Evaluation of the Recombinant Bacterial Chitinases as Anti-proliferative and Anti-migratory Agents for the Human Breast Cancer Cell Line, MCF-7.

Chitinases, a glycosyl hydrolase family 18 members, have a wide distribution in both prokaryotes and eukaryotes, including humans. Regardless of the absence of endogenous chitin polymer, various chitinases and chitinase-like proteins (CLPs) have been reported in mammals. However, several other carbohydrate polymers, such as hyaluronic acid and heparan sulfate, show structural similarities with chitin, which could be a potential target of chitinase and CLPs. Heparan sulfate is part of the integral membrane proteins and involves in cell adherence and migration. Hence, to demonstrate the effect of chitinase on cancer cell progression, we selected two chitinases from Serratia marcescens, ChiB and ChiC, which function as exo- and endo-chitinase, respectively. The ChiB and ChiC proteins were produced recombinantly by cloning chiB and chiC genes from Serratia marcescens. The cell viability of the Michigan Cancer Foundation-7 (MCF-7) cells was studied using different concentrations of the purified recombinant proteins. Cell viability assay was performed using 3-(4, 5-dimethyl thiazolyl-2)-2, 5-diphenyltetrazolium bromide and water-soluble tetrazolium salt, and the effect of ChiB and ChiC on cell proliferation was studied by clonogenic assay. The cell migration study was analysed by wound healing, transwell migration, and invasion assays. Cell cycle analysis of propidium iodide-stained cells and cell proliferation markers such as pERK1/2, pAKT, and SMP30 were also done. It was observed that both ChiB and ChiC were able to impede cell viability, cell migration, and invasion significantly. These observations and our in silico molecular docking analysis suggest that ChiC is a potential anticancer agent and is more efficient than ChiB. Since the ChiC is able to inhibit both cancer cell proliferation and migration, it could be a potential candidate for the treatment of metastatic cancer.

Burden of Typhoid and Paratyphoid Fever in India.

BACKGROUND: In 2017, more than half the cases of typhoid fever worldwide were projected to have occurred in India. In the absence of contemporary population-based data, it is unclear whether declining trends of hospitalization for typhoid in India reflect increased antibiotic treatment or a true reduction in infection. METHODS: From 2017 through 2020, we conducted weekly surveillance for acute febrile illness and measured the incidence of typhoid fever (as confirmed on blood culture) in a prospective cohort of children between the ages of 6 months and 14 years at three urban sites and one rural site in India. At an additional urban site and five rural sites, we combined blood-culture testing of hospitalized patients who had a fever with survey data regarding health care use to estimate incidence in the community. RESULTS: A total of 24,062 children who were enrolled in four cohorts contributed 46,959 child-years of observation. Among these children, 299 culture-confirmed typhoid cases were recorded, with an incidence per 100,000 child-years of 576 to 1173 cases in urban sites and 35 in rural Pune. The estimated incidence of typhoid fever from hospital surveillance ranged from 12 to 1622 cases per 100,000 child-years among children between the ages of 6 months and 14 years and from 108 to 970 cases per 100,000 person-years among those who were 15 years of age or older. Salmonella enterica serovar Paratyphi was isolated from 33 children, for an overall incidence of 68 cases per 100,000 child-years after adjustment for age. CONCLUSIONS: The incidence of typhoid fever in urban India remains high, with generally lower estimates of incidence in most rural areas. (Funded by the Bill and Melinda Gates Foundation; NSSEFI Clinical Trials Registry of India number, CTRI/2017/09/009719; ISRCTN registry number, ISRCTN72938224.).

Heart rate variability during head-up tilt shows inter-individual differences among healthy individuals of extreme Prakriti types.

Autonomic modulation is critical during various physiological activities, including orthostatic stimuli and primarily evaluated by heart rate variability (HRV). Orthostatic stress affects people differently suggesting the possibility of identification of predisposed groups to autonomic dysfunction-related disorders in a healthy state. One way to understand this kind of variability is by using Ayurvedic approach that classifies healthy individuals into Prakriti types based on clinical phenotypes. To this end, we explored the differential response to orthostatic stress in different Prakriti types using HRV. HRV was measured in 379 subjects(Vata = 97, Pitta = 68, Kapha = 68, and Mixed Prakriti = 146) from two geographical regions(Vadu and Delhi NCR) for 5 min supine (baseline), 3 min head-up-tilt (HUT) at 60°, and 5 min resupine. We observed that Kapha group had lower baseline HRV than other two groups, although not statistically significant. The relative change (%Δ1&2 ) in various HRV parameters in response to HUT was although minimal in Kapha group. Kapha also had significantly lower change in HR, LF (nu), HF (nu), and LF/HF than Pitta in response to HUT. The relative change (%Δ1 ) in HR and parasympathetic parameters (RMSSD, HF, SD1) was significantly greater in the Vata than in the Kapha. Thus, the low baseline and lower response to HUT in Kapha and the maximum drop in parasympathetic activity of Vata may indicate a predisposition to early autonomic dysfunction and associated conditions. It emphasizes the critical role of Prakriti-based phenotyping in stratifying the differential responses of cardiac autonomic modulation in various postures among healthy individuals across different populations.

An Overview of the Medicinally Important Plant Type III PKS Derived Polyketides.

Plants produce interesting secondary metabolites that are a valuable source of both medicines for human use, along with significant advantages for the manufacturer species. The active compounds which lead to these instrumental effects are generally secondary metabolites produced during various plant growth phases, which provide the host survival advantages while affecting human health inadvertently. Different chemical classes of secondary metabolites are biosynthesized by the plant type III polyketide synthases (PKSs). They are simple homodimeric proteins with the unique mechanistic potential to produce a broad array of secondary metabolites by utilizing simpler starter and extender units. These PKS derived products are majorly the precursors of some important secondary metabolite pathways leading to products such as flavonoids, stilbenes, benzalacetones, chromones, acridones, xanthones, cannabinoids, aliphatic waxes, alkaloids, anthrones, and pyrones. These secondary metabolites have various pharmaceutical, medicinal and industrial applications which make biosynthesizing type III PKSs an important tool for bioengineering purposes. Because of their structural simplicity and ease of manipulation, these enzymes have garnered interest in recent years due to their application in the generation of unnatural natural polyketides and modified products in the search for newer drugs for a variety of health problems. The following review covers the biosynthesis of a variety of type III PKS-derived secondary metabolites, their biological relevance, the associated enzymes, and recent research.

Geographically Weighted Regression Modeling of Spatial Clustering and Determinants of Focal Typhoid Fever Incidence.

BACKGROUND: Typhoid is known to be heterogenous in time and space, with documented spatiotemporal clustering and hotspots associated with environmental factors. This analysis evaluated spatial clustering of typhoid and modeled incidence rates of typhoid from active surveillance at 4 sites with child cohorts in India. METHODS: Among approximately 24 000 children aged 0.5-15 years followed for 2 years, typhoid was confirmed by blood culture in all children with fever >3 days. Local hotspots for incident typhoid cases were assessed using SaTScan spatial cluster detection. Incidence of typhoid was modeled with sociodemographic and water, sanitation, and hygiene-related factors in smaller grids using nonspatial and spatial regression analyses. RESULTS: Hotspot households for typhoid were identified at Vellore and Kolkata. There were 4 significant SaTScan clusters (P < .05) for typhoid in Vellore. Mean incidence of typhoid was 0.004 per child-year with the highest incidence (0.526 per child-year) in Kolkata. Unsafe water and poor sanitation were positively associated with typhoid in Kolkata and Delhi, whereas drinking untreated water was significantly associated in Vellore (P = .0342) and Delhi (P = .0188). CONCLUSIONS: Despite decades of efforts to improve water and sanitation by the Indian government, environmental factors continue to influence the incidence of typhoid. Hence, administration of the conjugate vaccine may be essential even as efforts to improve water and sanitation continue.

Antibiotics for Fever Among Children: Findings From the Surveillance for Enteric Fever in India Cohorts.

BACKGROUND: Acute febrile illness in children is frequently treated with antibiotics. However, the inappropriate use of antibiotics has led to the emergence of multidrug-resistant pathogens. METHODS: We measured use of antibiotics for fever in 4 pediatric cohorts that were part of the Surveillance for Enteric Fever in India (SEFI) network. In this network, 24 062 children were followed up weekly, capturing information on fever and other morbidity between October 2017 and December 2019. RESULTS: An antibiotic was given in 27 183 of the 76 027 (35.8%) episodes of fever. The incidence of fever-related antibiotic use was 58.0 (95% confidence interval [CI], 57.2-58.6) per 100 child-years. The median time to initiation of antibiotics was 4 days, and in 65% of those who received an antibiotic it was initiated by the second day. Antibiotics were continued for <3 days in 24% of the episodes. Higher temperature, younger age, male sex, joint family, higher education, internet access, and availability of personal conveyance were associated with antibiotic treatment for fever. CONCLUSIONS: In developing countries where antibiotic use is not regulated, broad-spectrum antibiotics are initiated early, and often inappropriately, in febrile illness. Frequent and inappropriate use of antibiotics may increase risk of antimicrobial resistance.

Western Indian Rural Gut Microbial Diversity in Extreme Prakriti Endo-Phenotypes Reveals Signature Microbes.

Heterogeneity amidst healthy individuals at genomic level is being widely acknowledged. This, in turn, is modulated by differential response to environmental cues and treatment regimens, necessitating the need for stratified/personalized therapy. We intend to understand the molecular determinants of Ayurvedic way (ancient Indian system of medicine) of endo-phenotyping individuals into distinct constitution types termed "Prakriti," which forms the basis of personalized treatment. In this study, we explored and analyzed the healthy human gut microbiome structure within three predominant Prakriti groups from a genetically homogenous cohort to discover differentially abundant taxa, using 16S rRNA gene based microbial community profiling. We found Bacteroidetes and Firmicutes as major gut microbial components in varying composition, albeit with similar trend across Prakriti. Multiple species of the core microbiome showed differential abundance within Prakriti types, with gender specific signature taxons. Our study reveals that despite overall uniform composition of gut microbial community, healthy individuals belonging to different Prakriti groups have enrichment of specific bacteria. It highlights the importance of Prakriti based endo-phenotypes to explain the variability amongst healthy individuals in gut microbial flora that have important consequences for an individual's health, disease and treatment.

Recapitulation of Ayurveda constitution types by machine learning of phenotypic traits.

In Ayurveda system of medicine individuals are classified into seven constitution types, "Prakriti", for assessing disease susceptibility and drug responsiveness. Prakriti evaluation involves clinical examination including questions about physiological and behavioural traits. A need was felt to develop models for accurately predicting Prakriti classes that have been shown to exhibit molecular differences. The present study was carried out on data of phenotypic attributes in 147 healthy individuals of three extreme Prakriti types, from a genetically homogeneous population of Western India. Unsupervised and supervised machine learning approaches were used to infer inherent structure of the data, and for feature selection and building classification models for Prakriti respectively. These models were validated in a North Indian population. Unsupervised clustering led to emergence of three natural clusters corresponding to three extreme Prakriti classes. The supervised modelling approaches could classify individuals, with distinct Prakriti types, in the training and validation sets. This study is the first to demonstrate that Prakriti types are distinct verifiable clusters within a multidimensional space of multiple interrelated phenotypic traits. It also provides a computational framework for predicting Prakriti classes from phenotypic attributes. This approach may be useful in precision medicine for stratification of endophenotypes in healthy and diseased populations.

Evaluation of enhanced thermostability and operational stability of carbonic anhydrase from Micrococcus species.

Carbonic anhydrase (CA) was purified from Micrococcus lylae and Micrococcus luteus with 49.90 and 53.8 % yield, respectively, isolated from calcium carbonate kilns. CA from M. lylae retained 80 % stability in the pH and temperature range of 6.0-8.0 and 35-45 °C, respectively. However, CA from M. luteus was stable in the pH and temperature range of 7.5-10.0 and 35-55 °C, respectively. Cross-linked enzyme aggregates (CLEAs) raised the transition temperature of M. lylae and M. luteus CA up to 67.5 and 74.0 °C, while the operational stability (T(1/20) of CA at 55 °C was calculated to be 7.7 and 12.0 h, respectively. CA from both the strains was found to be monomeric in nature with subunit molecular weight and molecular mass of 29 kDa. Ethoxozolamide was identified as the most potent inhibitor based on both IC(50) values and inhibitory constant measurement (K(i)). The K(m) and V(max) for M. lylae CA (2.31 mM; 769.23 µmol/mg/min) and M. luteus CA (2.0 mM; 1,000 µmol/mg/min) were calculated from Lineweaver-Burk plots in terms of esterase activity. Enhanced thermostability of CLEAs alleviates its role in operational stability for application at an on-site scrubber. The characteristic profile of purified CA from Micrococcus spp. advocates its effective application in biomimetic CO(2) sequestration.

Biomimetic CO2 sequestration using purified carbonic anhydrase from indigenous bacterial strains immobilized on biopolymeric materials.

The present study deals with immobilization of purified CA and whole cell of Pseudomonas fragi, Micrococcus lylae, and Micrococcus luteus 2 on different biopolymer matrices. Highest enzyme immobilization was achieved with P. fragi CA (89%) on chitosan-KOH beads, while maximum cell immobilization was achieved with M. lylae (75%) on chitosan-NH(4)OH beads. A maximum increase of 1.08-1.18 fold stability between 35 and 55°C was observed for M. lylae immobilized CA. The storage stability was improved by 2.02 folds after immobilization. FTIR spectra confirmed the adsorption of CA on chitosan-KOH beads following hydrophilic interactions. Calcium carbonate precipitation was achieved using chitosan-KOH immobilized P. fragi CA. More than 2 fold increase in sequestration potential was observed for immobilized system as compared to free enzyme. XRD spectra revealed calcite as the dominant phase in biomimetically produced calcium carbonate.

Characterization of carbonic anhydrase from diversified genus for biomimetic carbon-dioxide sequestration.

Diversified group of bacteria were screened for carbonic anhydrase (CA) activity. Significant CA activity was found in crude enzyme extracts of Enterobacter and Aeromonas isolates while minimal or negligible CA activity was observed in case of Shigella and Klebsiella spp. Optimization and characterization study of potent CA producing isolates revealed that the maximum enzyme activity of 3.86 EU/ml was observed in E. taylorae and the optimum pH range for enzyme stability was found to be 7.5-9.0 along with an optimum temperature range of 35-50 °C. The molecular mass of CA was 29-kDa indicating α-type with periplasmic and cytosolic location. Present investigation for the first time reports CA in diversified genus and optimized parameters for enhanced production of CA in Enterobacter sp. & Aeromonas sp. from fresh water bodies that inturn lay down grounds for exploitation of CA from E. taylorae as an efficient catalyst for CO(2) sequestration within a bioreactor.