Production of biohydrogen by recombinant expression of [NiFe]-hydrogenase 1 in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Hydrogenases catalyze reversible reaction between hydrogen (H₂) and proton. Inactivation of hydrogenase by exposure to oxygen is a critical limitation in biohydrogen production since strict anaerobic conditions are required. While [FeFe]-hydrogenases are irreversibly inactivated by oxygen, it was known that [NiFe]-hydrogenases are generally more tolerant to oxygen. The physiological function of [NiFe]-hydrogenase 1 is still ambiguous. We herein investigated the H₂production potential of [NiFe]-hydrogenase 1 of <it>Escherichia coli in vivo </it>and <it>in vitro</it>. The <it>hya</it>A and <it>hya</it>B genes corresponding to the small and large subunits of [NiFe]-hydrogenase 1 core enzyme, respectively, were expressed in BL21, an <it>E. coli </it>strain without H₂producing ability.</p> <p>Results</p> <p>Recombinant BL21 expressing [NiFe]-hydrogenase 1 actively produced H₂(12.5 mL H₂/(h·L) in 400 mL glucose minimal medium under micro-aerobic condition, whereas the wild type BL21 did not produce H₂even when formate was added as substrate for formate hydrogenlyase (FHL) pathway. The majority of recombinant protein was produced as an insoluble form, with translocation of a small fraction to the membrane. However, the membrane fraction displayed high activity (~65% of total cell fraction), based on unit protein mass. Supplement of nickel and iron to media showed these metals contribute essentially to the function of [NiFe]-hydrogenase 1 as components of catalytic site. In addition, purified <it>E. coli </it>[NiFe]-hydrogenase 1 using his₆-tag displayed oxygen-tolerant activity of ~12 nmol H₂/(min·mg protein) under a normal aeration environment, compared to [FeFe]-hydrogenase, which remains inactive under this condition.</p> <p>Conclusions</p> <p>This is the first report on physiological function of <it>E. coli </it>[NiFe]-hydrogenase 1 for H₂production. We found that [NiFe]-hydrogenase 1 has H₂production ability even under the existence of oxygen. This oxygen-tolerant property is a significant advantage because it is not necessary to protect the H₂production process from oxygen. Therefore, we propose that [NiFe]-hydrogenase can be successfully applied as an efficient biohydrogen production tool under micro-aerobic conditions.</p

Improved production of biohydrogen in light-powered Escherichia coli by co-expression of proteorhodopsin and heterologous hydrogenase

<p>Abstract</p> <p>Background</p> <p>Solar energy is the ultimate energy source on the Earth. The conversion of solar energy into fuels and energy sources can be an ideal solution to address energy problems. The recent discovery of proteorhodopsin in uncultured marine γ-proteobacteria has made it possible to construct recombinant <it>Escherichia coli </it>with the function of light-driven proton pumps. Protons that translocate across membranes by proteorhodopsin generate a proton motive force for ATP synthesis by ATPase. Excess protons can also be substrates for hydrogen (H₂) production by hydrogenase in the periplasmic space. In the present work, we investigated the effect of the co-expression of proteorhodopsin and hydrogenase on H₂production yield under light conditions.</p> <p>Results</p> <p>Recombinant <it>E. coli </it>BL21(DE3) co-expressing proteorhodopsin and [NiFe]-hydrogenase from <it>Hydrogenovibrio marinus </it>produced ~1.3-fold more H₂in the presence of exogenous retinal than in the absence of retinal under light conditions (70 μmole photon/(m²·s)). We also observed the synergistic effect of proteorhodopsin with endogenous retinal on H₂production (~1.3-fold more) with a dual plasmid system compared to the strain with a single plasmid for the sole expression of hydrogenase. The increase of light intensity from 70 to 130 μmole photon/(m²·s) led to an increase (~1.8-fold) in H₂production from 287.3 to 525.7 mL H₂/L-culture in the culture of recombinant <it>E. coli </it>co-expressing hydrogenase and proteorhodopsin in conjunction with endogenous retinal. The conversion efficiency of light energy to H₂achieved in this study was ~3.4%.</p> <p>Conclusion</p> <p>Here, we report for the first time the potential application of proteorhodopsin for the production of biohydrogen, a promising alternative fuel. We showed that H₂production was enhanced by the co-expression of proteorhodopsin and [NiFe]-hydrogenase in recombinant <it>E. coli </it>BL21(DE3) in a light intensity-dependent manner. These results demonstrate that <it>E. coli </it>can be applied as light-powered cell factories for biohydrogen production by introducing proteorhodopsin.</p

Essential and checkpoint functions of budding yeast ATM and ATR during meiotic prophase are facilitated by differential phosphorylation of a meiotic adaptor protein, Hop1

A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis

Pulmonary tuberculosis and lung cancer: simultaneous and sequential occurrence

OBJECTIVE: Lung cancer (LC) is the leading cause of cancer-related death and represents a major public health problem worldwide. Another major cause of morbidity and mortality, especially in developing countries, is tuberculosis. The simultaneous or sequential occurrence of pulmonary tuberculosis and LC in the same patient has been reported in various case series and case-control studies. The objective of this study was to describe the characteristics of patients developing tuberculosis and LC, either simultaneously or sequentially. METHODS: This was a cross-sectional study based on the review of medical charts. RESULTS: The study involved 24 patients diagnosed with tuberculosis and LC between 2009 and 2012. The diagnoses of tuberculosis and LC occurred simultaneously in 10 patients, whereas tuberculosis was diagnosed prior to LC in 14. The median time between the two diagnoses was 5 years (interquartile range: 1-30 years). Fourteen patients (58.3%) were male, 20 (83.3%) were White, and 22 (91.7%) were smokers or former smokers. The most common histological type was adenocarcinoma, identified in 14 cases (58.3%), followed by epidermoid carcinoma, identified in 6 (25.0%). Seven patients (29.2%) presented with distant metastases at diagnosis; of those 7 patients, 5 (71%) were diagnosed with LC and tuberculosis simultaneously. CONCLUSIONS: In the present study, most of the patients with tuberculosis and LC were smokers or former smokers, and tuberculosis was diagnosed either before or simultaneously with LC. Non-small cell lung cancer, especially adenocarcinoma, was the most common histological type

Temporal trends in prevalence and antithrombotic treatment among Asians with atrial fibrillation undergoing percutaneous coronary intervention: A nationwide Korean population-based study

<div><p>Background</p><p>We investigated the recent 10-year trends in the number of patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI) in relation to prescription patterns of antithrombotic therapy.</p><p>Methods</p><p>We analyzed the annual prevalence of PCI and patterns of antithrombotic therapy after PCI, including antiplatelets and oral anticoagulants (vitamin K antagonists and non-vitamin K antagonist oral anticoagulants [NOACs]), in patients with AF between 2006 and 2015 by using the Korean National Health Insurance Service database. Independent factors associated with triple therapy (oral anticoagulant plus dual antiplatelet) prescription were assessed using multivariable logistic regression analysis.</p><p>Results</p><p>The number of patients with AF undergoing PCI increased gradually from 2006 (n = 2,140) to 2015 (n = 3,631) (p_trend<0.001). In 2006, only 22.7% of patients received triple therapy after PCI although 96.2% of them were indicated for anticoagulation (CHA₂DS₂-VASc score ≥2). The prescription rate of triple therapy increased to 38.3% in 2015 (p_trend<0.001), which was mainly attributed to a recent increment of NOAC-based triple therapy from 2013 (17.5% in 2015). Previous ischemic stroke or systemic embolism, old age, hypertension, and congestive heart failure were significantly associated with a higher triple therapy prescription rate, whereas previous myocardial infarction, PCI, and peripheral arterial disease were associated with triple therapy underuse.</p><p>Conclusions</p><p>From 2006 to 2015, the number of patients with AF undergoing PCI and the prescription rate of triple therapy increased gradually with a recent increment of NOAC-based antithrombotic therapy from 2013. Previous myocardial infarction, peripheral artery disease, and PCI were associated with underuse of triple therapy.</p></div

Impact of Treadmill Running and Sex on Hippocampal Neurogenesis in the Mouse Model of Amyotrophic Lateral Sclerosis

Hippocampal neurogenesis in the subgranular zone (SGZ) of dentate gyrus (DG) occurs throughout life and is regulated by pathological and physiological processes. The role of oxidative stress in hippocampal neurogenesis and its response to exercise or neurodegenerative diseases remains controversial. The present study was designed to investigate the impact of oxidative stress, treadmill exercise and sex on hippocampal neurogenesis in a murine model of heightened oxidative stress (G93A mice). G93A and wild type (WT) mice were randomized to a treadmill running (EX) or a sedentary (SED) group for 1 or 4 wk. Immunohistochemistry was used to detect bromodeoxyuridine (BrdU) labeled proliferating cells, surviving cells, and their phenotype, as well as for determination of oxidative stress (3-NT; 8-OHdG). BDNF and IGF1 mRNA expression was assessed by in situ hybridization. Results showed that: (1) G93A-SED mice had greater hippocampal neurogenesis, BDNF mRNA, and 3-NT, as compared to WT-SED mice. (2) Treadmill running promoted hippocampal neurogenesis and BDNF mRNA content and lowered DNA oxidative damage (8-OHdG) in WT mice. (3) Male G93A mice showed significantly higher cell proliferation but a lower level of survival vs. female G93A mice. We conclude that G93A mice show higher hippocampal neurogenesis, in association with higher BDNF expression, yet running did not further enhance these phenomena in G93A mice, probably due to a ‘ceiling effect’ of an already heightened basal levels of hippocampal neurogenesis and BDNF expression