Systematic Review and Geospatial Modeling of Molecular Markers of Resistance to Artemisinins and Sulfadoxine-Pyrimethamine in Plasmodium falciparum in India.

Surveillance for genetic markers of resistance can provide valuable information on the likely efficacy of antimalarials but needs to be targeted to ensure optimal use of resources. We conducted a systematic search and review of publications in seven databases to compile resistance marker data from studies in India. The sample collection from the studies identified from this search was conducted between 1994 and 2020, and these studies were published between 1994 and 2022. In all, Plasmodium falciparum Kelch13 (PfK13), P. falciparum dihydropteroate synthase, and P. falciparum dihydrofolate reductase (PfDHPS) genotype data from 2,953, 4,148, and 4,222 blood samples from patients with laboratory-confirmed malaria, respectively, were extracted from these publications and uploaded onto the WorldWide Antimalarial Resistance Network molecular surveyors. These data were fed into hierarchical geostatistical models to produce maps with a predicted prevalence of the PfK13 and PfDHPS markers, and of the associated uncertainty. Zones with a predicted PfDHPS 540E prevalence of >15% were identified in central, eastern, and northeastern India. The predicted prevalence of PfK13 mutants was nonzero at only a few locations, but were within or adjacent to the zones with >15% prevalence of PfDHPS 540E. There may be a greater probability of artesunate-sulfadoxine-pyrimethamine failures in these regions, but these predictions need confirmation. This work can be applied in India and elsewhere to help identify the treatments most likely to be effective for malaria elimination.

Safety and efficacy of primaquine in patients with Plasmodium vivax malaria from South Asia: a systematic review and individual patient data meta-analysis.

BACKGROUND: The optimal dosing of primaquine to prevent relapsing Plasmodium vivax malaria in South Asia remains unclear. We investigated the efficacy and safety of different primaquine regimens to prevent P. vivax relapse. METHODS: A systematic review identified P. vivax efficacy studies from South Asia published between 1 January 2000 and 23 August 2021. In a one-stage meta-analysis of available individual patient data, the cumulative risks of P. vivax recurrence at day 42 and 180 were assessed by primaquine total mg/kg dose and duration. The risk of recurrence by day 180 was also determined in a two-stage meta-analysis. Patients with a >25% drop in haemoglobin to <70 g/L, or an absolute drop of >50 g/L between days 1 and 14 were categorised by daily mg/kg primaquine dose. RESULTS: In 791 patients from 7 studies in the one-stage meta-analysis, the day 180 cumulative risk of recurrence was 61.1% (95% CI 42.2% to 80.4%; 201 patients; 25 recurrences) after treatment without primaquine, 28.8% (95% CI 8.2% to 74.1%; 398 patients; 4 recurrences) following low total (2 to <5 mg/kg) and 0% (96 patients; 0 recurrences) following high total dose primaquine (≥5 mg/kg). In the subsequent two-stage meta-analysis of nine studies (3529 patients), the pooled proportions of P. vivax recurrences by day 180 were 12.1% (95% CI 7.7% to 17.2%), 2.3% (95% CI 0.3% to 5.4%) and 0.7% (95% CI 0% to 6.1%), respectively. No patients had a >25% drop in haemoglobin to <70 g/L. CONCLUSIONS: Primaquine treatment led to a marked decrease in P. vivax recurrences following low (~3.5 mg/kg) and high (~7 mg/kg) total doses, with no reported severe haemolytic events. PROSPERO REGISTRATION NUMBER: CRD42022313730.

A comprehensive review on spatial and temporal variation of arsenic contamination in Ghaghara basin and its relation to probable incremental life time cancer risk in the local population.

Spatial and temporal variations have been found in the levels of arsenic (As) throughout the groundwater of the Ghaghara basin. Fifteen out of twenty-five districts in this basin are reported to be affected by As, where the levels of As in groundwater and soil exceed the permissible limits set by the WHO (10 µgl-1) and FAO (20 mgkg-1) respectively. These districts include a total of four municipalities in Nepal and eighty-six blocks in India, all of which have varying degrees of As contamination. Approximately 17 million people are at risk of As poisoning, with more than two orders of magnitude higher potential lifetime incremental cancer risk, constituting over 153 thousand potential additional cases of cancer due to As-contaminated drinking water. Out of the 90 As-contaminated blocks in the Ghaghara basin, 4 blocks have about 7-fold higher potential risk of developing cancer, 49 blocks have 8-37-fold higher risk, and 37 blocks have up to 375-fold higher risk compared to the upper limit of the USEPA acceptable range, which is 1 × 10-6-1 × 10-4. High accumulation of As has been reported in the nails, hair, and urine of local inhabitants, with higher levels observed in females than males. The toxicity of As is manifested in terms of a higher occurrence of various diseases. Reproductive endpoints, such as increased incidences of preterm birth, spontaneous abortion, stillbirth, low-birth weight, and neonatal death, have also been reported in the basin. The level of As in tube wells has been found to be negatively correlated with the depth (r = -0.906), and tube wells with high levels of As (>150 µgl-1) are generally located within close proximity (<10 km) to abandoned or present meander channels in the floodplain areas of the Ghaghara river. In addition to As contamination, the water quality index (WQI) in the Ghaghara basin is poor according to the BIS standards for drinking water. Groundwater in six out of fifteen districts is unsuitable for drinking purposes, with a WQI exceeding 100. The levels of As in agricultural soil in many villages of Ballia, Bahraich, and Lakhimpur Kheri districts have exceeded the FAO limit. Water from deep tube wells has been found to be relatively safe in terms of As content, and thus can be recommended for drinking purposes. However, the use of surface water needs to be encouraged for irrigation purposes in order to preserve soil health and reduce As contamination in the food chain, thereby minimizing the risk of cancer.

A critical analysis of various post-harvest arsenic removal treatments of rice and their impact on public health due to nutrient loss.

Rice (Oryza sativa L.) is particularly susceptible to arsenic (As) accumulation. Currently, to decrease the level of As accumulated in rice, various post-harvest methods, i.e., polishing, parboiling, pH-dependent soaking, washing, and cooking at different rice-to-water ratios (r/w), are being focused, because it removes significant amount of As from rice grain. Depending upon the rice variety and type, i.e., rough (with husk), husked (without husk/brown), or polished rice, these methods can remove 39-54% As by parboiling, 38-55% by polishing, 37-63% by soaking, and 6-80% by washing and cooking. Infants are highly vulnerable to As exposure; thus, these methods can be helpful for the production of rice-based infant foods. Although concern arises during the use of these methods that apart from decreasing the level of As in rice grain, they also lead to a significant loss of nutrients, such as macro- and micro-elements present in rice. Among these discussed methods, parboiling curtails 5-59%, polishing curtails 6-96%, soaking curtails 33-83%, and washing and cooking in different r/w reduce 8-81% of essential nutrients resulting in 2-90% reduction in contribution to the RDI of these nutrients through rice-based diet. Thus, these post-harvest arsenic removal methods, although reduce arsenic induced health hazard, but may also lead to malnutrition and compromised health in the population based on rice diet. There is a need to explore another way to reduce As from rice without compromising the nutrient availability or to supplement these nutrients through grain enrichment or by introducing additional dietary sources by changing eating habits; however, this may impose an extra economic burden on people.

HighAltitudeOmicsDB, an integrated resource for high-altitude associated genes and proteins, networks and semantic-similarities.

Millions of people worldwide visit, live or work in the hypoxic environment encountered at high altitudes and it is important to understand the biomolecular responses to this stress. This would help design mitigation strategies for high altitude illnesses. In spite of a number of studies spanning over 100 years, still the complex mechanisms controlling acclimatization to hypoxia remain largely unknown. To identify potential diagnostic, therapeutic and predictive markers for HA stress, it is important to comprehensively compare and analyse these studies. Towards this goal, HighAltitudeOmicsDB is a unique resource that provides a comprehensive, curated, user-friendly and detailed compilation of various genes/proteins which have been experimentally validated to be associated with various HA conditions, their protein-protein interactions (PPIs) and gene ontology (GO) semantic similarities. For each database entry, HighAltitudeOmicsDB additionally stores the level of regulation (up/down-regulation), fold change, study control group, duration and altitude of exposure, tissue of expression, source organism, level of hypoxia, method of experimental validation, place/country of study, ethnicity, geographical location etc. The database also collates information on disease and drug association, tissue-specific expression level, GO and KEGG pathway associations. The web resource is a unique server platform that offers interactive PPI networks and GO semantic similarity matrices among the interactors.These unique features help to offer mechanistic insights into the disease pathology. Hence, HighAltitudeOmicsDBis a unique platform for researchers working in this area to explore, fetch, compare and analyse HA-associated genes/proteins, their PPI networks, and GO semantic similarities. The database is available at http://www.altitudeomicsdb.in .

Evaluating the efficacy and feasibility of post harvest methods for arsenic removal from rice grain and reduction of arsenic induced cancer risk from rice-based diet.

Food-chain arsenic (As) contamination is a severe environmental and health problem worldwide, and its intake through rice affects billions of people. In this review, we have summarized the post harvest As removal methods from rice and their efficacy and feasibility. Rice grain subspecies (indica and japonica), size (short, medium and long), type (husked, parboiled or polished), soaking time, temperature and rice to water ratio (r/w) during washing and cooking are the major factors that affect the removal of total arsenic (tAs) from rice grain. The reduction in tAs was greater in japonica than indica rice and was directly proportional to As in husked rice. For the removal of As, a low water volume (1:2 r/w) was more effective during washing due to friction between rice grains, while high water (≥4 times water) during cooking was more effective. Up to 80 % As was removed by cooking in 1:10 (rice: water). Soaking rice in edible acids such as vinegar, acetic and ascorbic acid was not effective, except citric acid, which removes tAs up to 63 %. Human-health risk assessment showed that these post harvest and cooking methods reduce the non-carcinogenic and incremental lifetime cancer risk by up to 5-fold, as calculated on the basis of bioaccessible inorganic As. These post harvest methods also remove nutrient elements and vitamins. The recommended dietary intake (RDI) of Zn and Cu was particularly affected (up to 40 and 83 %). The levels of P, Mo, Mn and Co were still sufficient to meet the RDI through the rice-based diet, while rice is already poor in the RDI of Ca, K, Fe and Se, and their levels were further reduced by 0.22-44 %. In conclusion, these post harvest and cooking methods may significantly reduce As induced health risks; however, other dietary sources of nutrients need to be carefully evaluated and supplemented.

Cardiovascular diseases display etiological and seasonal trend in human population: Evidence from seasonal cardiovascular comorbid diseases (SCCD) index.

Seasonal changes in the human cardiovascular system are known to play an important role in the onset of many diseases. Confounding variables include behavioral and environmental factors; failing to address such variables makes measuring the true temporal impact of these diseases difficult. On the other hand, numerous clinical studies imply that only specific groups of people are more seasonal sensitive and that their maladaptation might contribute to various illnesses. As a result, it is critical to evaluate the etiological and seasonal sensitive patterns of cardiovascular diseases (CVD), which impact the majority of the human population. The hypothesis for this study formulated that cardiovascular and associated illnesses had substantial connections with seasonal and etiological variations. Thus in the present study, 4519 systematic screen-eligible studies were analyzed using data mining to uncover 852 disease association relationships between cardiovascular and associated disorders. A disease ontology-based semantic similarity network (DSN) analysis was performed to narrow down the identified CVDs. Further, topological analysis was used to predict the seven CVDs, including myocardial infarction (MI), in three clusters. Following that, Mann-Kendall and Cox-Stuart analyses were used to investigate the seasonal sensitivity and temporal relationship of these seven CVDs. Finally, temporal relationships were confirmed using LOESS and TBATS, as well as seasonal breakdown utilizing autocorrelation and fast Fourier transform results. The study provides indirect evidence of a severe etiological association among the three cardiovascular diseases, including MI, atrial fibrillation, and atherosclerosis, which are winter season sensitive in most of the world population. Hypertension has two seasonal falls and peaks due to its seasonal nature, that is, summer and winter hypertension. While, heart failure was also identified, with minor temporal trends. Hence, all five diseases could be classified as seasonal cardiovascular comorbid diseases (SCCD). Furthermore, these diseases could be studied for potential common risk factors such as biochemical, genetic, and physiological factors.

High-density lipoproteins may play a crucial role in COVID-19.

BACKGROUND: Lipids play a central role in the virus life cycle and are a crucial target to develop antiviral therapeutics. Importantly, among the other lipoproteins, the 'good cholesterol' high-density lipoprotein (HDL) has been widely studied for its role in not only cardiovascular but several infectious diseases as well. Studies have suggested a role of serum lipids and lipoproteins including HDL, total cholesterol (TC), triglycerides (TG), and low-density lipoproteins (LDL) in several viral infections including COVID-19. This disease is currently a major public health problem and there is a need to explore the role of these host lipids/lipoproteins in virus pathogenesis. METHODOLOGY: A total of 75 retrospective COVID-19 positive serum samples and 10 COVID-19 negative controls were studied for their lipid profiles including TC, HDL, LDL, and very-low-density lipoproteins (VLDL), and TG. RESULTS: Systematic literature search on dyslipidemia status in India shows that low HDL is the most common dyslipidemia. In this cohort, 65% (49) of COVID-19 patients had severely low HDL levels whereas 35% (26) had moderately low HDL and none had normal HDL levels. On the other hand, ~ 96% of samples had normal TC (72) and LDL (72) levels. VLDL and TG levels were also variable. In the controls, 100% of samples had moderately low HDL but none severely low HDL levels. CONCLUSION: HDL likely plays a crucial role in COVID-19 infection and outcomes. The causal relationships between HDL levels and COVID-19 need to be studied extensively for an understanding of disease pathogenesis and management.

A network-biology led computational drug repurposing strategy to prioritize therapeutic options for COVID-19.

The alarming pandemic situation of novel Severe Acute Respiratory Syndrome Coronavirus 2 (nSARS-CoV-2) infection, high drug development cost and slow process of drug discovery have made repositioning of existing drugs for therapeutics a popular alternative. It involves the repurposing of existing safe compounds which results in low overall development costs and shorter development timeline. In the present study, a computational network-biology approach has been used for comparing three candidate drugs i.e. quercetin, N-acetyl cysteine (NAC), and 2-deoxy-glucose (2-DG) to be effectively repurposed against COVID-19. For this, the associations between these drugs and genes of Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS) diseases were retrieved and a directed drug-gene-gene-disease interaction network was constructed. Further, to quantify the associations between a target gene and a disease gene, the shortest paths from the target gene to the disease genes were identified. A vector DV was calculated to represent the extent to which a disease gene was influenced by these drugs. Quercetin was quantified as the best among the three drugs, suited for repurposing with DV of -70.19, followed by NAC with DV of -39.99 and 2-DG with DV of -13.71. The drugs were also assessed for their safety and efficacy balance (in terms of therapeutic index) using network properties. It was found that quercetin was a forerunner than other two drugs.

HAHmiR.DB: a server platform for high-altitude human miRNA-gene coregulatory networks and associated regulatory circuits.

Around 140 million people live in high-altitude (HA) conditions! and even a larger number visit such places for tourism, adventure-seeking or sports training. Rapid ascent to HA can cause severe damage to the body organs and may lead to many fatal disorders. During induction to HA, human body undergoes various physiological, biochemical, hematological and molecular changes to adapt to the extreme environmental conditions. Several literature references hint that gene-expression-regulation and regulatory molecules like miRNAs and transcription factors (TFs) control adaptive responses during HA stress. These biomolecules are known to interact in a complex combinatorial manner to fine-tune the gene expression and help in controlling the molecular responses during this stress and ultimately help in acclimatization. High-Altitude Human miRNA Database (HAHmiR.DB) is a unique, comprehensive and curated collection of miRNAs that have been experimentally validated to be associated with HA stress, their level of expression in different altitudes, fold change, experiment duration, biomarker association, disease and drug association, tissue-specific expression level, Gene Ontology (GO) and Kyoto Encyclopaedia of Gene and Genomes (KEGG) pathway associations. As a server platform, it also uniquely constructs and analyses interactive miRNA-TF-gene coregulatory networks and extracts regulatory circuits/feed-forward loops (FFLs). These regulatory circuits help to offer mechanistic insights into complex regulatory mechanisms during HA stress. The server can also build these regulatory networks between two and more miRNAs of the database and also identify the regulatory circuits from this network. Hence, HAHmiR.DB is the first-of-its-kind database in HA research, which is a reliable platform to explore, compare, analyse and retrieve miRNAs associated with HA stress, their coregulatory networks and FFL regulatory-circuits. HAHmiR.DB is freely accessible at http://www.hahmirdb.in.

MyomirDB: A unified database and server platform for muscle atrophy myomiRs, coregulatory networks and regulons.

Muscular atrophy or muscle loss is a multifactorial clinical condition during many critical illnesses like cancer, cardiovascular diseases, diabetes, pulmonary diseases etc. leading to fatigue and weakness and contributes towards a decreased quality of life. The proportion of older adults (>65 y) in the overall population is also growing and aging is another important factor causing muscle loss. Some muscle miRNAs (myomiRs) and their target genes have even been proposed as potential diagnostic, therapeutic and predictive markers for muscular atrophy. MyomirDB (http://www.myomirdb.in/) is a unique resource that provides a comprehensive, curated, user- friendly and detailed compilation of various miRNA bio-molecular interactions; miRNA-Transcription Factor-Target Gene co-regulatory networks and ~8000 tripartite regulons associated with 247 myomiRs which have been experimentally validated to be associated with various muscular atrophy conditions. For each database entry, MyomirDB compiles source organism, muscle atrophic condition, experiment duration, its level of expression, fold change, tissue of expression, experimental validation, disease and drug association, tissue-specific expression level, Gene Ontology and KEGG pathway associations. The web resource is a unique server platform which uses in-house scripts to construct miRNA-Transcription Factor-Target Gene co-regulatory networks and extract tri-partite regulons also called Feed Forward Loops. These unique features helps to offer mechanistic insights in disease pathology. Hence, MyomirDB is a unique platform for researchers working in this area to explore, fetch, compare and analyse atrophy associated miRNAs, their co-regulatory networks and FFL regulons.

Analysis of Hypoxiamir-Gene Regulatory Network Identifies Critical MiRNAs Influencing Cell-Cycle Regulation Under Hypoxic Conditions.

BACKGROUND: Hypoxia is a pathophysiological condition which arises due to low oxygen concentration in conditions like cardiovascular diseases, inflammation, ascent to higher altitude, malignancies, deep sea diving, prenatal birth, etc. A number of microRNAs (miRNAs), Transcription Factors (TFs) and genes have been studied separately for their role in hypoxic adaptation and controlling cell-cycle progression and apoptosis during this stress. OBJECTIVE: We hypothesize that miRNAs and TFs may act in conjunction to regulate a multitude of genes and play a crucial and combinatorial role during hypoxia-stress-responses and associated cellcycle control mechanisms. METHOD: We collected a comprehensive and non-redundant list of human hypoxia-responsive miRNAs (also known as hypoxiamiRs). Their experimentally validated gene-targets were retrieved from various databases and a comprehensive hypoxiamiR-gene regulatory network was built. RESULTS: Functional characterization and pathway enrichment of genes identified phospho-proteins as enriched nodes. The phospho-proteins which were localized both in the nucleus and cytoplasm and could potentially play important role as signaling molecules were selected; and further pathway enrichment revealed that most of them were involved in NFkB signaling. Topological analysis identified several critical hypoxiamiRs and network perturbations confirmed their importance in the network. Feed Forward Loops (FFLs) were identified in the subnetwork of enriched genes, miRNAs and TFs. Statistically significant FFLs consisted of four miRNAs (hsa-miR-182-5p, hsa- miR-146b-5p, hsa-miR-96, hsa-miR-20a) and three TFs (SMAD4, FOXO1, HIF1A) both regulating two genes (NFkB1A and CDKN1A). CONCLUSION: Detailed BioCarta pathway analysis identified that these miRNAs and TFs together play a critical and combinatorial role in regulating cell-cycle under hypoxia, by controlling mechanisms that activate cell-cycle checkpoint protein, CDKN1A. These modules work synergistically to regulate cell-proliferation, cell-growth, cell-differentiation and apoptosis during hypoxia. A detailed mechanistic molecular model of how these co-regulatory FFLs may regulate the cell-cycle transitions during hypoxic stress conditions is also put forth. These biomolecules may play a crucial and deterministic role in deciding the fate of the cell under hypoxic-stress.

Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli.

Microbial production of value-added chemicals from biomass is a sustainable alternative to chemical synthesis. To improve product titer, yield, and selectivity, the pathways engineered into microbes must be optimized. One strategy for optimization is dynamic pathway regulation, which modulates expression of pathway-relevant enzymes over the course of fermentation. Metabolic engineers have used dynamic regulation to redirect endogenous flux toward product formation, balance the production and consumption rates of key intermediates, and suppress production of toxic intermediates until later in the fermentation. Most cases, however, have utilized a single strategy for dynamically regulating pathway fluxes. Here we layer two orthogonal, autonomous, and tunable dynamic regulation strategies to independently modulate expression of two different enzymes to improve production of D-glucaric acid from a heterologous pathway. The first strategy uses a previously described pathway-independent quorum sensing system to dynamically knock down glycolytic flux and redirect carbon into production of glucaric acid, thereby switching cells from "growth" to "production" mode. The second strategy, developed in this work, uses a biosensor for myo-inositol (MI), an intermediate in the glucaric acid production pathway, to induce expression of a downstream enzyme upon sufficient buildup of MI. The latter, pathway-dependent strategy leads to a 2.5-fold increase in titer when used in isolation and a fourfold increase when added to a strain employing the former, pathway-independent regulatory system. The dual-regulation strain produces nearly 2 g/L glucaric acid, representing the highest glucaric acid titer reported to date in Escherichia coli K-12 strains.

Therapeutic Implications of Superoxide Dismutase And Its Importance in Kinase Drug Discovery.

In recent years, several scientific investigations have reported the therapeutic implications of superoxide dismutase (SOD) against oxidative stress and -induced pathology in clinical and preclinical trials. Indeed, various kinase, molecular signaling and physiological process has altered by reactive oxygen species. In spite of the abundant available literature reports, patents, clinical trials and commercialized products, the therapeutic application of SOD as a potential drug still remains unclear. Owing to the technical challenges associated with the utilization of SOD as a drug, we revisited the structural arrangement and cellular signaling, significant association with kinase, exploring the new target sites and introducing new formulation strategies such as gene modulation, nano-formulations and click chemistry is a prerequisite. In-addition to gene modulation strategies, encapsulated formulation within a nano-carrier for producing promising SOD therapeutic effects, application of click chemistry including bioconjugation and cyclo-addition are the most prominent methods to produce highly efficient SOD formulations. Thus, the present review enlightens the foremost technique which may have better interaction with kinase and other cellular signaling for regulating the physiological process.

Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit.

Metabolic engineering of microorganisms to produce desirable products on an industrial scale can result in unbalanced cellular metabolic networks that reduce productivity and yield. Metabolic fluxes can be rebalanced using dynamic pathway regulation, but few broadly applicable tools are available to achieve this. We present a pathway-independent genetic control module that can be used to dynamically regulate the expression of target genes. We apply our module to identify the optimal point to redirect glycolytic flux into heterologous engineered pathways in Escherichia coli, resulting in titers of myo-inositol increased 5.5-fold and titers of glucaric acid increased from unmeasurable to >0.8 g/L, compared to the parent strains lacking dynamic flux control. Scaled-up production of these strains in benchtop bioreactors resulted in almost ten- and fivefold increases in specific titers of myo-inositol and glucaric acid, respectively. We also used our module to control flux into aromatic amino acid biosynthesis to increase titers of shikimate in E. coli from unmeasurable to >100 mg/L.

Improvement of glucaric acid production in E. coli via dynamic control of metabolic fluxes.

D-glucaric acid can be used as a building block for biopolymers as well as in the formulation of detergents and corrosion inhibitors. A biosynthetic route for production in E. coli has been developed (Moon et al., 2009), but previous work with the glucaric acid pathway has indicated that competition with endogenous metabolism may limit carbon flux into the pathway. Our group has recently developed an E. coli strain where phosphofructokinase (Pfk) activity can be dynamically controlled and demonstrated its use for improving yields and titers of the glucaric acid precursor myo-inositol on glucose minimal medium. In this work, we have explored the further applicability of this strain for glucaric acid production in a supplemented medium more relevant for scale-up studies, both under batch conditions and with glucose feeding via in situ enzymatic starch hydrolysis. It was found that glucaric acid titers could be improved by up to 42% with appropriately timed knockdown of Pfk activity during glucose feeding. The glucose feeding protocol could also be used for reduction of acetate production in the wild type and modified E. coli strains.

Screening and modular design for metabolic pathway optimization.

Biological conversion of substrate sugars to a variety of products is an increasingly popular option for chemical transformation due to its high specificity and because of significant interest in the use of renewable feedstocks. However, pathway optimization through metabolic engineering is often needed to make such molecules economically at a relevant scale. Employing effective methods to search and narrow the immense pathway parameter space is essential to meet performance metrics such as high titer, yield and productivity with efficiency. This review focuses on two practices that increase the likelihood of finding a more advantageous pathway solution: implementing a screen to identify high producers and utilizing modular pathway design to streamline engineering efforts. While screens seek to couple product titer with a high-throughput measurement output, modular design aims to rationally construct pathways to allow parallel optimization of various units. Both of these methodologies have proven widely successful in metabolic engineering, with combinations of them resulting in synergistic enhancements to pathway optimization. This review will particularly highlight their utility for microbially derived acid and alcohol products, which are of interest as fuels and value added products.

Effect of Oxidative Phytochemicals on Nicotine-stressed UMNSAH/DF-1 Cell Line.

Nicotine is a parasympathomimetic alkaloid found in the nightshade family of plants (Solanaceae) and is a cholinergic drug. It acts directly by stimulating the nicotinic or muscarinic receptors or indirectly by inhibiting cholinesterase, promoting acetylcholine release, or by other mechanisms. 3% of tobacco or one cigarette yields 1 mg of nicotine. As nicotine enters the body, it disturbs the healthy functioning of the body. In this study, we isolated UMNSAH/DF-1 cell line from Gallus gallus. For this, 9 ± 2 day old chicken embryo was taken. This was followed by the extraction of nicotine (1 mg/ml) from cigarette. The cells were then given nicotine stress and were observed for blackening after 24 h of incubation under 40× resolution of microscope. It was found that this blackening of the cells was permanent even after a wash with 1× phosphate-buffered saline (PBS) followed by replenishing the medium. The phytochemicals extracted were from the dried powder, which included Curcuma longa ( Jiang Huáng; Turmeric) 40 mg/ml, Azadirachta indica (neem) 50 mg/ml, Cinnamomum tamala (bay leaf) 30 mg/ml, Camellia sinensis ( Lǜ Chá; Green Tea) 100 mg/ml, and Ocimum sanctum (tulsi) 30 mg/ml. When applied to nicotine-stressed cells, it was observed that ursolic acid in neem recovered 70%, followed by 65% recovery by tulsi (having triterpenoid), 50% recovery by the catechins in Ca. sinensis, and very little recovery shown by Ci. tamala. Due to the yellow coloration of the cells by Cu. longa, much could not be inferred, although it was inferable that it had resulted in little effects. Mixtures of these phytochemicals were used, and it was found that neem: tulsi diluted in 3:1 ratio was highly effective and cell recovery was almost 80%. 68% was recovered by tulsi: green tea in a ratio 1:3 and 42% by turmeric:green tea in a ratio of 1:5.

Encapsulated fusion protein confers "sense and respond" activity to chitosan-alginate capsules to manipulate bacterial quorum sensing.

We demonstrate that "nanofactory"-loaded biopolymer capsules placed in the midst of a bacterial population can direct bacterial communication. Quorum sensing (QS) is a process by which bacteria communicate through small-molecules, such as autoinducer-2 (AI-2), leading to collective behaviors such as virulence and biofilm formation. In our approach, a "nanofactory" construct is created, which comprises an antibody complexed with a fusion protein that produces AI-2. These nanofactories are entrapped within capsules formed by electrostatic complexation of cationic (chitosan) and anionic (sodium alginate) biopolymers. The chitosan capsule shell is crosslinked by tripolyphosphate (TPP) to confer structural integrity. The capsule shell is impermeable to the encapsulated nanofactories, but freely permeable to small molecules. In turn, the capsules are able to take in substrates from the external medium via diffusion, and convert these via the nanofactories into AI-2, which then diffuses out. The exported AI-2 is shown to stimulate QS responses in vicinal Escherichia coli. Directing bacterial population behavior has potential applications in next-generation antimicrobial therapy and pathogen detection. We also envision such capsules to be akin to artificial "cells" that can participate in native biological signaling and communicate in real-time with the human microbiome. Through such interaction capabilities, these "cells" may sense the health of the microbiome, and direct its function in a desired, host-friendly manner.

Biological nanofactories target and activate epithelial cell surfaces for modulating bacterial quorum sensing and interspecies signaling.

In order to control the behavior of bacteria present at the surface of human epithelial cells, we have created a biological "nanofactory" construct that "coats" the epithelial cells and "activates" the surface to produce the bacterial quorum sensing signaling molecule, autoinducer-2 (AI-2). Specifically, we demonstrate directed modulation of signaling among Escherichia coli cells grown over the surface of human epithelial (Caco-2) cells through site-directed attachment of biological nanofactories. These "factories" comprise a fusion protein expressed and purified from E. coli containing two AI-2 bacterial synthases (Pfs and LuxS), a protein G IgG binding domain, and affinity ligands for purification. The final factory is fabricated ex vivo by incubating with an anti-CD26 antibody that binds the fusion protein and specifically targets the CD26 dipeptidyl peptidase found on the outer surface of Caco-2 cells. This is the first report of the intentional "in vitro" synthesis of bacterial autoinducers at the surface of epithelial cells for the redirection of quorum sensing behaviors of bacteria. We envision tools such as this will be useful for interrogating, interpreting, and disrupting signaling events associated with the microbiome localized in human intestine and other environments.