Characterization and applications of glutaminase free L-asparaginase from indigenous Bacillus halotolerans ASN9.

L-asparaginase (L-ASNase) is a versatile anticancer and acrylamide reduction enzyme predominantly used in medical and food industries. However, the high specificity of L-asparaginase formulations for glutamine, low thermostability, and blood clearance are the major disadvantages. Present study describes production, characterization, and applications of glutaminase free extracellular L-asparaginase from indigenous Bacillus halotolerans ASN9 isolated from soil sample. L-asparaginase production was optimized in M9 medium (containing 0.2% sucrose and 1% L-asparagine) that yielded maximum L-ASNase with a specific activity of 256 U mg-1 at pH 6 and 37°C. L-asparaginase was purified through acetone precipitation and Sephadex G-100 column, yielding 48.9 and 24% recovery, respectively. Enzyme kinetics revealed a Vmax of 466 mM min-1 and Km of 0.097 mM. Purified L-ASNase showed no activity against glutamine. The purified glutaminase free L-ASNase has a molecular mass of 60 kDa and an optimum specific activity of 3083 U mg-1 at pH 7 and 37°C. The enzyme retains its activity and stability over a wide range of pH and temperature, in the presence of selected protein inhibitors (SDS, ß-mercaptoethanol), CoCl2, KCl, and NaCl. The enzyme also exhibited antioxidant activity against DPPH radical (IC50 value 70.7 µg mL-1) and anticancer activity against U87 human malignant glioma (IC50 55 µg mL-1) and Huh7 human hepatocellular carcinoma (IC50 37 µg mL-1) cell lines. Normal human embryonic kidney cells (HEK293) had greater than 80% cell viability with purified L-ASNase indicating its least cytotoxicity against normal cells. The present work identified potent glutaminase free L-ASNase from B. halotolerans ASN9 that performs well in a wide range of environmental conditions indicating its suitability for various commercial applications.

Insights into the microbiological and virulence characteristics of bacteria in orthopaedic implant infections: A study from Pakistan.

The exponential increase in the prevalence of multidrug resistant bacteria has resulted in limiting surgical treatment options globally, potentially causing biofilm-related complications, implant failure, and severe consequences. This study aims to isolate and characterize bacteria from post-surgical orthopaedic implant infections and screening for multiple antibiotic resistance. A cross-sectional study was conducted, involving isolation of forty-four dominant pathogenic bacterial isolates from 16 infected implant samples from across Islamabad and Rawalpindi. Out of forty-four, 38% cocci and 61% bacilli were obtained. Approximately 90% of isolates showed multiple antibiotic resistance (MAR) index of more than 0.2. Eleven strains were identified via 16S rRNA gene sequencing as Pseudomonas aeruginosa, Bacillus spp., Planococcus chinensis, Staphylococcus, Escherichia coli and Enterobacter cloacae. The bacterial strain E. coli MB641 showed sensitivity to Polymyxin only, and was resistant to all other antibiotics used. Maximum biofilm forming ability 0.532 ± 0.06, 0.55 ± 0.01 and 0.557 ± 0.07 was observed in Pseudomonas aeruginosa MB663, Pseudomonas aeruginosa MB664 and Bacillus spp. MB647 respectively after 24 hours of incubation. EPS production of bacterial strains was assessed, the polysaccharides and protein content of EPS were found to be in the range of 11-32 µg/ml and 2-10 µg/ml, respectively. Fourier transform infrared spectroscopic analysis of EPS showed the presence of carbohydrates, proteins, alkyl halides, and nucleic acids. X-ray diffraction analysis revealed crystalline structure of EPS extracted from biofilm forming bacteria. These findings suggest a high prevalence of antibiotic-resistant bacteria in orthopaedic implant-associated surgeries, highlighting the urgent need for ongoing monitoring and microorganism testing in infected implants.

Unveiling the Antibiotic Susceptibility and Antimicrobial Potential of Bacteria from Human Breast Milk of Pakistani Women: An Exploratory Study.

Background: Human life quality and expectancy have increased dramatically over the past 5 decades because of improvements in nutrition and antibiotic's usage fighting against infectious diseases. Yet, it was soon revealed that the microbes adapted to develop resistance to any of the drugs that were used. Recently, there is great concern that commensal bacteria from food and the gastrointestinal tract of humans and animals could act as a reservoir for antibiotic resistance genes. Methodology. This study was intended for evaluating the phenotypic antibiotic resistance/sensitivity profiles of probiotic bacteria from human breast milk and evaluating the inhibitory effect of the probiotic bacteria against both Gram-negative and Gram-positive bacteria. Results: The results point out that some of the isolated bacteria were resistant to diverse antibiotics including gentamycin, imipenem, trimethoprim sulfamethoxazole, and nalidixic acid. Susceptibility profile to certain antibiotics like vancomycin, tetracycline, ofloxacin, chloramphenicol, streptomycin, rifampicin, and bacitracin was also observed. The antimicrobial qualities of cell-free supernatants of some probiotic bacteria inhibited the growth of indicator bacteria. Also, antimicrobial properties of the probiotic bacteria from the present study attributed to the production of organic acid, bacterial adhesion to hydrocarbons (BATH), salt aggregation, coaggregation with pathogens, and bacteriocin production. Some isolated bacteria from human milk displayed higher hydrophobicity in addition to intrinsic probiotic properties like Gram-positive classification, catalase-negative activity, resistance to gastric juice (pH 2), and bile salt (0.3%) concentration. Conclusion: This study has added to the data of the antibiotic and antimicrobial activity of some probiotic bacteria from some samples of Pakistani women breast milk. Probiotic bacteria are usually considered to decrease gastrointestinal tract diseases by adhering to the gut epithelial and reducing population of pathogens and in the case of Streptococcus lactarius MB622 and Streptococcus salivarius MB620 in terms of hydrophobicity and exclusion of indicator pathogenic strains.

Isolation and Characterization of Lactobacillus crispatus, Lactococcus lactis, and Carnobacterium divergens as Potential Probiotic Bacteria from Fermented Black and Green Olives (Olea europaea): An Exploratory Study.

Background: Table olives are becoming well recognized as a source of probiotic bacteria that might be used to create a health-promoting fermented food product by traditional procedures based on the activities of indigenous microbial consortia present in local environments. Methodology. In the present study, the characterization of probiotic bacteria isolated from mince, chunks, and brine of fermented green and black olives (Olea europaea) was done based on morphological, biochemical, and physiological characteristics. Results: Bacterial isolates demonstrated excellent survival abilities at 25, 37, and 45°C and at a variable range of pH. However, the optimum temperature is 37 and the optimum pH is 7 for all three isolates. An antimicrobial susceptibility pattern was found among these isolates through the disc diffusion method. Most of the isolates were susceptible to streptomycin, imipenem, and chloramphenicol, whereas, amoxicillin showed resistance to these isolates, and variable results were recorded for the rest of the antibiotics tested. The growth of the isolates was optimum with the supplementation of 3% NaCl and 0.3% bile salt. The isolated bacteria were able to ferment skimmed milk into yogurt, hence making it capable of producing organic acid. Conclusion: Isolates of Lactobacillus crispatus MB417, Lactococcus lactis MB418 from black olives, and Carnobacterium divergens MB421 from green olives were characterized as potential candidates for use as starter cultures to induce fermentation of other probiotic food products.

Leveraging molecular docking to understand Congo red degradation by Staphylococcus caprae MB400.

Congo red (CR) is a genotoxic, sulphonated azo dye and poses significant pollution problem. We hereby report its degradation by Staphylococcus caprae MB400. The bacterium initially propagated as a suspected contaminant upon CR dye supplemented nutrient agar plates, forming zones of clearance around its growth area. The bacterium was purified, gram stained and identified as Staphylococcus caprae via 16S rRNA gene sequencing. Dye decolourization was analysed in liquid culture, and Fourier-transform infrared spectroscopy (FTIR) was conducted for analysis of degraded product/metabolites. A decolourization of ~ 96.0% at 100 µg/ml concentration and pH 7 after 24 h of incubation was observed. Structure of the azoreductase enzyme, responsible for breakage of the bond in the dye and ultimately decolourization, was predicted, and molecular docking was harnessed for understanding the mechanism behind the reduction of azo bond (-N=N-) and conversion to metabolites. Our analysis revealed 12 residues critical for structural interaction of the azoreductase enzyme with this dye. Among these, protein backbone region surrounding four residues, i.e. Lys65, Phe122, Ile166 and Phe169, showed major displacement changes, upon binding with the dye. However, overall the conformational changes were not large.

Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk reduce chemokine IL-8 production in response to TNF-α in Caco-2 cell line, an exploratory study.

Streptococci are a predominant genera of the human milk microbiome. Among different lactic acid bacteria (LAB) a few Streptococcal strains are also considered as probiotics. Probiotic bacteria are reported to modulate immunity when consumed in adequate amount and bacterial hydrophobicity can be considered as a preliminary experiment for the adhesive capability of probiotic bacteria to the epithelial cells. The present study aimed to investigate the probiotic, hydrophobic and immune modulation property of Streptococcus lactarius MB622 and Streptococcus salivarius MB620, isolated from human milk. S. lactarius MB622 and S. salivarius MB620 displayed higher hydrophobicity (78 % and 59 % respectively) in addition to intrinsic probiotic properties such as gram positive classification, catalase negative activity, resistance to artificially stimulated gastric juice and gastrointestinal bile salt concentration. In conclusion Streptococcus lactarius MB622 and Streptococcus salivarius MB620 isolated from human milk when administered in sufficient amount and for certain duration could be used to reduce inflammation inside the colon by reducing the production of inflammatory booster (IL-8) in diseased state.

Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria.

Present study was aimed at screening and characterizing thermostable amylase-producing bacteria from water and sediment samples of unexplored hot spring of Tatta Pani Kotli Azad Kashmir. Four thermophilic isolates were characterized on morphological, biochemical, physiological basis and were authenticated by molecular analysis. By 16S rDNA sequencing, isolates were identified as Anoxybacillus mongoliensis (MBT001), Anoxybacillus flavithermus (MBT002), Bacillus (MBT004). Among all identified strains, MBT003 showed maximum homology with both Anoxybacillus mongoliensis and Anoxybacillus flavithermus. Amylase activity was analyzed qualitatively in starch agar and quantitatively by DNS method. The optimal enzyme production was observed and authenticated by Response Surface Methodology at 7 pH, 70 °C, 1.25% substrate concentration, 300 µL of inocula volume after 48 h of incubation. Optimum amylase activity (4.4 U/mL) and stability (3.3 U/mL) was observed with 1.5% soluble starch at 70 °C. Maximum activity (3.7 U/mL) and stability (1.5 U/mL) was found at pH 8. Enzyme activity was increased in the presence of MgSO4 and CaCl2. Amylase was stable with surfactants and commercial detergents for 30 min. Supplementation of the enzyme with commercial detergent improved the washing ability of the detergent. This investigation has revealed that these thermostable bacteria are excellent source of amylase which can be used commercially for generating economic activity on sustainable basis.

Functionalization of ampicillin and gentamicin with biogenic copper nanoparticles (CuNPs) remodel antimicrobial and cytotoxic outcome against MDR clinical isolates.

The present study reports the functionalization of antibiotic-conjugated Alternanthera pungens and Trichodesma indicum copper nanoparticles (CuNPs). Initially, antibiotic profiling of multi-drug resistant (MDR) clinical isolates against five antibiotics was verified and then gentamicin and ampicillin conjugates of CuNPs were prepared. Biosynthesized nanostructures were characterized through UV-visible spectroscopy, Fourier-transformed infrared spectroscopy, X-ray diffraction and scanning electron microscope. Biogenic synthesized CuNPs displayed highest antibacterial activity (24.0-31.3 mm inhibition zones) when capped with gentamicin as compared to the ampicillin-conjugated NPs which showed resistance against most of the bacterial species. A. pungens-derived conjugates of gentamicin (CuAp-GNT) along with the vehicle revealed 4.86 ± 0.20% and 4.25 ± 2.96% hemolytic potential and highest MDA production in S. typhimurium (3.18 ± 1.52 µg/mL and 6.31 ± 3.49 µg/mL) and K. pneumoniae (2.99 ± 0.90 µg/mL and 4.06 ± 1.20 µg/mL). Similarly, CuAp-GNT also showed highest DNA protection ability by displaying 1342.99 ± 11.87 band intensity. All-inclusive, CuAp showed more promising effects when conjugated with gentamicin indicating that capping of gentamicin with the active components of the plant-based copper nanostructures increases the antibacterial capacity of the drug. Hence, conjugation of antibiotics with bio-based sources offers great potential for identifying potent drug leads.

Cancer and Disease Diagnosis - Biosensor as Potential Diagnostic Tool for Biomarker Detection.

Analysis of cancer biomarkers has enormous promise for advancing our molecular understanding of illness and facilitating more precise and timely diagnosis and follow-up care. MicroRNA, exosomes, ctDNA, CTCs, and proteins are only some of the circulating biomarkers that can be detected by liquid biopsy instead of the more intrusive and time-consuming process of doing a tissue biopsy. As the cancer diagnosis bio-markers reveal ultra-low levels in the early stages of the disease, highly sensitive approaches are urgently required. Researchers have taken an interest in a optical biosensor for detecting cancer biomarkers as a potential tool for early disease diagnosis. These techniques have the potential to aid in the development of effective treatments, ultimately leading to a higher rate of patient survival. This review briefly discuss the i) understanding of cancer and biomarkers for early diagonosis purpose ii) Molecular methods and ii) biosensor-based diagnostics. The reseach primary focus on advancement in biosensor design using various concepts ie., Electrochemical, Chemiluminescence and Colorimetric, Surface plasmons (SP), Surface plasmon resonance (SPR), localized surface plasmon resonance (LSPR), Fluorescence, Fiber-based sensors, Terahertz based biosensors, and Surface enhanced Raman spectroscopy (SERS). As a result of the local electric field amplification around plasmonic (usually gold and silver) nanostructures, surface-enhanced Raman spectroscopy (SERS) has emerged as a rapid, selective, and sensitive alternative to conventional laboratory analytical methods, making significant strides in a number of biosensing applications but still under developing stage to be used as diagnostic tool in clinical research.

Metabolites produced by inoculated Vigna radiata during bacterial assisted phytoremediation of Pb, Ni and Cr polluted soil.

Phytoremediation assisted with plant growth promoting bacteria (PGPB) is a green technology to remediate metal contaminated soils. Plants usually produce secondary metabolites to tolerate metal toxicity. Present study was designed to explore the phytoremediation potential of Vigna radiata var. NM-II in the presence of metal resistant PGPB and comparison of metabolites produced under heavy metal stresses (Pb, Ni, Cr). Three PGPB selected for present study include Bacillus pumilus MB246, Serratia nematodiphila MB307 and Delftia Lacustris MB322. Pot experiments were conducted with inoculated V. radiata NM-II seeds grown in soil artificially contaminated with lead (Pb), Nickle (Ni) and chromium (Cr) at a concentration of 300, 200 and 100 mg/kg respectively. After harvesting various growth parameters were studied (root length, shoot length, fresh weight and dry weight). Bacterial colonization on root surfaces of harvested plants was observed through Scanning electron microscopy (SEM) and Elemental composition was recorded through Energy dispersive X-ray spectroscopy (EDX) attached with SEM. Metabolic response of harvested plants was studied through Gas chromatography Mass spectrophotometry (GC-MS) analysis. Metal accumulation in roots, shoots and soil was analysed by acid digestion method from which Bioaccumulation factor (BF) and Translocation factor (TF) of metal from soil to plant was calculated. Results revealed stimulatory effect of PGPB on growth and phytoextraction ability of V. radiata. Soil metal removal efficiency was in the order Pb>Ni>Cr, whereas metal distribution in each part of plant was root>stem>leaf. The BF and TF values suggested V. radiata as Pb and Ni excluder while moderate accumulator for Cr. Elemental analysis through Energy Dispersive X- ray spectroscopy (EDX) found potassium (K+)and calcium (Ca+)as highly abundant nutrients with least accumulation of sulphur (S). Metabolites study through GC-MS revealed variety of compounds (carbohydrates, amino acids, fatty acids, steroids etc) detected differentially under each metal treatment and their concentration was influenced by different bacterial inoculations. Overall 9-Octadecenamide was found as commonly present lipid compound in most of the treatments which is required for detoxification in plants. The study concluded beneficial role of PGPB for successful phytoremediation of heavy metals and differential response of metabolites towards each metal stress that is related to metal tolerance ability of V. radiata.

Impact of Azo Dyes and Ibuprofen on the Proteome of Serratia nematodiphila sp. MB307.

BACKGROUND: Micropollutants comprise organic/mineral substances that cause an undesirable impact on the environment, by affecting life at all scales. In this study, we explored the changes they impart on the global proteome of a soil bacterium Serratia nematodiphila MB307, for two classes of pollutants, i.e., Azo dyes (Methyl orange, Congo red) and a pharmaceutical (Ibuprofen). METHODS: The 100 µg pollutant supplemented alteration of pure S. nematodiphila MB307 culture after 24 hours of incubation at 37 °C and its control was analyzed using a differential proteomics approach. MaxQuant software with the Perseus package was used for data analysis purposes. RESULTS: Prominently, ribosomal proteins and chaperones were up or downregulated in the whole cell and membranous fraction. CONCLUSION: This illustrates dynamic protein production adaptation of bacteria, to cope with stress and cell growth/division trade-off for survival. A collective pattern of survival under stress or pollution resistance could not be decrypted for all classes of pollutants, portraying dissimilar mechanisms of coping with differently structured pollutant moieties.

DAT and PRX1 gene expression modulates vincristine production in Catharanthus roseus L. propagates using Cu, Fe and Zn nano structures.

Underlying mechanism of nanostructures upon monoterpene induction in Catharanthus roseus has not been explored yet. In the current study, Copper, Iron and Zinc nanoparticles were biosynthesized by Eriobotrya japonica seed extract and capped with reduced glutathione. Biosynthesized nanoparticles and their capped analogues were characterized by UV-visible spectrophotometer, FTIR, XRD and SEM. Selected concentration of nanostructures were used in plant tissue culture media which instigated the production of alkaloids, tannins and flavonoids without significantly affecting the growth index of propagated calli and shoots cultures of C. roseus. Accelerated vincristine production was noticed in propagated calli and shoots under copper and zinc nanostress (1645-1865 µg/ml respectively) with the least effect by iron nanostructure. Highest concentration of calcium was recorded in in vitro shoots under capped (3.42 mg/ml ± 7.16) and uncapped (4.41 mg/ml ± 20.44) Zn nanoparticles compared to control (2.82 mg/ml ± 13.41). Real time PCR depicts nano-zinc mediated increased expression of DAT and PRX1 genes of TIA pathway. Significant correlation among PRX1/DAT gene expression with vincristine production and calcium accumulation in the presence of nanostress validate by PCA. This study paved way the opportunities of metal biogenic nanomaterials as an ideal drug modulator in plant tissue culture studies.

Genetic Evidences of Biosurfactant Production in Two Bacillus subtilis Strains MB415 and MB418 Isolated From Oil Contaminated Soil.

Biosurfactants are a diverse group of amphiphilic compounds obtained from microbes. In the present study, the genomic analysis of biosurfactant-producing Bacillus subtilis MB415 and MB418 obtained from oil-contaminated soil was performed. Initially, the strains were screened for biosurfactant production by hemolytic assay, emulsification index, and oil displacement. Further FTIR analysis of extracted biosurfactants revealed the presence of lipopeptides. The sequenced genomes of MB415 and MB418 were of 4.2 Mbps with 43% GC content. Among more than 4,500 protein-coding genes, many were involved in virulence, metal/multidrug resistances, flagella assembly, chemotactic response, and aromatic ring hydroxylating dioxygenases. An annotation analysis revealed that both genomes possessed non-ribosomal synthetase gene clusters for the lipopeptide synthetases srf and fen responsible for surfactin and fengycin production. Comparative studies of both genomes highlighted variability in gene operons mainly for surfactin biosynthesis.

Sulphonated azo dye decolorization by Alcaligenes faecalis subsp. phenolicus MB207: Insights from laboratory and computational analysis.

Microbial decolorization of azo dyes, mediated by an enzymatic mechanism is an intricate cost-effective, and eco-friendly treatment method of genotoxic azo pollutants. Scientists are on the constant lookout for microbes, enzymes, and mechanisms that could aid remediation of the environment at a fast pace. Alcaligenes faecalis subsp. phenolicus MB207 is one such bacteria, consisting of azoreductase (AzoR) and laccase/multicopper oxidase enzyme responsible for sulphonated mono-azo dye (Methyl orange) and di-azo dye (Congo red) degradation. AzoR degrades dyes by a ping-pong setup while multicopper oxidase achieves this through a non-specified radical approach. We have coupled experimental analysis with bioinformatics for deciphering intricacies of this procedure in tiny scale enzymatic machines of this biotope. The degradation assays were followed by molecular docking of the enzyme-substrate complexes. Key anchoring bonds were detected and mapped H-bonding, electron exchange, ionic interactions, as well as hydrophobic interactions, provided insights into dye-enzyme and NADH-enzyme binding. This study establishes a foundation of the molecular basis of dye interaction with azoR and multicopper oxidase in A. facealis subsp. phenolicus MB207.

Biotransformation of dimethoate into novel metabolites by bacterial isolate Pseudomonas kilonensis MB490.

Bacterial strain (Pseudomonas kilonensis MB490) isolated from agricultural fields of Mianwali, was selected to check its potential to degrade Organophosphate insecticide dimethoate (DM). Strain MB490 was able to degrade dimethoate equally well at given pH range (6.0, 7.0 and 8.0), thus showing its pH independence for dimethoate degradation. Optimum temperature for dimethoate degradation varied from 25-30 °C. There was more dimethoate degradation under shaking conditions with optimum growth. Strain MB490 showed 90% dimethoate degradation in M-9 broth and 90.6% in soil slurry, while exhibited 81.5% dimethoate degradation in soil microcosm within 9 days, based on HPLC analysis of bacterial samples supplemented with 200 mg/L dimethoate. The average half-life (t 1/2) of dimethoate after bacterial degradation ranged from 1.95 days in 1st phase to 5 days in 2nd phase in M-9 broth, soil slurry and soil microcosm, while in control media without bacteria, it ranged from 30 to 64.3 days. GCMS investigation revealed the transformation of dimethoate into 5 metabolic products namely Methyl diethanol amine, Aspartylglycine ethyl ester, Phosphonothioic acid propyl-O, S-dimethyl ester, O, O, O-Trimethyl thiophosphate and omethoate which were ultimately mineralized by the strain MB490, providing energy for its growth.

Correlation of ERCC5 polymorphisms and linkage disequilibrium associated with overall survival and clinical outcome to chemotherapy in breast cancer.

Purpose: ERCC5 is a DNA endonuclease and nucleotide excision repair gene; its mutations lead to a lack of activity by this enzyme, causing oxidative DNA damage. This study aimed to assess the role of four selected single nucleotide polymorphisms (SNPs) in ERCC5 and their linkage disequilibrium associated with survival analysis and clinical outcomes in breast cancer. Patients and methods: Four SNPs (rs751402, rs17655, rs2094258, and rs873601) of the ERCC5 gene were analyzed using the PCR-RFLP technique, followed by sequencing in 430 breast cancer (BC) cases and 430 cancer-free individuals. Statistical analysis was performed using MedCalc 17 and SPSS version 24, while bioinformatic analysis of linkage disequilibrium was performed using Haploview software 4.2. Results: Multivariate analysis showed that the rs751402 and rs2094258 polymorphisms were significantly associated with an elevated risk of BC (P < 0.001), while the other two SNPs, rs17655 and rs873601, did not show any association (P > 0.001). Survival analysis revealed that rs751402 and rs2094258 had longer overall survival periods (P <0.001) than rs17655 and rs873601. Moreover, rs751402 and rs2094258 also had significantly longer overall survival (log-rank test, P < 0.005) for all three survival functions (positive family history, ER+PR status, and use of contraceptives), while rs17655 and rs873601 did not show any significant association. Only rs873601 showed a strong negative correlation with all the chemotherapeutic groups. Conclusion: The current results suggest that variations in ERCC5 may contribute to BC development and that their genetic anomalies may be associated with cancer risk and may be used as a biomarker of clinical outcome.

Annotation of chemotaxis gene clusters and proteins involved in chemotaxis of Bacillus subtilis strain MB378 capable of biodecolorizing different dyes.

This study explores the chemotactic potential of Bacillus subtilis MB378 against industrial dyes. Initial screening with swim plate assay showed significant movement of Bacillus subtilis MB378 towards test compounds. According to quantitative capillary assay, B. subtilis MB378 exhibited high chemotaxis potential towards Acid Orange 52 (CI: 9.52), followed by Direct Red 28 (CI: 8.39) and Basic Green 4 (CI: 5.21) in glucose-supplemented medium. Sequencing and gene annotation results evidently showed presence of chemotaxis genes and flagellar motor proteins in Bacillus subtilis draft genome. Methyl-accepting proteins (involved in chemotaxis regulation) belonged to pfam00672, pfam00072, and pfam00015 protein families. Annotated chemotaxis machinery of MB378 comprised 8 Che genes, 5 chemoreceptor genes, associated flagellar proteins, and rotary motors. Chemotaxis genes of B. subtilis MB378 were compared with genes of closely related Bacillus strains (168, WK1, and HTA426), depicting highly conserved regions showing evolutionary relation between them. Considering results of present study, it can be speculated that test compounds triggered chemotactic genes, which made these compounds bioavailable to the bacterium. Hence, the bacterium recognized and approached these compounds and facilitated biodegradation and detoxification of these compounds.

Litchi chinensis inspired nanoformulations: a synergy guided approach for unraveling promising cytotoxic attributes of metal and nonmetal conjugates.

In present study, diverse Litchi chinensis-mediated nanostructures in combination with 5-fluorouracil drug were fabricated viz. Au, Se, Ag, Ag-Se, Ag-Au, 5-FU Ag-Se and 5-FU Ag-Au with subsequent characterization and scrutinization of their anticarcinogenic capabilities. UV-Visible spectroscopic analysis confirmed the state transition for each precursor salt. XRD and transmission electron microscopy analysis revealed spherical/quasispherical nanostructures with monoclinic crystalline organization ranged between 18 nm and 38 nm. FTIR analysis revealed fabricated nanoparticles to be capped with various phytoconstituents. DLS and Zeta potential analysis of unloaded and drug-loaded bielemental nanoparticles (BNPs) showed comparatively large hydrodynamic particle size distribution and sufficient stability of nanoparticles. BNPs showed promising lethality concentrations for brine shrimp (LC50 < 2 µg/ml) and antitumor (LC50 < 10 µg/ml) assessments. These findings were in positive correlation with the antioxidant inhibitory concentrations IC50 (74.2-180.1 µg/ml) of the tested entities. Ag-Se and Ag-Au were loaded with 5-FU (loading efficiency of 47% ± 1.14 and 25% ± 0.32, respectively) in light of their promising cytotoxic actions. All nanostructures showed profound hemocompatibility with maximum hemolytic activity as low as 2.4%. Highly significant difference (P < 0.01) was observed in antineoplastic potentials of unloaded and 5-FU loaded BNPs against HepG2 and HT144, with most substantial IC50 for 5-FU Ag-Au (8.95 ± 2.86 µg/ml). 5-FU Ag-Au was identified as a significant inducer of DNA fragmentation with maximum relative tail moment (HepG2: 3.45 ± 0.21) among all treatments.

Identification of bacterial communities in extreme sites of Pakistan using high throughput barcoded amplicon sequencing.

Microorganisms thrive nearly everywhere including extreme environments where few other forms of life can exist. Geochemistry of extreme sites plays a major role in shaping these microbial communities and microbes thriving in such harsh conditions are untapped sources of novel biomolecules. To understand the structure and composition of such microbial communities, culture-independent bacterial diversity was characterised for two extreme sites in Pakistan, Khewra salt range and Murtazaabad hot spring. Barcoded amplicon sequencing technique was used to study the microbial communities. Physicochemical analysis of these sites was also conducted to study the dynamics of microbial communities under stressed conditions. Metagenomic sequencing of salt range soil samples yielded of 40,433 16S rRNA sequences, while hot spring sediments produced 76,449 16S rRNA sequence reads. Proteobacteria were predominant in saline soil while Firmicutes were most abundant in hot spring sediment. The taxonomic analysis of saline samples revealed 914 operational taxonomic units (OTUs) while that of hot spring sequences were clustered into 726 distinct OTUs. OTUs from genus Alkalibacillus were most abundant in hot spring sediments, whereas Haloarcula were more prevalent in saline soil. Some unidentified sequences were also present at each taxonomic level. Multivariate analysis indicated that electrical conductivity and pH are the major environmental factors involved in modelling microbial communities. This study revealed a poly-extremophilic microbial community in the Murtazaabad hot spring and characterised the unexplored halophilic microbial diversity of saline soil of Pakistan.

Genome sequence analysis and bioactivity profiling of marine-derived actinobacteria, Brevibacterium luteolum, and Cellulosimicrobium funkei.

Genome analysis gives important insights into the biosynthetic potential of marine actinobacteria. The genomes of two marine actinomycetes Brevibacterium luteolum MOSEL-ME10a and Cellulosimicrobium funkei MOSEL-ME6 were sequenced to identify the biosynthetic gene clusters (BGCs). Additionally, anti-proliferative, antioxidant, and enzyme inhibitory activities were studied in vitro. We report a total genome size of 2.77 Mb with GC content of 67.8% and 6.81 Mb with GC content of 69% for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Biosynthetic gene clusters (BGCs) encoding different classes of natural products were predicted including terpenes, peptides, siderophores, ectoines, and bacteriocins. The bioactivity potential of crude extracts derived from these strains was evaluated. Notable anti-proliferative activity was observed against HepG2 cell line (hepatocellular carcinoma) with an IC50 value of 182 µg/mL for Brevibacterium sp. MOSEL-ME10a. Furthermore, antioxidant activity was assessed with IC50 values of 48.91 µg/mL and 102.5 µg/mL for Brevibacterium sp. MOSEL-ME10a and Cellulosimicrobium sp. MOSEL-ME6, respectively. Protein kinase inhibition potential was observed only for Brevibacterium sp. MOSEL-ME10a. Our study also reports lower amylase enzyme inhibition potential for both strains. Moreover, both crude extracts showed only slight-to-no toxic effect on erythrocytes at 400 µg/mL and below, indicating erythrocyte membrane stability. Our data present the genomic features revealing biosynthetic potential of marine actinobacteria as well as biological activities found in vitro.