Model-assisted metabolic engineering of Escherichia coli for long chain alkane and alcohol production

Biologically-derived hydrocarbons are considered to have great potential as next-generation biofuels owing to the similarity of their chemical properties to contemporary diesel and jet fuels. However, the low yield of these hydrocarbons in biotechnological production is a major obstacle for commercialization. Several genetic and process engineering approaches have been adopted to increase the yield of hydrocarbon, but a model driven approach has not been implemented so far. Here, we applied a constraint-based metabolic modeling approach in which a variable demand for alkane biosynthesis was imposed, and co-varying reactions were considered as potential targets for further engineering of an E. coli strain already expressing cyanobacterial enzymes towards higher chain alkane production. The reactions that co-varied with the imposed alkane production were found to be mainly associated with the pentose phosphate pathway (PPP) and the lower half of glycolysis. An optimal modeling solution was achieved by imposing increased flux through the reaction catalyzed by glucose-6-phosphate dehydrogenase (zwf) and iteratively removing 7 reactions from the network, leading to an alkane yield of 94.2% of the theoretical maximum conversion determined by in silico analysis at a given biomass rate. To validate the in silico findings, we first performed pathway optimization of the cyanobacterial enzymes in E. coli via different dosages of genes, promoting substrate channelling through protein fusion and inducing substantial equivalent protein expression, which led to a 36-fold increase in alka(e)ne production from 2.8 mg/L to 102 mg/L. Further, engineering of E. coli based on in silico findings, including biomass constraint, led to an increase in the alka(e)ne titer to 425 mg/L (major components being 249 mg/L pentadecane and 160 mg/L heptadecene), a 148.6-fold improvement over the initial strain, respectively; with a yield of 34.2% of the theoretical maximum. The impact of model-assisted engineering was also tested for the production of long chain fatty alcohol, another commercially important molecule sharing the same pathway while differing only at the terminal reaction, and a titer of 1506 mg/L was achieved with a yield of 86.4% of the theoretical maximum. Moreover, the model assisted engineered strains had produced 2.54 g/L and 12.5 g/L of long chain alkane and fatty alcohol, respectively, in the bioreactor under fed-batch cultivation condition. Our study demonstrated successful implementation of a combined in silico modeling approach along with the pathway and process optimization in achieving the highest reported titers of long chain hydrocarbons in E. coli

A clinical study of arrhythmias associated with acute coronary syndrome: a hospital based study of a high risk and previously undocumented population

Background: ACS represents a global epidemic. Arrhythmia in ACS is common. Careful investigation may lead to further improvement of prognosis. Retrospectively analyzed the year- round data of our center. Study was undertaken to analyze the incidence, frequency and type of arrhythmias in ACS. This is to aid timely intervention and to modify the outcome. Identification of the type of arrhythmia is of therapeutic and prognostic importance.Methods: This cross sectional analytical study was conducted in the Department of Cardiology, Apollo Hospitals Dhaka, from January 2019 to January 2020 with ACS patients. Enrolled consecutively and data analyzed.Results: There were 500 patients enrolled considering inclusion and exclusion criteria. Sample was subdivided into 3 groups on the type of ACS. Group-I with UA, Group-II with NSTE - ACS and Group-III with STE - ACS. Different types of arrhythmia noted. Types of arrhythmia were correlated with type of ACS. 500 patients included. Mean age 55.53±12.70, 71.6% male and 28.4% female. 60.4% hypertensive, 46.2% diabetic, 20.2% positive family history of CAD, 32.2% current smoker, 56.4% dyslipidaemic and 9.6% asthmatic. 31.2% UA, 39.2% NSTE-ACS and 29.6% STE-ACS. Type of arrhythmias noted. 22% sinus tachycardia, 20.2% sinus bradycardia, 9% atrial fibrillation, 5.2% ventricular ectopic, 4.8% supra ventricular ectopic, 2.8% bundle branch block, 2.2% atrio-ventricular block, 1% broad complex tachycardia, 0.4% narrow complex tachycardia, 0.2% sinus node dysfunction and 32.2% without any arrhythmia. Significant incidences of arrhythmia detected - respectively 29.8%, 39.2% and 31%, p<0.001.Conclusions: In conclusion, arrhythmias in ACS are common. More attention should be paid to improve their treatment and prognosis

Strategies for prevention of gastrointestinal cancers in developing countries: a systematic review

BACKGROUND: Gastrointestinal cancers account for one third of total cancer incidence and mortality in developing countries. To date, there is no systematic synthesis of evidence regarding strategies to prevent gastrointestinal cancers in developing countries. We aimed to provide a systematic overview of studies evaluating strategies for prevention or early detection of the three most common gastrointestinal cancers (gastric, liver and colorectal cancer) in developing countries. METHODS: We searched MEDLINE, Web of Science and WHO Global Index Medicus databases for relevant articles published until October 2016 using combinations of the search terms "gastrointestinal", "digestive system", "gastric", "liver", "colorectal", "cancer", "prevention", "early detection" and "developing country" (including names). RESULTS: Overall, 73 articles met the inclusion criteria, providing information on short- and long-term outcomes (up to 30 years) from various intervention studies (∼45% randomized). Trials on hepatitis B vaccination consistently showed vaccine efficacy over time and indicated long-term preventive effects on liver cancer incidence that start to become measurable at the population level. Studies on anti-H. pylori treatment suggested a reduction in gastric cancer incidence reaching statistical significance after long-term follow-up, while evidence regarding a preventive effect in persons with precancerous lesions is still inconclusive. The studies regarding colorectal cancer focused on early detection, ∼90% of which were restricted to intermediate endpoints. CONCLUSION: In conclusion, there were a number of studies on gastric and liver cancer prevention in developing countries showing promising results after long-term follow-up. Important next steps include pooled meta-analyses as far as possible given the heterogeneity between studies as well as implementation research

A computational study of drag reduction and vortex shedding suppression of flow past a square cylinder in presence of small control cylinders

This article presents a two-dimensional numerical study of the unsteady laminar flow from a square cylinder in presence of multiple small control cylinders. The cylinders are placed in an unconfined medium at low Reynolds numbers (Re = 100 and 160). Different flow phenomena are captured for the gap spacings (g = s/D, where s is the surface-to-surface distance between the main cylinder and small control cylinders and D is the size of the main cylinder) between 0.25 – 3 and angle of attack (θ) ranging from 300 to 1800. Numerical calculations are performed by using a lattice Boltzmann method. In this paper, the important flow physics of different observed flow patterns in terms of instantaneous vorticity contours visualization, time-trace analysis of drag and lift coefficients and power spectra analysis of lift coefficient are presented and discussed. Drag reduction and suppression of vortex shedding is also discussed in detail and compared with the available experimental and numerical results qualitatively as well as quantitatively. In addition, the mean drag coefficient, Strouhal number, root-mean-square values of the drag and lift coefficients are determined and compared with a single square cylinder without small control cylinders. We found that the drag is reduced 99.8% and 97.6% for (θ, g) = (300, 3) at Re = 100 and 160, respectively

Governing heatwaves in Europe: comparing health policy and practices to better understand roles, responsibilities and collaboration

Background: The expectation that climate change will further exacerbate extreme weather events such as heatwavesis of primary concern to policymakers and scientists. Effective governance is fundamental to preparedness for and response to such threats. This paper explores the governance structures of European heat health action plans and provides insights into key stakeholders, roles, responsibilities and collaboration. Methods: This was a two-phase qualitative study, in which we complemented a desk review of 15 European national heat health action plans (NHHAPs) with, after obtaining informed consent, 68 interviews in nine countries with key informants involved in the development, implementation and/or evaluation of these NHHAPs. A thematic analysis was used to analyze the NHHAPs inductively. This analysis focused on three themes: identifying key stakeholders, defining and assigning roles and collaboration among stakeholders. The iteratively created codebook was then applied to the analysis of the key informant interviews. All analyses were done using NVivo 10 qualitative analysis software. Results: The majority of the NHHAPs have governance as one of their main objectives, to support the coordination of actions and collaboration among involved stakeholders. There are, however, significant differences between plan and practice. On the basis of the available data, we have little insight into the process of stakeholder identification, but we do find that most countries involve the same types of stakeholders. Roles are mainly defined and assigned in relation to the alert levels of the warning system, causing other role aspects and other roles to be vague and ambiguous. Collaboration is key to many NHHAP elements and is mainly experienced positively, though improvements and new collaborations are considered. Conclusions: Our findings show a need for a more deliberate and structured approach to governance in the context of NHHAPs. A cross-sectoral approach to the identification of key stakeholders can facilitate a broader preparedness and response to heatwaves. Roles and responsibilities of stakeholders should be defined and assigned more clearly to avoid confusion and to improve effective implementation. To this extent, we identify and describe seven key roles and potential stakeholders to which these roles are usually assigned. Finally, also collaboration among stakeholders can benefit from a cross-sectoral approach, but also formal structures can be beneficial

Core genome mediated potential vaccine targets prioritization against Clostridium difficile via reverse vaccinology - an immuno-informatics approach

The emergence of multi-drug resistance in clostridium difficile is an urgent bio-threat associated with high morbidity and mortality. The availability of core genome sequences for pathogen strains provides the opportunity to rationally approach novel therapeutic targets. No preventive vaccine against C. difficile infections is available commercially to date. The core genome data of 63 complete genome sequences of C. difficile was utilized in the current study. A precise immune informatics layout has been followed for the identification of potential vaccine target proteins that are essential for the pathogen and show no homology to human as well as human gut flora proteome. Two antigenic outer membrane proteins were prioritized for the lead epitopes prediction based on the reverse vaccinology approach. The multi-epitope chimeric vaccines were engineered using lead T cell and B cell epitopes in combination with the adjuvant peptides and linker sequences involved in enhancing the immunogenic responses. Four vaccine constructs were designed from overlapping epitopes. Based on multiple physiochemical properties, including the antigenicity, allergenicity, solubility and, molecular docking scores, the vaccine construct V2 was finalized for further analysis. The in silico restriction and cloning analysis inferred the V2 vaccine construct cloning capability within the bacterial expression system. The molecular modeling and molecular dynamic simulation showed favorable molecular interaction of V2 in the host cell and ensured the molecular stability of the V2 prioritized construct. Multi-epitope-based vaccine designing is an effective approach to develop potential therapeutics with better safety, increased potency, and logistically viable. The chimeric vaccine construct prioritized in the current study speculates to effectively bind with human immune receptor proteins and possibly triggers a potent immune response against C. difficile invasion

Engineered Production of Short Chain Fatty Acid in <i>Escherichia coli</i> Using Fatty Acid Synthesis Pathway

<div><p>Short-chain fatty acids (SCFAs), such as butyric acid, have a broad range of applications in chemical and fuel industries. Worldwide demand of sustainable fuels and chemicals has encouraged researchers for microbial synthesis of SCFAs. In this study we compared three thioesterases, i.e., TesAT from <i>Anaerococcus tetradius</i>, TesBF from <i>Bryantella formatexigens</i> and TesBT from <i>Bacteroides thetaiotaomicron</i>, for production of SCFAs in <i>Escherichia coli</i> utilizing native fatty acid synthesis (FASII) pathway and modulated the genetic and bioprocess parameters to improve its yield and productivity. <i>E</i>. <i>coli</i> strain expressing <i>tesBT</i> gene yielded maximum butyric acid titer at 1.46 g L^-1, followed by <i>tesBF</i> at 0.85 g L^-1 and <i>tesAT</i> at 0.12 g L^-1. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. The modulation of genetic factors that are known to influence long chain fatty acid production, such as deletion of the <i>fadD</i> and <i>fadE</i> that initiates the fatty acid degradation cycle and overexpression of <i>fadR</i> that is a global transcriptional activator of fatty acid biosynthesis and repressor of degradation cycle, did not improve the butyric acid titer significantly. Use of chemical inhibitor cerulenin, which restricts the fatty acid elongation cycle, increased the butyric acid titer by 1.7-fold in case of TesBF, while it had adverse impact in case of TesBT. <i>In vitro</i> enzyme assay indicated that cerulenin also inhibited short chain specific thioesterase, though inhibitory concentration varied according to the type of thioesterase used. Further process optimization followed by fed-batch cultivation under phosphorous limited condition led to production of 14.3 g L^-1 butyric acid and 17.5 g L^-1 total free fatty acid at 28% of theoretical yield. This study expands our understanding of SCFAs production in <i>E</i>. <i>coli</i> through FASII pathway and highlights role of genetic and process optimization to enhance the desired product.</p></div

Catharanthus roseus extract attenuates E. coli toxin-induced short circuit current in isolated jejunal epithelium of goat and buffalo

Enterotoxigenic E. coli-derived heat-stable enterotoxin not only alters intestinal barrier functions and produces secretory diarrhea but also causes production losses in livestock. This study aimed to elucidate the ameliorative effect of Catharanthus roseus extract on Cl- secretion in an in vitro model. Isolated jejunal epithelia from goats (N=10) and buffaloes (N=4) were divided into four groups: A) control; B) 1.5% C. roseus extract; C) 10% E. coli toxin, and D) 10% E. coli toxin and 1.5% C. roseus extract. The jejunal epithelia were mounted on Ussing chambers with an exposed surface area of 0.95 cm2 and treated with E. coli toxin on the mucosal side to invoke Cl- channels. Transepithelial potential difference (Pdt), tissue conductance (Gt), and short circuit current (Isc) were determined under voltage-clamped conditions. E. coli toxin produced a significant increase in the Pdt, Gt, and Isc for both goat and buffalo jejunal epithelia, whereas C. roseus extract on the mucosal side alone or followed by the E. coli toxin decreased these parameters, which were comparable with the control. The change in Isc was more pronounced in goats than buffaloes (ΔIscgoat > ΔIscbuff). In experiments with the goat jejunal epithelia, replacement of Cl- from the mucosal buffer decreased Isc, similar to a decrease observed for the C. roseus extract groups. In conclusion, the results of this study suggest that C. roseus extract has a potential to attenuate E. coli-induced Cl- secretion in isolated jejunal epithelia of goats and buffaloes.Keywords: Catharanthus roseus extract; Electrophysiology; Secretory diarrhea; Small intestine, Ruminant

Effect of oxygen availability on production of butyric acid.

<p>(A) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in 100 ml flask containing different volume of culture medium to vary oxygen availability and checked for butyric acid production. (B) <i>E</i>. <i>coli</i> MG1655 transformed with pZA-tesBT plasmid was grown in the bioreactor containing 350 ml culture medium with different oxygen saturation level and checked for butyric acid production.</p

Impact of genotype of <i>E</i>. <i>coli</i> on butyric acid production using different thioesterases.

<p>Different <i>E</i>. <i>coli</i> strains were transformed with plasmid pQE-tesAT, pQE-tesBF and pQE-tesBT for expression of thioesterase TesAT (represented as AT), TesBF (represented as BF) and TesBT (represented as BT), respectively, and used for butyric acid production.</p