Engineering of a specific CYP450 for an industrial process shows 700-fold increase in activity with Kcat of 6.2 s-1 - Residues causing Hydrogen Migration and Double Hydrogen Abstraction at Δx Carbon identified by Quantum Mechanics revealed to be the game

V3 2 32.7 and 33.8 3.5±0.5 Figure 1A. – Cyp450 complexed with heme and substrate integrated in the yeast hypo membrane B. Shows the rate limiting transitions state of the enzymatic reaction Table 1 – Energies of the crucial transition states and the experimental Kcat values A CYP450 catalyzed desaturation reaction for the synthesis of an Active Pharmaceutical Ingredient (API) via biocatalytic route that is estimated to top up the revenue of the company to 8.5 million dollars was identified in this study. Currently this route is being tested for the industrial scale production of the API. This was achieved by engineering a specific membrane bound CYP450 enzyme that was initially inactive towards the substrate. Extensive modelling studies were carried out to obtain an initial promiscuous activity (0.53/min). Here the substrate enters through the transmembrane region of the enzyme that acts as a tunnel and moves to the Heme Binding Site in the CYP450 during which it undergoes rotation of ~180°. The substrate entry was identified as the first rate limiting step in the reaction. Using a grid-based path optimization method this path was engineered to facilitate the easy entry and movement of substrate in to the active site. The second rate limiting step was observed in the formation of two transition states of the reaction which was identified using Quantum Mechanics hybridized with Molecular Mechanics (QM/MM) simulations. The enzyme was engineered using quantum polarized grid technology to reduce the energy of these transition states. The mutations introduced, improved the enzyme activity \u3e 700 folds giving a Kcat value of 6.2s-1 which translated to a good yield of the product in the lab scale fermenter. A greater finding in this project is the desaturation reaction mechanisms involving the hydrogen migration and double hydrogen abstracted from the same ΔX Carbon. This was obtained using extensive QM/MM simulations studies and a high-resolution grid energy evaluation method which revealed transition and intermediate states of the reaction, not reported before in any of the desaturation mechanisms solved so far. Some important aspects of this study include, modelling yeast membrane with different lipids and varying substrate concentrations, enzyme behavior at high substrate concentrations and process optimization. In conclusion, this is a cost effective route with a conversion rate that is ~10 times more than the chemical route. This process can be applied to engineer CYP450 for any chemical routes that involve a desaturation reaction Please click Additional Files below to see the full abstract

7D-Grid-AI-Technology: A technology that translates enzymes from a computer to business with limited lab experiments

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7D QSAR based grid maps generated using quantum mechanic probes to identify hotspots and predict activity of mutated enzymes for enzyme engineering

Use of Quantum Mechanics hybridized with Molecular Mechanics (QM/MM) in Enzyme studies have greatly accelerated the finding of intermediate states of enzymatic reactions. The gaps in the conventional methods are in the identification of hot spots and screening enzyme variants. As a proof of concept, for the first time, receptor dependent – 4D Quantitative Structure Activity Relationship (RD-4D-QSAR) to predict kinetic properties of enzymes was demonstrated by Pravin Kumar et al, presented in Enzyme Engineering XXII, 2013, Toyama. We have extended this methodology to study enzymes using 7D-QSAR based grid maps. Induced-fit scenarios were explored using QM/MM simulations, which was then placed in a grid that stores interactions energies derived from QM parameters (QMgrid). The novelty of this method is that the mutated enzymes are immersed completely inside the QMgrid and this is combined with solvation models to predict descriptors; the grid captures the accurate electronic details of the reaction at very high resolution. Every grid point here is a probe, which are atoms that mimic atoms of the substrate interacting with the atoms of the enzyme, also atoms of the enzyme interacting with itself. The probes with its reaction coordinates are mapped on the ES complex conformations derived from ES, enzyme-transition and enzyme-product stages. The statistically relevant conformations are derived after screening using knowledge-based energy scoring matrices. The grid map shows high energy and low energy reactions across the ES system, which is used to pick hotspots. A substitution matrix is automatically constructed on the chosen hotspots using an evolutionary based scoring matrix coupled with statistical modelling process that gives the suited amino acids for a specific hot spot. We have tested this on a specific transaminase and QSAR models showed \u3e90% specificity and \u3e85% sensitivity towards the experimental activity with enzyme variants. Mapping descriptors on the enzyme structure revealed hotspots important to enhance the enantioselectivity of the enzyme. The method is efficient to design enzymes and proteins with minimum of double extending upto seven mutations on its own. Please click Additional Files below to see the full abstract

Addressing the challenges of Visually Impaired using IoT

Internet of Things is a new revolution of the Internet. IoT allows networked objects to be sensed and controlled remotely, creating opportunities for more direct integration of the physical world into computer-based systems, resulting in reduced human intervention. People with complete blindness or low vision often have a difficult time self-navigating in unusual environments. In fact, mobility is one of the biggest challenges for visually Impaired. IoT offers the assistance and support to the visually impaired people to achieve a quality life allowing them to involve in social activities. Assistive IoT technologies are powerful tools to increase independence and improve participation. Therefore, the purpose of this paper is to analyze how people with visual impairment can interact with and benefit from the IoT

ROLE OF GANDUSHA IN PITTAJA MUKHAPAKA (APHTHOUS ULCER) - A RANDOMIZED CONTROLLED TRIAL

Background: Pittaja Mukhapaka or the Aphthous ulcer is a painful and often recurrent inflammatory process of the oral mucosa that can appear secondary to various well-defined disease processes. Idiopathic recurrent aphthous stomatitis is referred to as recurrent aphthous stomatitis. Objectives: To evaluate the effect of Patoladi kashaya gandusha in Toda, Daha, Asyavairasyata and the pH of Pittaja mukhapaka (Aphthous ulcer) and to standardize the frequency of Gandusha (An Ayurvedic intervention). Design: This was a randomized controlled study with thirty patients divided into two groups, 15 in each. In Group A, Patoladi kashaya gandusha (gargling) was given once a day for 7 days and in Group B, Patoladi kashaya gandusha (gargling) was carried out twice a day for 7 days. After the completion of treatment, follow up was done once in fifteen days for a period of 2 months. Results: Statistically significant results were seen in reduction of Toda with p<0.001, Group A (61.53%) compared to Group B (81.81%), Daha with p<0.001 Group A (73.07%) compared to Group B (81.81%), Asyavairasyata with p<0.001 Group A (73.91%) compared to Group B (100%) and also in relieving the pH of saliva with p<0.001 and Group A (100%) compared to Group B (100%). Conclusions: Group B (Patoladi kashaya gandusha given twice a day) showed statistically significant results compared to Group A (Patoladi kashaya gandusha given once a day) in reduction of Toda, Daha, Asya vairasyata and the pH of Pittaja Mukhapaka (Aphthous ulcer)

Insilico guided CRISPR-Cas driven enzyme engineering framework: An automated and efficient enzyme engineering method

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Comparison of pattern of self-medication among urban and rural population of Telangana state, India

Background: Self-medication is one of the components of self-care, which may treat the disease or result in worsening of the condition due to irrational use of drug.1 In developing countries like India, self-medication is a common practice as it provides a low-cost alternative for people who cannot afford the high cost of clinical service, and is time efficient.Methods: A total of 110 participants completed the study. A printed questionnaire was given to those who were willing to participate in the study and came to buy medicines without consulting a doctor to various pharmacy outlets.Results: Among the group of drugs used antibiotics were the common drugs used in rural area (74%) and cough suppressants (50%) in urban area. Symptoms for opting self-medication were fever and common cold in both the groups. Individuals in both areas took self-medication based on their previous prescriptions (rural 42% vs urban41.6%) and advertisements. Rural individuals preferred self-medication with the opinion of saving time and urban people felt that it was less expensive.Conclusions: There is a difference in the pattern self-medication among rural and urban individuals. It is also to be noted that use of antibiotics may result in problems related to drug resistance. So, it would be advisable to restrict the sale of antibiotics as over the counter drugs

Genetic evidence that SMAD2 is not required for gonadal tumor development in inhibin-deficient mice

<p>Abstract</p> <p>Background</p> <p>Inhibin is a tumor-suppressor and activin antagonist. Inhibin-deficient mice develop gonadal tumors and a cachexia wasting syndrome due to enhanced activin signaling. Because activins signal through SMAD2 and SMAD3 in vitro and loss of SMAD3 attenuates ovarian tumor development in inhibin-deficient females, we sought to determine the role of SMAD2 in the development of ovarian tumors originating from the granulosa cell lineage.</p> <p>Methods</p> <p>Using an inhibin α null mouse model and a conditional knockout strategy, double conditional knockout mice of Smad2 and inhibin alpha were generated in the current study. The survival rate and development of gonadal tumors and the accompanying cachexia wasting syndrome were monitored.</p> <p>Results</p> <p>Nearly identical to the controls, the Smad2 and inhibin alpha double knockout mice succumbed to weight loss, aggressive tumor progression, and death. Furthermore, elevated activin levels and activin-induced pathologies in the liver and stomach characteristic of inhibin deficiency were also observed in these mice. Our results indicate that SMAD2 ablation does not protect inhibin-deficient females from the development of ovarian tumors or the cachexia wasting syndrome.</p> <p>Conclusions</p> <p>SMAD2 is not required for mediating tumorigenic signals of activin in ovarian tumor development caused by loss of inhibin.</p

Dangers of Polypharmacy

Although the definition of polypharmacy has evolved over time, it has been and remains to be an issue in healthcare. With the prevalence of polypharmacy increasing, those in the health care field must remain vigilant of the adverse effects of medications and work to coordinate care and maintain appropriate prescribing practices. Here we present a clinical vignette that describes an encounter of a patient on multiple medications and the individual, provider, and systems‐level issues that may have contributed to an adverse event resulting in a hospital stay. We will discuss the definition of polypharmacy, review the prevalence and economic implications of drug prescription practices, and examine the consequences and complications of polypharmacy in a number of different patient populations. We will discuss a number of scenarios involving polypharmacy that lead to medication errors, decreased quality of life, and patient harm, and then review evidence‐based methods of interventions aimed at reducing the prevalence of polypharmacy and its associated complications

Association of Long-Term Exposure to Fine Particulate Matter and Cardio-Metabolic Diseases in Low- and Middle-Income Countries: A Systematic Review.

: Background: Numerous epidemiological studies indicated high levels of particulate matter less than2.5 μm diameter (PM2.5) as a major cardiovascular risk factor. Most of the studies have been conducted in high-income countries (HICs), where average levels of PM2.5 are far less compared to low- and middle- income countries (LMICs), and their socio-economic profile, disease burden, and PM speciation/composition are very different. We systematically reviewed the association of long-term exposure to PM2.5 and cardio-metabolic diseases (CMDs) in LMICs. METHODS: Multiple databases were searched for English articles with date limits until March 2018. We included studies investigating the association of long-term exposure to PM2.5 (defined as an annual average/average measure for 3 more days of PM2.5 exposure) and CMDs, such as hospital admissions, prevalence, and deaths due to CMDs, conducted in LMICs as defined by World Bank. We excluded studies which employed exposure proxy measures, studies among specific occupational groups, and specific episodes of air pollution. RESULTS: A total of 5567 unique articles were identified, of which only 17 articles were included for final review, and these studies were from Brazil, Bulgaria, China, India, and Mexico. Outcome assessed were hypertension, type 2 diabetes mellitus and insulin resistance, and cardiovascular disease (CVD)-related emergency room visits/admissions, death, and mortality. Largely a positive association between exposure to PM2.5 and CMDs was found, and CVD mortality with effect estimates ranging from 0.24% to 6.11% increased per 10 μg/m3 in PM2.5. CVD-related hospitalizations and emergency room visits increased by 0.3% to 19.6%. Risk factors like hypertension had an odds ratio of 1.14, and type 2 diabetes mellitus had an odds ratio ranging from 1.14-1.32. Diversity of exposure assessment and health outcomes limited the ability to perform a meta-analysis. CONCLUSION: Limited evidence on the association of long-term exposure to PM2.5 and CMDs in the LMICs context warrants cohort studies to establish the association