achieved when buffalo are bred artificially 24 h after the onset of standing heat.(AU)

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing.

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66.

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p ≤ 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Identification and characterization of rhizospheric microbial diversity by 16S ribosomal RNA gene sequencing

In the present study, samples of rhizosphere and root nodules were collected from different areas of Pakistan to isolate plant growth promoting rhizobacteria. Identification of bacterial isolates was made by 16S rRNA gene sequence analysis and taxonomical confirmation on EzTaxon Server. The identified bacterial strains were belonged to 5 genera i.e. Ensifer, Bacillus, Pseudomona, Leclercia and Rhizobium. Phylogenetic analysis inferred from 16S rRNA gene sequences showed the evolutionary relationship of bacterial strains with the respective genera. Based on phylogenetic analysis, some candidate novel species were also identified. The bacterial strains were also characterized for morphological, physiological, biochemical tests and glucose dehydrogenase (gdh) gene that involved in the phosphate solublization using cofactor pyrroloquinolone quinone (PQQ). Seven rhizoshperic and 3 root nodulating stains are positive for gdh gene. Furthermore, this study confirms a novel association between microbes and their hosts like field grown crops, leguminous and non-leguminous plants. It was concluded that a diverse group of bacterial population exist in the rhizosphere and root nodules that might be useful in evaluating the mechanisms behind plant microbial interactions and strains QAU-63 and QAU-68 have sequence similarity of 97 and 95% which might be declared as novel after further taxonomic characterization.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.

Bioinformatics based structural characterization of glucose dehydrogenase (gdh) gene and growth promoting activity of Leclercia sp. QAU-66

Glucose dehydrogenase (GDH; EC 1.1. 5.2) is the member of quinoproteins group that use the redox cofactor pyrroloquinoline quinoine, calcium ions and glucose as substrate for its activity. In present study, Leclercia sp. QAU-66, isolated from rhizosphere of Vigna mungo, was characterized for phosphate solubilization and the role of GDH in plant growth promotion of Phaseolus vulgaris. The strain QAU-66 had ability to solubilize phosphorus and significantly (p < 0.05) promoted the shoot and root lengths of Phaseolus vulgaris. The structural determination of GDH protein was carried out using bioinformatics tools like Pfam, InterProScan, I-TASSER and COFACTOR. These tools predicted the structural based functional homology of pyrroloquinoline quinone domains in GDH. GDH of Leclercia sp. QAU-66 is one of the main factor that involved in plant growth promotion and provides a solid background for further research in plant growth promoting activities.