Extensive Structural Variations Between Mitochondrial Genomes of CMS and Normal Peppers (Capsicum Annuum L.) Revealed by Complete Nucleotide Sequencing

Cytoplasmic male sterility (CMS) is an inability to produce functional pollen that is caused by mutation of the mitochondrial genome. Comparative analyses of mitochondrial genomes of lines with and without CMS in several species have revealed structural differences between genomes, including extensive rearrangements caused by recombination. However, the mitochondrial genome structure and the DNA rearrangements that may be related to CMS have not been characterized in Capsicum spp. Results: We obtained the complete mitochondrial genome sequences of the pepper CMS line FS4401 (507,452 bp) and the fertile line Jeju (511,530 bp). Comparative analysis between mitochondrial genomes of peppers and tobacco that are included in Solanaceae revealed extensive DNA rearrangements and poor conservation in non-coding DNA. In comparison between pepper lines, FS4401 and Jeju mitochondrial DNAs contained the same complement of protein coding genes except for one additional copy of an atp6 gene (psi atp6-2) in FS4401. In terms of genome structure, we found eighteen syntenic blocks in the two mitochondrial genomes, which have been rearranged in each genome. By contrast, sequences between syntenic blocks, which were specific to each line, accounted for 30,380 and 17,847 bp in FS4401 and Jeju, respectively. The previously-reported CMS candidate genes, orf507 and psi atp6-2, were located on the edges of the largest sequence segments that were specific to FS4401. In this region, large number of small sequence segments which were absent or found on different locations in Jeju mitochondrial genome were combined together. The incorporation of repeats and overlapping of connected sequence segments by a few nucleotides implied that extensive rearrangements by homologous recombination might be involved in evolution of this region. Further analysis using mtDNA pairs from other plant species revealed common features of DNA regions around CMS-associated genes. Conclusions: Although large portion of sequence context was shared by mitochondrial genomes of CMS and male-fertile pepper lines, extensive genome rearrangements were detected. CMS candidate genes located on the edges of highly-rearranged CMS-specific DNA regions and near to repeat sequences. These characteristics were detected among CMS-associated genes in other species, implying a common mechanism might be involved in the evolution of CMS-associated genes.Golden Seed ProjectMinistry of Agriculture, Food and Rural Affairs (MAFRA)Ministry of Oceans and Fisheries (MOF)Rural Development Administration (RDA)Korea Forest Service (KFS)Vegetable Breeding Research Center through the R&D Convergence Center Support Program, Ministry of Agriculture, Food and Rural Affairs (MAFRA) Republic of Korea 710001-07Molecular Bioscience

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

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

Comparative analysis of outcomes after multiport and single-port laparoscopic colectomy in emergency situations: Is single-port laparoscopic colectomy safe and feasible?

SummaryBackground/ObjectiveAlthough consensus has been reached on the superiority of laparoscopy for a majority of conditions underlying acute abdominal pain, the safety and feasibility of single-port laparoscopic colectomy (SPLC) in emergency situations have not been determined.MethodsA prospective electronic database of all emergency patients who underwent either multiport laparoscopic colectomy (MPLC) or SPLC between April 2006 and December 2014 was used to compare the surgical outcomes of these operative methods.ResultsDuring the study period, 31 MPLCs and 76 SPLCs were performed. These two operative methods resulted in similar operating times, transfusion amounts, lengths of stay, postoperative complications, attainment of lymph nodes, and proximal and distal cut margins. However, the SPLC group had a shorter time to first flatus (2.8±1.9 days vs. 3.8±1.5 days, p=0.005), earlier reinitiation of free oral fluids (3.2±2.1 days vs. 4.4±1.8 days, p=0.002), and lesser requirement of narcotic analgesics (2.5±3.9 times vs. 4.7±4.8 times, p=0.017).ConclusionSPLC could be a safe and effective alternative to MPLC, even in emergency situations when performed by surgeons who have overcome the learning curve associated with single-port laparoscopic techniques. The tendency toward earlier returns to bowel function and decreased incidence of postoperative analgesic use would be potential benefits of SPLC in emergency situations

Cerebral Perfusion Monitoring Using Near-Infrared Spectroscopy During Head-Up Tilt Table Test in Patients With Orthostatic Intolerance

The head-up tilt table test (HUT) is one of the primary clinical examinations for evaluating orthostatic intolerance (OI). HUT can be divided into three phases: dynamic tilt phase (supine to tilt up), static tilt phase (remain tilted at 70°), and post tilt phase (tilt down back to supine position). Commonly, blood pressure (BP) and heart rate (HR) are monitored to observe for OI symptoms, but are indirect measurements of cerebral perfusion and can lead to inaccurate HUT evaluation. In this study, we implemented a 108-channel near-infrared spectroscopy (NIRS) probe to characterize HUT performance by monitoring cerebral hemodynamic changes for healthy controls (HCs), OI patients with normal HUT results, and OI patients with positive HUT results: vasovagal syncope (VS), postural orthostatic tachycardia syndrome (POTS), orthostatic hypotension (OH), and orthostatic hypertension (OHT). By the end of the static tilt phase, OI patients typically did not show a complete recovery back to baseline cerebral oxygenation and total blood volume compared to HCs. We characterized the return to cerebral homeostasis by polynomial fitting total blood volume changes and determining the inflection point. The OI patients with normal HUT results, VS, OH, or OHT showed a delay in the return to cerebral homeostasis compared to the HC group during HUT