L-Lysine production independent of the oxidative pentose phosphate pathway by Corynebacterium glutamicum with the Streptococcus mutans gapN gene

Published online 29 March 2016We have recently developed a Corynebacterium glutamicum strain that generates NADPH via the glycolytic pathway by replacing endogenous NAD-dependent glyceraldehyde 3-phosphate dehydrogenase (GapA) with a nonphosphorylating NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (GapN) from Streptococcus mutans. Strain RE2, a suppressor mutant spontaneously isolated for its improved growth on glucose from the engineered strain, was proven to be a high-potential host for l-lysine production (Takeno et al., 2010). In this study, the suppressor mutation was identified to be a point mutation in rho encoding the transcription termination factor Rho. Strain RE2 still showed retarded growth despite the mutation rho696. Our strategy for reconciling improved growth with a high level of l-lysine production was to use GapA together with GapN only in the early growth phase, and subsequently shift this combination-type glycolysis to one that depends only on GapN in the rest of the growth phase. To achieve this, we expressed gapA under the myo-inositol-inducible promoter of iolT1 encoding a myo-inositol transporter in strain RE2. The resulting strain RE2Aiol was engineered into an l-lysine producer by introduction of a plasmid carrying the desensitized lysC, followed by examination for culture conditions with myo-inositol supplementation. We found that as a higher concentration of myo-inositol was added to the seed culture, the following fermentation period became shorter while maintaining a high level of l-lysine production. This finally reached a fermentation period comparable to that of the control GapA strain, and yielded a 1.5-fold higher production rate compared with strain RE2. The transcript level of gapA, as well as the GapA activity, in the early growth phase increased in proportion to the myo-inositol concentration and then fell to low levels in the subsequent growth phase, indicating that improved growth was a result of increased GapA activity, especially in the early growth phase. Moreover, blockade of the pentose phosphate pathway through a defect in glucose 6-phosphate dehydrogenase did not significantly affect l-lysine production in the engineered GapN strains, while a drastic decrease in l-lysine production was observed for the control GapA strain. Determination of the intracellular NADPH/NADP+ ratios revealed that the ratios in the engineered strains were significantly higher than the ratio of the control GapA strain irrespective of the pentose phosphate pathway. These results demonstrate that our strain engineering strategy allows efficient l-lysine production independent of the oxidative pentose phosphate pathway.ArticleMETABOLIC ENGINEERING. 37:1-10 (2016)journal articl

Real-time In Situ Electron Spin Resonance Measurements on Fungal Spores of Penicillium digitatum during Exposure of Oxygen Plasmas

We report the kinetic analysis of free radicals on fungal spores of Penicillium digitatum interacted with atomic oxygen generated plasma electric discharge using real time in situ electron spin resonance (ESR) measurements. We have obtained information that the ESR signal from the spores was observed and preliminarily assignable to semiquinone radical with a g-value of around 2.004 and a line width of approximately 5G. The decay of the signal is possibly linked to the inactivation of the fungal spore. The real-time in situ ESR has proven to be a useful method to elucidate plasma-induced surface reactions on biological specimens.Comment: 11 pages, 5 figure

Annually-resolved water isotope measurements in a shallow ice core drilled in a vicinity of Dome Fuji station, East Antarctica

第3回極域科学シンポジウム/第35回極域気水圏シンポジウム 11月29日（木） 国立国語研究所 ２階ロビ

Reduction of lipid accumulation rescues Bietti’s crystalline dystrophy phenotypes

眼の難病クリスタリン網膜症の発症メカニズムを解明 --治療薬の有力候補発見により創薬研究の進展に期待--. 京都大学プレスリリース. 2018-03-27.Bietti’s crystalline dystrophy (BCD) is an intractable and progressive chorioretinal degenerative disease caused by mutations in the CYP4V2 gene, resulting in blindness in most patients. Although we and others have shown that retinal pigment epithelium (RPE) cells are primarily impaired in patients with BCD, the underlying mechanisms of RPE cell damage are still unclear because we lack access to appropriate disease models and to lesion-affected cells from patients with BCD. Here, we generated human RPE cells from induced pluripotent stem cells (iPSCs) derived from patients with BCD carrying a CYP4V2 mutation and successfully established an in vitro model of BCD, i.e., BCD patient-specific iPSC-RPE cells. In this model, RPE cells showed degenerative changes of vacuolated cytoplasm similar to those in postmortem specimens from patients with BCD. BCD iPSC-RPE cells exhibited lysosomal dysfunction and impairment of autophagy flux, followed by cell death. Lipidomic analyses revealed the accumulation of glucosylceramide and free cholesterol in BCD-affected cells. Notably, we found that reducing free cholesterol by cyclodextrins or δ-tocopherol in RPE cells rescued BCD phenotypes, whereas glucosylceramide reduction did not affect the BCD phenotype. Our data provide evidence that reducing intracellular free cholesterol may have therapeutic efficacy in patients with BCD

A multicenter randomized controlled trial to evaluate the efficacy and safety of nelfinavir in patients with mild COVID-19

Nelfinavir, an orally administered inhibitor of human immunodeficiency virus protease, inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. We conducted a randomized controlled trial to evaluate the clinical efficacy and safety of nelfinavir in patients with SARS-CoV-2 infection. We included unvaccinated asymptomatic or mildly symptomatic adult patients who tested positive for SARS-CoV-2 infection within 3 days before enrollment. The patients were randomly assigned (1:1) to receive oral nelfinavir (750 mg; thrice daily for 14 days) combined with standard-of-care or standard-of-care alone. The primary endpoint was the time to viral clearance, confirmed using quantitative reverse-transcription PCR by assessors blinded to the assigned treatment. A total of 123 patients (63 in the nelfinavir group and 60 in the control group) were included. The median time to viral clearance was 8.0 (95% confidence interval [CI], 7.0 to 12.0) days in the nelfinavir group and 8.0 (95% CI, 7.0 to 10.0) days in the control group, with no significant difference between the treatment groups (hazard ratio, 0.815; 95% CI, 0.563 to 1.182; P = 0.1870). Adverse events were reported in 47 (74.6%) and 20 (33.3%) patients in the nelfinavir and control groups, respectively. The most common adverse event in the nelfinavir group was diarrhea (49.2%). Nelfinavir did not reduce the time to viral clearance in this setting. Our findings indicate that nelfinavir should not be recommended in asymptomatic or mildly symptomatic patients infected with SARS-CoV-2. The study is registered with the Japan Registry of Clinical Trials (jRCT2071200023). IMPORTANCE The anti-HIV drug nelfinavir suppresses the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. However, its efficacy in patients with COVID-19 has not been studied. We conducted a multicenter, randomized controlled trial to evaluate the efficacy and safety of orally administered nelfinavir in patients with asymptomatic or mildly symptomatic COVID-19. Compared to standard-of-care alone, nelfinavir (750 mg, thrice daily) did not reduce the time to viral clearance, viral load, or the time to resolution of symptoms. More patients had adverse events in the nelfinavir group than in the control group (74.6% [47/63 patients] versus 33.3% [20/60 patients]). Our clinical study provides evidence that nelfinavir, despite its antiviral effects on SARS-CoV-2 in vitro, should not be recommended for the treatment of patients with COVID-19 having no or mild symptoms

Xylitol Affects the Intestinal Microbiota and Metabolism of Daidzein in Adult Male Mice

This study examined the effects of xylitol on mouse intestinal microbiota and urinary isoflavonoids. Xylitol is classified as a sugar alcohol and used as a food additive. The intestinal microbiota seems to play an important role in isoflavone metabolism. Xylitol feeding appears to affect the gut microbiota. We hypothesized that dietary xylitol changes intestinal microbiota and, therefore, the metabolism of isoflavonoids in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 5% xylitol diet (XD group) and those fed a 0.05% daidzein-containing control diet (CD group) for 28 days. Plasma total cholesterol concentrations were significantly lower in the XD group than in the CD group (p < 0.05). Urinary amounts of equol were significantly higher in the XD group than in the CD group (p < 0.05). The fecal lipid contents (% dry weight) were significantly greater in the XD group than in the CD group (p < 0.01). The cecal microbiota differed between the two dietary groups. The occupation ratios of Bacteroides were significantly greater in the CD than in the XD group (p < 0.05). This study suggests that xylitol has the potential to affect the metabolism of daidzein by altering the metabolic activity of the intestinal microbiota and/or gut environment. Given that equol affects bone health, dietary xylitol plus isoflavonoids may exert a favorable effect on bone health