Statistical optimization of process variables for agarase production using Microbacterium sp. SS5 strain from non-marine sources.

Agar oligosaccharides are thought to be valuable biomolecules with high bioactivity potential, along with a wide range of applications and advantages. The current study aimed to optimize the culture parameters required to produce agarase enzyme and agar oligosaccharides from industrial waste agar. Microbacterium spp. strain SS5 was isolated from a non-marine source and could synthesize oligo derivatives for use in a variety of industries ranging from food to pharmaceuticals. In addition, the strain and culture conditions were optimized to maximize extracellular agarase production. The bacterium grew best at pH 5.0 - 9.0, with an optimal pH of 7.5 - 8.0; temperatures ranging from 25 to 45 °C, with an optimal of 35 °C; and carbon and nitrogen concentrations of 0.5% each. Plackett-Burman experimental design and response surface methods were used to optimize various process parameters for agarase production by Microbacterium spp. strain SS5. Using the Plackett-Burman experimental design, eleven process factors were screened, and agar, beef extract, CaCl2, and beginning pH were found as the most significant independent variables affecting agarase production with confidence levels above 90%. To determine the optimal concentrations of the identified process factors on agarase production, the Box- Behnken design was used. Agarase production by Microbacterium spp. strain SS5 after optimization was 0.272 U/mL, which was determined to be greater than the result obtained from the basal medium (0.132 U/mL) before screening using Plackett-Burman and BBD with a fold increase of 2.06.

Screening of potential phytomolecules against MurG as drug target in nosocomial pathogen Pseudomonas aeruginosa: perceptions from computational campaign.

The nosocomial infection outbreak caused by Pseudomonas aeruginosa remains a public health concern. Multi-drug resistant (MDR) strains of P. aeruginosa are rapidly spreading leading to a huge mortality rate because of the unavailability of promising antimicrobials. MurG glycotransferase [UDP-N-acetylglucosamine-N-acetylmuramyl (pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase] is located at the plasma membrane and plays a key role in murein (peptidoglycan) biosynthesis in bacteria. Since MurG is required for bacterial cell wall synthesis and is non-homologous to Homo sapiens; it can be a potential target for the antagonist to treat P. aeruginosa infection. The discovery of high-resolution crystal structure of P. aeruginosa MurG offers an opportunity for the computational identification of its prospective inhibitors. Therefore, in the present study, the crystal structure of MurG (PDB ID: 3S2U) from P. aeruginosa was selected, and computational docking analyses were performed to search for functional inhibitors of MurG. IMPPAT (Indian medicinal plants, phytochemicals and therapeutic) phytomolecule database was screened by computational methods with MurG catalytic site. Docking results identified Theobromine (-8.881 kcal/mol), demethoxycurcumin (-8.850 kcal/mol), 2-alpha-hydroxycostic acid (-8.791 kcal/mol), aurantiamide (-8.779 kcal/mol) and petasiphenol (-8.685 kcal/mol) as a potential inhibitor of the MurG activity. Further, theobromine and demethoxycurcumin were subjected to MDS (molecular dynamics simulation) and free energy (MM/GBSA) analysis to comprehend the physiological state and structural stability of MurG-phytomolecules complexes. The outcomes suggested that these two phytomolecules could act as most favorable natural hit compounds for impeding the enzymatic action of MurG in P. aeruginosa, and thus it needs further validation by both in vitro and in vivo analysis. HIGHLIGHTSThe top phytomolecules such as theobromine, demethoxycurcumin, 2-alpha-hydroxycostic acid, aurantiamide and petasiphenol displayed promising binding with MurG catalytic domain.MurG complexed with theobromine and demethoxycurcumin showed the best interaction and stable by MD simulation at 100 ns.The outcome of MurG binding phytomolecules has expanded the possibility of hit phytomolecules validation.Communicated by Ramaswamy H. Sarma.

Nigella sativa and its nano-mediated approach toward management of neurodegenerative disorders: A review.

Nigella sativa L., also known as black seed or black cumin, is a plant that has been used for centuries. In the past, this flowering plant was used as a food preservative and medicinal herb. A vital component of Nigella sativa, thymoquinone (TQ), plays a significant therapeutic role in the management of most diseases, including cancer, diabetes mellitus, hypertension, inflammation, gastrointestinal disorders, and neurodegenerative disorders. Neurodegenerative disorders are primarily caused by neurotransmitter hypoactivity, particularly insufficient serotonin activity. It has been discovered that many medicinal herbs and their active compounds have therapeutic value. Black cumin seeds have been used to heal ailments and its history traces back to ancient times such as ancient Babylonia. They can be used applied to alleviate edema, hair loss, and bruising, and consumd to treat stomach issues. It is one of the most feasible and effective medicinal plants. The use of nanoformulations based on Nigella sativa and TQ to treat neurodegenerative diseases (NDs) has yielded promising outcomes. Customized administration of nanoparticle (NP) systems and nanomedicine are two of the many options for drug delivery to the central nervous system (CNS) that are attracting increasing interest. Delivering a therapeutic and diagnostic substance to a particular location is the core target of NPs. Because of their distinct cell uptake and trafficking mechanisms, NPs can reduce the amount that accumulates in undesirable organs. The focus of the current review is on recent studies on the various neuroprotective properties of Nigella sativa as well as nanoformulations for NDs and the brain's uptake of NPs. The review summarizes the In vivo, In vitro, and In silico studies on the protective effects of black cumin against neurodegenerative disorders.

Screening and characterization of agarolytic bacteria from different sources.

According to the results of our investigation, distinct bacterial isolates capable of breaking down agar were found in various nonmarine environments. The deficiency of reducing sugar in the control media demonstrates that the agar in the experiment is broken down by the bacteria to produce various oligosaccharides because the viscosity of the medium containing the agar was found to have been extremely high before inoculation, reducing with incubation duration and attaining a maximum after 48 hours. These isolates were subsequently used in tests along with additional investigation since they could create reducing sugar. Interestingly, the deterioration of agar appears to be mainly caused by Gram-negative bacteria. In order to study the agarase properties, the relative quantity of the enzyme secreted by the bacteria that hydrolyze the agar was used. The detection of extracellular agarase surrounding the colonies and the absence of stained halos on iodine-treated agar plates show that the agarase diffusing from the bacteria impacted the characteristics of the gel. Inconclusion, these agarsase-producing bacteria can be exploited for industrial applications. Waste agar from the plant tissue culture business can be utilized for a range of applications and this degraded agar can be explored for reliable and ecologically safe alternatives.

Neurodegenerative disorders: Assessing the impact of natural vs drug-induced treatment options.

Neurodegenerative illnesses refer to the gradual, cumulative loss of neural activity. Neurological conditions are considered to be the second leading cause of mortality in the modern world and the two most prevalent ones are Parkinson's disease and Alzheimer's disease. The negative side effects of pharmaceutical use are a major global concern, despite the availability of many different treatments for therapy. We concentrated on different types of neurological problems and their influence on targets, in vitro, in vivo, and in silico methods toward neurological disorders, as well as the molecular approaches influencing the same, in the first half of the review. The bulk of the second half of the review focuses on the many categories of treatment possibilities, including natural and artificial. Nevertheless, herbal treatment solutions are piquing scholarly attention due to their anti-oxidative properties and accessibility. However, more quality investigations and innovations are undoubtedly needed to back up these conclusions.

Computational docking investigation of phytocompounds from bergamot essential oil against Serratia marcescens protease and FabI: Alternative pharmacological strategy.

The rapid development of multi-drug resistant (MDR) pathogens adds urgency to search for novel and safe drugs having promising action on new and re-emerging infectious pathogens. Serratia marcescens is an MDR pathogen that causes several-healthcare associated infections. Curbing bacterial virulence, rather than inhibiting its growth, is a promising strategy to diminish the pathogenesis of infectious bacteria, reduce the development of antimicrobial resistance, and boost the host immune power to eradicate infections. Bergamot essential oil (BEO) is a remarkable source of promising therapeutics against pathogens. Therefore, the present investigation aimed to analyze the major phytocompounds from BEO against S. marcescens virulent proteins using in silico studies. The analysis of BEO phytocompounds was achieved by Gas chromatography-mass spectrometry (GC-MS) method. The molecular docking was carried out using the SP and XP docking protocol of the Glide program. The drug-likeness and pharmacokinetics properties (ADMET properties) were analyzed with SwissADME and pkCSM server. The results revealed that the major compounds present in BEO are Linalool (8.17%), D-Limonene (21.26%), and Linalyl acetate (26.91%). Molecular docking analysis revealed that these compounds docked strongly within the binding cavities of Serratia protease and FabI model which in turn curb the pathogenesis of this bacteria. Linalool interacted with the Serratia protease and FabI with a binding energy of - 3.130 kcal/mol and - 3.939 kcal/mol, respectively. Based on the pharmacokinetics findings all lead BEO phytocompounds appear to be promising drug candidates. Overall, these results represent a significant step in the development of plant-based compounds as a promising inhibitor of the virulent proteins of the MDR S. marcescens.

We are all aging, and here's why.

Here, through this review, we aim to serve this purpose by first discussing the statistics and aging demographics, including the life expectancy of the world and India, along with the gender life expectancy gap observed throughout the world, followed by explaining the hallmarks and integral causes of aging, along with the role played by senescent cells in controlling inflammation and the effect of senescence associated secretory phenotype on longevity. A few of the molecular pathways which are crucial in modulating the process of aging, such as the nutrient-sensing mTOR pathway, insulin signaling, Nrf2, FOXO, PI3-Akt, Sirtuins, and AMPK, and their effects are also covered in paramount detail. A diverse number of ingenious research methodologies are used in the modern era of longevity exploration. We have attempted to cover these methods under the umbrella of three broad categories: in vitro, in vivo, and in silico techniques. The drugs developed to attenuate the aging process, such as rapamycin, metformin, resveratrol, etc. and their interactions with the above-mentioned molecular pathways along with their toxicity have also been reviewed in detail.

Hepatitis B virus DNA polymerase gene polymorphism based prediction of genotypes in chronic HBV patients from Western India.

BACKGROUND: Hepatitis B Virus (HBV) infection is one of the major causes of liver cirrhosis, hepatocellular carcinoma and deaths due to the acute or chronic consequences worldwide. HBV is distributed into various genotypes based on nucleic acid sequence variation. OBJECTIVES: To develop a method of HBV genotyping and drug resistance interpretation using partial sequencing of polymerase gene. METHODS: This study was performed on 98 HBV infected patients' serum samples from Western India. A nested PCR protocol was designed for amplification of pol gene from HBV genome and Sanger's sequencing of the gene fragment. Sequences were aligned with HBV reference sequences for phylogenetic analysis and for characterization of genetic diversity. Drug resistance mutations were screened using HBVSeq program from Stanford University. RESULTS: Distribution of HBV genotypes showed predominance of genotype D, circulating in 76 (77.55%) patients (p < 0.05). Genotypes A and C were less prevalent and were identified in 4 (4.08%) and 18 (18.37%) patients, respectively. Anti-retroviral drug resistance mutations were not detected in any patient. CONCLUSION: A method for determination of HBV genotypes using pol gene sequencing which simultaneously detects major drug resistance mutations has been established. HBV genetic diversity may play an important role in treatment decision.

In vitro callus induction and estimation of plumbagin content from Plumbago auriculata Lam.

The medicinal plant Plumbago contains a very potent secondary metabolite, plumbagin having many therapeutic properties. Callus culture was induced using explants, leaf, stem and shoot apex, from P. auriculata. Murashige and Skoog media fortified with various growth hormones like NAA, IAA, IBA and 2, 4-D individually and in various combinations were checked for callus induction. Among the growth hormones used, 1 mg/L 2, 4-D showed best callusing. The hormonal combinations of 1 mg/L IAA and 1.5 mg/L NAA in the media exhibited best callus induction using stem internode as an explant. Plumbagin content from root, stem, leaf and callus was analyzed by using thin layer chromatographic technique. The callus derived from stem showed comparable plumbagin content to the in vivo plant parts. Quantitative spectrophotometric analysis of plumbagin from plant samples and callus indicated that plumbagin content was maximum in roots which was followed by callus, stem and leaf samples respectively. Generation of in vitro sources for p!umbagin, for therapeutic applications will serve as a continuous supply and will contribute to preserve the natural plant recourses.

Development, validation and clinical evaluation of a low cost in-house HIV-1 drug resistance genotyping assay for Indian patients.

Human Immunodeficiency Virus-1 (HIV-1) drug resistance genotyping assay is a part of clinical management of HIV-1 positive individuals under treatment with highly active antiretroviral therapy (HAART). Routine monitoring of drug resistance mutations in resource limited settings like India is not possible due to high cost of commercial drug resistance assays. In this study we developed an in-house, cost effective HIV-1 drug resistance genotyping assay for Indian patients and validated it against the US-FDA-approved ViroSeq HIV-1 drug resistance testing system. A reference panel of 20 clinical samples was used to develop and validate the assay against ViroSeq HIV-1 drug resistance testing system which was subsequently used to genotype a clinical panel of 225 samples. The Stanford HIV database was used to identify drug resistant mutations. The analytical sensitivity of the assay was 1000 HIV-1 RNA copies/ml of plasma sample while precision and reproducibility was 99.68 ± 0.16% and 99.76 ± 0.18% respectively. One hundred and one drug resistant mutations were detected by the in-house assay compared to 104 by ViroSeq system in the reference panel. The assay had 91.55% success rate in genotyping the clinical panel samples and was able to detect drug resistant mutations related to nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse-transcriptase inhibitor (NNRTI) as well as protease inhibitor (PI) classes of antiretroviral drugs. It was found to be around 71.9% more cost effective compared to ViroSeq genotyping system. This evaluation of the assay on the clinical panel demonstrates its potential for monitoring clinical HIV-1 drug resistance mutations and population-based surveillance in resource limited settings like India.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting.

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000-25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

A robust HIV-1 viral load detection assay optimized for Indian sub type C specific strains and resource limiting setting

BACKGROUND: Human Immunodeficiency Virus Type 1 (HIV-1) viral load testing at regular intervals is an integral component of disease management in Acquired Immunodeficiency Syndrome (AIDS) patients. The need in countries like India is therefore an assay that is not only economical but efficient and highly specific for HIV-1 sub type C virus. This study reports a SYBR Green-based HIV-1 real time PCR assay for viral load testing and is designed for enhanced specificity towards HIV-1 sub type C viruses prevalent in India. RESULTS: Linear regression of the observed and reference concentration of standards used in this study generated a correlation coefficient of 0.998 (p<0.001). Lower limit of detection of the test protocol was 50 copies/ml of plasma. The assay demonstrated 100% specificity when tested with negative control sera. The Spearman coefficient of the reported assay with an US-FDA approved, Taqman probe-based commercial kit was found to be 0.997. No significant difference in viral load was detected when the SYBR Green based assay was used to test infected plasma stored at -20°C and room temperature for 7 days respectively (Wilcoxon signed rank test, p=0.105). In a comparative study on 90 pretested HIV-1 positive samples with viral loads ranging from 5,000 - 25,000 HIV-1 RNA copies/ml and between two commercial assays it was found that the later failed to amplify in 13.33% and 10% samples respectively while in 7.77% and 4.44% samples the copy number values were reduced by >0.5 log value, a figure that is considered clinically significant by physicians. CONCLUSION: The HIV-1 viral load assay reported in this study was found to be robust, reliable, economical and effective in resource limited settings such as those existing in India. PCR probes specially designed from HIV-1 Subtype C-specific nucleotide sequences originating from India imparted specificity towards such isolates and demonstrated superior results when compared to two similar commercial assays widely used in India.

Distinct geographical clustering of HIV-1 and a signature amino acid at position 41 of the p24 unveiled by gag variability in India.

A portion of the gag gene cDNA for p24 protein from 30 Indian HIV-1 proviral DNA was amplified by PCR and sequenced. Phylogenetic analysis with reference samples of A1, A2, B, C, D, F1, F2, G, H, J, K, N and O subtypes revealed that 29 test samples aligned with subtype C reference strain while 1 matched with HIV-1 subtype A. Multiple alignment of predicted amino acid sequence of the Indian test samples and reference C subtype of HIV-1 samples from other countries indicated a molecular signature by way of rigid conservation of the amino acid 'S' at position 41 of the gag p24 protein in all Indian HIV-1 samples analyzed in this study as opposed to 'T' in the same position in C subtype sequences from other parts of the world. A phylogenetic analysis and visualization of the resulting tree in radial position showed distinct clubbing of all Indian C subtypes and formation of a cluster when compared to C subtype sequences from other countries with a single Chinese sample as an exception which was found in the Indian cluster. The use of a portion of p24 gene sequence as tool for subtyping as well as phylogenetic grouping with special reference to its geographical location is discussed.