Mentha piperita silver nanoparticle-loaded hydrocolloid film for enhanced diabetic wound healing in rats.

OBJECTIVE: To investigate the role of Mentha piperita silver nanoparticle-loaded carbopol gel for enhanced wound healing in a diabetic rat model. This research further aims to explore bioactive compounds derived from Mentha piperita obtained from high altitude. METHOD: Methanolic extracts of Mentha piperita (MP), Mentha spicata (MS) and Mentha longifolia (ML) were used to synthesise silver nanoparticles (AgNP). AgNP synthesis was confirmed by ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antioxidant activity was assessed by 2, 2-diphenyl-1-picrylhydrazyl (DDPH) assay. Antiglycation potential was determined by measuring the fluorescent advanced glycation end products. The bioactive compound identified in the Mentha piperita methanolic (MPM) fraction through electrospray ionisation tandem mass spectrometric analysis (ESI-MS) was responsible for the highest antiglycation. The effects of MPM and MPM.AgNP-loaded Carbopol (Sanare Lab, India) on wound healing were compared in male, alloxan-induced, diabetic albino rats (200-250g), divided into control and treated groups. Effects on wound healing were assessed via histopathology. RESULTS: UV-Vis and FTIR confirmed NP synthesis with peaks for flavonoids and polyphenols. SEM and XRD explored the cubical, 30-63nm crystalline NP. The maximum antioxidant and antiglycation potential was observed in order of; MP.AgNP>MS.AgNP>ML.AgNP. The highest antioxidant activity was observed by methanolic and aqueous MP.AgNPs (88.55% and 83.63%, respectively) at 2mg.ml-1, and (75.16% and 69.73%, respectively) at 1mg.ml-1, compared to ascorbic acid (acting as a positive control, 90.01%). MPM.AgNPs demonstrated the best antiglycation potential of 75.2% and 83.3% at 1mg.ml-1 and 2mg.ml-1, respectively, comparable to positive control (rutin: 88.1%) at 14 days post-incubation. A similar trend was observed for antimicrobial activity against Bacillus subtilis, Micrococcus luteus and Escherichia coli with an inhibition zone of 21mm, 21.6mm and 24.6mm. Rosmarinic acid was the active compound present in Mentha piperita, as identified by ESI-MS. MPM.AgNP-loaded Carbopol resulted in 100% wound closure compared with control at 20 days post-wounding. In the treatment group, re-epithelialisation was achieved by day 18, compared with 25 days for the positive control group. CONCLUSION: MPM.AgNP-loaded Carbopol demonstrated safer and more effective biological properties, hence accelerating the diabetic excision wound healing process in alloxan-induced diabetic rats.

Expression and immobilization of trypsin-like domain of serine protease from Pseudomonas aeruginosa for improved stability and catalytic activity.

Protein engineering and enzyme immobilization strategies have produced numerous biocatalysts for modern industrial applications. In this study, we have also used these two strategies for improving the operational stability and catalytic efficiency of serine protease from Pseudomonas aeruginosa. The enzyme serine protease was truncated to separate its trypsin-like domain from the PDZ1 and PDZ2 domains. The truncated trypsin-like domain was expressed in Escherichia coli BL21, and its catalytic activity and thermostability were estimated. Later this trypsin-like domain was immobilized with 2% Na-alginate. The immobilized domain showed 10°C increase in optimum temperature compared to its free counterpart. Kinetic studies showed two-folds increased Vmax of the immobilized domain. Likewise, the Km value of this domain was 11.5 folds lower compared to the free trypsin-like domain. The catalytic efficiency (Kcat /Km ) of the immobilized enzyme also elevated to 311 folds. Additionally, the immobilized trypsin-like domain remained active in the presence of surfactants (Triton-X 100, SDS, and Tween-40) and metal ions (Mg2+ , Ca2+ , Na+ , and Zn2+ ). It also efficiently removes gelatin layer from X-ray film and hair from sheepskin. Thus, the immobilized trypsin-like domain of serine protease, with increased thermostability and catalytic efficiency, is operationally more stable than the soluble truncated trypsin-like domain.

Characterization of recombinant endo-1,4-ß-xylanase of Bacillus halodurans C-125 and rational identification of hot spot amino acid residues responsible for enhancing thermostability by an in-silico approach.

Increased demand of enzymes for industrial use has led the scientists towards protein engineering techniques. In different protein engineering strategies, rational approach has emerged as the most efficient method utilizing bioinformatics tools to produce enzymes with desired reaction kinetics; physiochemical (temperature, pH, half life, etc) and biological (selectivity, specificity, etc.) characteristics. Xylanase is one of the widely used enzymes in paper and food industry to degrade xylan component present in plant pulp. In this study endo 1,4-ß-xylanase (Xyl-11A) from Bacillus halodurans C-125 was cloned in pET-22b (+) vector and expressed in Escherichia coli BL21 (DE3) expression strain. The enzyme had Michaelis constant Km of 1.32 mg ml-1 birchwoodxylan (soluble form) and maximum reaction velocity (Vmax) 73.53 mmol min-1 mg-1 with an optimum temperature of 75 °C and pH 9.0. The thermostability analysis showed that enzyme retained more than 80% of its residual activity when incubated at 75 °C for 2 h. In addition, to increase Xyl-11A thermostability, an in-silico analysis was performedto identify the hot spot amino acid residues. Consensus-based amino acid substitution was applied to evaluate multiple sequence alignment of homologs and identified 20 amino acids positions by following Jensen-Shnnon Divergence method. 3D models of 20 selected mutants were analyzed for conformational transition in protein structures by using NMSim server. Two selected mutants T6K and I17M of Xyl-11A retained 40, 60% residual activity respectively, at 85 °C for 120 min as compared to wild type enzyme which retained 37% initial activity under same conditions, confirming the enhanced thermostability of mutants. The present study showed a good approach for the identification of promising amino acid residues responsible for enhancing the thermostability of enzymes of industrial importance.

Prevalence of molecular markers of sulfadoxine-pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan.

BACKGROUND: In Pakistan, artesunate (AS) in combination with sulfadoxine-pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan. METHODS: A total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing. RESULTS: The prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance. CONCLUSION: The absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

Biosynthesis of silver nanoparticles from leaf extract of Litchi chinensis and its dynamic biological impact on microbial cells and human cancer cell lines.

Green synthesis of metallic nanoparticles has attracted a great deal of attention from scientific community due to its biocompatibility and environment friendly nature. In the present study, silver nanoparticles were biologically synthesized using leave extracts of Litchi chinensis. Biosynthesized silver nanoparticles were characterized and their applications were observed by different methodologies. Bio-reduction reaction was confirmed by the surface plasmon resonance of silver nanoparticles at 417 nm through UV-VIS spectrophotometer. FTIR analysis revealed that the amine groups present in the leaf extracts were responsible for the reduction of silver ions to silver nanoparticles. X-ray diffraction analysis was used to determine the crystalline nature of silver nanoparticles and their diameter was noted in the range of 41-55 nm by scanning electron microscopy. Antibacterial activity was observed against gram positive and gram negative strains of bacteria. Furthermore, human epithelial type 2 cancer cells (HEp-2) and Human breast adenocarcinoma cells lines (MCF-7) were treated with the biosynthesized silver nanoparticles using MTT assay. The resulting cell death rate was noted up to 40.91+1.99%. This study concludes that plant mediated biosynthesis of nanoparticles is the superior alternative compared to chemical and physical approaches, to utilize them as drug delivery tool and need to conjugate apoptosis inducing biological agents with silver nanoparticles to suppress the uncontrolled division of cancer cells.

Potential involvement of mi-RNA 574-3p in progression of prostate cancer: A bioinformatic study.

Aberrant gene expression is a hallmark of prostate cancer (PCa), the second deadliest disease affecting males worldwide. Dysregulation of miRNA has been associated with the progression of PCa and in recent studies, miRNA 574-3p was found to be upregulated in cancerous prostate tissue. In this study, we characterize the effects of upregulated miRNA 574-3p on gene expression in the tumor microenvironment through different bioinformatic tools such as Diana-Tools, the KEGG Pathway Database, and the Reactome Database. We have identified nine regulatory genes that are targeted by miRNA 574-3p and downregulated in prostate cells. Pathway analysis of these genes shows that they are involved in the regulation of the Notch signaling pathway, Wnt signaling pathway, apoptosis, DNA damage response, G1 to S cell-cycle control, inflammatory response pathway, angiogenesis, translation factors, and the expression of oncogenes. Our results show the oncogenic potential of miRNA 574-3p in PCa progression and metastasis. Moreover, this study highlights the complex molecular mechanisms and pathways affected by the upregulation of miRNA 574-3p in prostate cells. In future studies, the presented data may aid in designing new therapies for PCa with improved efficacy.

Approach towards sustainable leather: Characterization and effective industrial application of proteases from Bacillus sps. for ecofriendly dehairing of leather hide.

Tanneries are one of the most polluted industries known for production of massive amount of solid and liquid wastes without proper management and disposal. In this project we demonstrated the ecofriendly single step dehairing of leather hides with minimum pollution load. In this study, Bacillus species (Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P) capable of producing proteases was successfully isolated by employing the new optimized selective media named M9-PEA as confirmed by 16sRNA genes sequencing. Sequence of 1493 bp long 16S rRNA genes of Bacillus paralicheniformis strain BL.HK and Bacillus cereus strain BS. P was submitted to GenBank under the accession number OP612692.1, OP612721.1 respectively The Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P produced extracellur proteases of 28 and 37 KDa as resolved by SDS-PAGE respectively. The enzymes showed temperature optima at 50 °C and 55 °C and pH optima at 8.5, 9.5 respectively. The Proteases of Bacillus paralicheniformis strain BL.HK, Bacillus cereus strain BS.P were employed for dehairing of animal hides. The process resulted in significant removal of interfibriller substances without damage to collagen layer after one hour treatment, which was confirmed by histology, scanning electron microscopy. The quantification of various skin constituents (collagen, uronic acid, hexosamines, and GAGs) and pollution load parameters revealed that enzymatic treatment are more reliable. The results of skin application trials at industrial level with complete elimination of chemicals remark the biotechnological potential of these proteases for ecofriendly dehairing of animal hides without affecting the quality of the leathers produced.

Tragacanth gum-based copper oxide nanoparticles: Comprehensive characterization, antibiofilm, antimicrobial and photocatalytic potentials.

Hereunder, we pioneered the synthesis of Copper Oxide nanoparticles (CuO NPs) utilizing Tragacanth gum (TG). The NPs were characterized using advanced techniques and assessed for different pharmaceutical and environmental perspectives. The successful formation of a colloidal NPs solution was confirmed by the appearance of a distinct black color and a distinct peak at 260 nm in UV-Visible spectrophotometry. The FTIR analysis unveiled a spectrum of functional groups responsible for the reduction and stabilization of CuO NPs. Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) revealed size of NPs as 36.24 nm and 28 ± 04 nm respectively. Energy Dispersive X-ray (EDX) Analysis indicated weight percentages of 70.38 % for Cu and 18.88 % for O, with corresponding atomic percentages. The X-ray Diffraction (XRD) analysis revealed the orthorhombic crystal structure of the prepared CuO NPs. Antimicrobial assessments through disc-diffusion assays demonstrated significant zones of inhibition (ZOI) against gram-positive bacterial strains (Bacillus Halodurans and Micrococcus leutus) and a gram-negative bacterial strain (E. coli). Against the fungal strain Aspergillus niger, a ZOI of 18.5 ± 0.31 mm was observed. The NPs exhibited remarkable antioxidant potential determined through 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and H2O2 scavenging assays. At a concentration of 3 mg/mL, the NPs demonstrated biofilm inhibition rates of 96 %, 90 %, 89.60 %, and 72.10 % against Micrococcus luteus, Bacillus halodurans, MRSA and E.coli respectively. Furthermore, the CuO NPs showed a high photocatalytic potential towards the degradation of safranin dye under sunlight irradiation. In conclusion, the findings underline the promising multifunctional properties of TG-based CuO NPs for different practical applications.

GC-MS metabolomics profile of methanol extract of Acacia modesta gum and gum-assisted fabrication and characterization of gold nanoparticles through green synthesis approach.

Hereunder, for the first time, we reported phytocompounds in the methanolic extract of Acacia modesta (AM) gum through Gas chromatography-mass spectrometry (GS-MS). Further, the AM gum aqueous solution was used for gold nanoparticles (AuNPs) synthesis through a simple, swift, eco-friendly, and less costly green synthesis approach. A total of 108 phytocompounds (63 with nonpolar, 45 with polar column) were identified in the gum extract, which includes fatty acids, alcohols, sterols, aldehyde/ketones, furans, aromatic compounds, esters, phenols, terpenes, sugar derivatives, alkaloids, and flavones. From three used concentrations (5, 10, and 15 mg/mL) of the AM gum aqueous solution, the 15 mg/mL gum solution resulted in more successful AuNP synthesis with a smaller size, which was visualized by a rusty red color appearance. UV-Visible absorption spectroscopy revealed the characteristic surface plasmon resonance (SPR) of AuNPs in aqueous solution at 540 nm. Dynamic light scattering (DLS) measurement of NPs solution revealed a hydrodynamic diameter of 162 ± 02 nm with the highest gum concentration where core AuNPs diameter was 22 ± 03 nm, recorded by Transmission electron microscopy. Zeta potential revealed fair stability of AuNPs that was not decreased with time. Catalytic activity experiments revealed that AM gum-based AuNPs can increase the rate of the reduction of methylene blue 10 times in comparison with AM gum extract alone. Results from this study showed that a diverse array of phytocompounds in AM gum can successfully reduce gold ions into gold nanoparticles, which can be used further in different pharmaceutical and industrial applications.

Production of Sitobion avenae-resistant Triticum aestivum cvs using laccase as RNAi target and its systemic movement in wheat post dsRNA spray.

Among the wheat biotic stresses, Sitobion avenae is one of the main factors devastating the wheat yield per hectare. The study's objective was to find out the laccase (lac) efficacy; as a potential RNAi target against grain aphids. The Sitobion avenae lac (Salac) was confirmed by Reverse Transcriptase-PCR. Gene was sequenced and accession number "ON703252" was allotted by GenBank. ERNAi tool was used to design 143 siRNA and one dsRNA target. 69% mortality and 61% reduction in lac expression were observed 8D-post lac DsRNA feeding. Phylogenetic analysis displayed the homology of grain aphid lac gene with peach potato, pea, and Russian wheat aphids. While Salac protein was found similar to the Russian grain, soybean, pea, and cedar bark aphid lac protein multi-copper oxidase. The dsRNAlac spray-induced silencing shows systematic translocation from leaf to root; with maximum lac expression found in the root, followed by stem and leaf 9-13D post-spray; comparison to control. RNAi-GG provides the Golden Gate cloning strategy with a single restriction ligation reaction used to achieve lac silencing. Agrobacterium tumefaciens mediated in planta and in-vitro transformation was used in the study. In vitro transformation, Galaxy 2012 yielded a maximum transformation efficiency (1.5%), followed by Anaj 2017 (0.8%), and Punjab (0.2%). In planta transformation provides better transformation efficiencies with a maximum in Galaxy 2012 (16%), and a minimum for Punjab (5%). Maximum transformation efficiency was achieved for all cultivars with 250 µM acetosyringone and 3h co-cultivation. Galaxy 2012 exhibited maximum transformation efficiency, and aphid mortality post-feeding transgenic wheat.

Assessment of Microscopic Detection of Malaria with Nested Polymerase Chain Reaction in War-Torn Federally Administered Tribal Areas of Pakistan.

INTRODUCTION: Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria-endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. METHODS: Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016-December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. RESULTS: Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. Plasmodium vivax was found in 623, Plasmodium falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. CONCLUSION: The conventional microscopy method has low sensitivity to detect the mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR make this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.

Environmental impact and diversity of protease-producing bacteria in areas of leather tannery effluents of Sialkot, Pakistan.

Massive discharge of wastes produced by the processing of leather so far confers the most important environmental challenge facing the tanneries worldwide. Waste material from tanneries mostly consists of skin remnants and proteinaceous substances as by-products of leather processing. In these conditions, protease-producing bacteria play a vital role in degrading wastes in this sludge. Therefore, an investigation was made to study the effect of long-term tannery sludge contamination on the diversity of both protease-producing microbes and of bacterial extracellular proteases near tanneries of Sambrial and Sialkot. The high amount of carbon and nitrogen in the soil samples reflected their effect on the diversity of the microbial communities in these areas. Phylogenetic analysis based on 16S rRNA gene sequences suggest that the isolated proteolytic bacteria belonged to 9 different genera including Pseudomonas (26.19%), Proteus (19.04%), Serratia (16.66%), Klebsiella (14.28%), Providencia (9.52%), Achromobacter (7.14%), Enterobacter (2.38%), Myroides (2.38%), and Acinetobacter (2.38%). Enzyme activity showed that among all Pseudomonas and Proteus showed relatively high protease production, and inhibition studies revealed that proteases produced by all isolates were strongly inhibited by serine and/or metalloprotease inhibitors, and a smaller proportion was inhibited by inhibitors of cysteine and/or aspartic proteases. Furthermore, isolated bacteria revealed promising degradation activities against casein and/or gelatin with only a few that could hydrolyze elastin, suggesting proteases produced by these isolated bacteria belong to different classes of proteases, i.e., serine and metalloproteases. This study provided new insights on the community structure of cultivable protease-producing bacteria near tannery sludge of Sambrial and Sialkot. This study would be beneficial not only for establishing the way for effective degradation of tannery slugs but also for questing the novel properties of proteases for a future technological application.

Surveillance of molecular markers of antimalarial drug resistance in Plasmodium falciparum and Plasmodium vivax in Federally Administered Tribal Area (FATA), Pakistan.

This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.

Antioxidant and antiglycation activities of traditional plants and identification of bioactive compounds from extracts of Hordeum vulgare by LC-MS and GC-MS.

Glycation has been involved in Schiff base reaction lead to hyperglycemia at cellular level. The current study aimed to identify the bioactive compounds from selected folkloric plants for their antiglycation and antioxidant potential. Methanol extracts demonstrated the highest activities, therefore, it was further fractionated using n-hexane, dichloromethane, ethyl acetate, and methanol solvents to isolate the nonpolar compounds from the Hordeum vulgare. Moreover, n-hexane and dichloromethane fractions of H. vulgare demonstrated the best antioxidant (61.58% and 62.89%) and antiglycation activities (72.52% and 61.52%) at 2 mg/ml, respectively. Analytical techniques of LC-MS and GC-MS were employed for identification of bioactive compounds; Biochanin A in dichloromethane (DCM) and Vitamin E in n-hexane fractions. There was a strong correlation between antioxidant and antiglycation activities (r = 0.97 and r = 0.96) of DCM & n-hexane fractions of H. vulgare. Findings of this study established the role of Biochanin A and Vit E from H. vulgare as potent antiglycation agents. PRACTICAL APPLICATIONS: The results of this study confirmed the potential role of Black Barley has involved in the inhibition of protein glycation, which can be the potential treatment to reduce the complications of Diabetic Patients. The Black Barley has a rich source of identified compounds Biochanin A and Vitamin E. We can use this plant as a staple food in curing the severity of diabetes. The other practical approach is to use this plant as an ingredient of different food products. The extraction of identified bioactive compounds from the plant will be a good and cheap source of the treatment.

Engineering of serine protease for improved thermostability and catalytic activity using rational design.

The study involves the isolation and characterization of a serine peptidase, named SP, from Pseudomonas aeruginosa. In addition to basic characterization, the protein was engineered, by site-directed mutagenesis of selected non-catalytic residues, to increase its thermal stability and catalytic activity. Among the eight-point mutations, predicted by FireProt, two mutants, A29G and V336I, yielded a positive impact. The Tm of A29G and V336I showed an increase by 5 °C and also a substantial increase in residual activity of the enzyme at elevated temperature. Moreover, the catalytic activity of A29G and V336I also showed an increase of 1.4-fold activity, compared to the wild-type (WT). Moreover, molecular docking simulations also predicted better substrate affinity of the mutants. We have also performed molecular dynamics (MD) simulations at 315 and 345 K, and the MD data at 345 K demonstrates improved thermostability for the mutants, compared to the WT. Our findings not only contribute to a better understanding of the structure-stability-activity relationship of SP but also highlights, that modification of non-catalytic residues could also promote favourable catalytic behaviour.

Mutational screening of GABRG2 gene in Pakistani population of Punjab with generalized tonic clonic seizures and children with childhood absence epilepsy.

BACKGROUND: Epilepsy is a multifaceted and multistep disorder that disrupts the proper functioning of neurons. It is becoming increasingly clear that the responsiveness of neurons depends on the appropriate trafficking of ions across the channels in the membrane of neurons. In line with this notion, impairment among these ion channels due to mutations has gain increasing attention in molecular neuroscience. METHODS: Mutation analysis of the coding exons (exon 3, 5 and 9) was performed by sequencing GABRG2 to identify any complex biological entities among two different types of epilepsies. RESULTS: Sequencing of the candidate gene "GABRG2" revealed a single polymorphic site in exon 3 in the children with absence epilepsy and generalized tonic clonic seizures. However, this single nucleotide alteration was more common in the patients with childhood absence epilepsy patients compared to the generalized cases. CONCLUSION: A silent mutation was identified at locus 27,909 C > T in 30.66% of the total screened or analyzed cases. However, no single nucleotide polymorphism was identified in exon 5 of GABRG2 in a Pakistani population, in contrast to a study of Chinese patients with childhood absence epilepsy.

Plant-bacterium interactions analyzed by proteomics.

The evolution of the plant immune response has resulted in a highly effective defense system that is able to resist potential attack by microbial pathogens. The primary immune response is referred to as pathogen associated molecular pattern (PAMP) triggered immunity and has evolved to recognize common features of microbial pathogens. In response to the delivery of pathogen effector proteins, plants acquired R proteins to fight against pathogen attack. R-dependent defense response is important in understanding the biochemical and cellular mechanisms and underlying these interactions will enable molecular and transgenic approaches for crops with increased biotic resistance. Proteomic analyses are particularly useful for understanding the mechanisms of host plant against the pathogen attack. Recent advances in the field of proteome analyses have initiated a new research area, i.e., the analysis of more complex microbial communities and their interaction with plant. Such areas hold great potential to elucidate, not only the interactions between bacteria and their host plants, but also of bacteria-bacteria interactions between different bacterial taxa, symbiotic, pathogenic bacteria, and commensal bacteria. During biotic stress, plant hormonal signaling pathways prioritizes defense over other cellular functions. Some plant pathogens take advantage of hormone dependent regulatory system by mimicking hormones that interfere with host immune responses to promote virulence (vir). In this review, it is discussed the cross talk that plays important role in response to pathogens attack with different infection strategies using proteomic approaches.

Heterologous expression and enhanced production of ß-1, 4-glucanase of Bacillus halodurans C-125 in Escherichia coli

Background: Recombinant DNA technology enables us to produce proteins with desired properties and insubstantial amount for industrial applications. Endo-1, 4-ß-glucanases (Egl) is one of the major enzyme involved in degradation of cellulose, an important component of plant cell wall. The present study was aimed at enhancing the production of endo-1, 4-ß-glucanases (Egl) of Bacillus halodurans in Escherichia coli. Results: A putative Egl gene of Bacillus Halodurans was expressed in E. coli by cloning in pET 22b (+). On induction with isopropyl-b-D-1-thiogalactopyranoside, the enzyme expression reached upto ~20% of the cell protein producing 29.2 mg/liter culture. An increase in cell density to 12 in auto-inducing LB medium (absorbance at 600 nm) enhanced ß-glucanase production up to 5.4 fold. The molecular mass of the enzyme was determined to be 39 KDa, which is nearly the same as the calculated value. Protein sequence was analyzed by CDD, Pfam, I TASSER, COACH, PROCHECK Servers and putative amino acids involved in the formation of catalytic, substrate and metal binding domains were identified. Phylogenetic analysis of the ß-glucanases of B. halodurans was performed and position of Egl among other members of the genus Bacillus producing endo-glucanases was determined. Temperature and pH optima of the enzyme were found to be 60°C and 8.0, respectively, under the assay conditions. Conclusion: Production of endo-1, 4 ß-glucanase enzymes from B. halodurans increased several folds when cloned in pET vector and expressed in E. coli. To our knowledge, this is the first report of high-level expression and characterization of an endo-1, 4 ß-glucanases from B. halodurans.