Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli.

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l-1 of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (µ) and the 1,3-PDO yield from glucose (YP/S ) in the disruptant were higher than those in the non-disruptant (ΔglpF, µ = 0·08 ± 0·00 h-1 , YP/S  = 0·06 mol mol-glucose-1 ; BW25113, µ = 0·06 ± 0·00 h-1 , YP/S  = 0·02 mol mol-glucose-1 ). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.

Caprylic acid enhances hydroxyhexylitaconic acid production in Aspergillus niger S17-5.

AIM: Aspergillus niger S17-5 produces two alkylitaconic acids, 9-hydroxyhexylitaconic acid (9-HHIA) and 10-hydroxyhexylitaconic acid (10-HHIA), which have cytotoxic and polymer building block properties. In this study, we characterized the production of 9-HHIA and 10-HHIA by addition of their expected precursor, caprylic acid, to a culture of A. niger S17-5, and demonstrated batch fermentation of 9-HHIA and 10-HHIA in a jar fermenter with DO-stat. METHODS AND RESULTS: Production titres of 9-HHIA and 10-HHIA from 3% glucose in a flask after 25 days cultivation were 0·35 and 1·01 g l-1 respectively. Addition of 0·22 g l-1 of caprylic acid to a suspension of resting cells of A. niger S17-5 led to 32% enhancement of total 9-HHIA and 10-HHIA production compared to no addition. No enhancement of the production of 9-HHIA or 10-HHIA by the addition of oxaloacetic acid was observed. Addition of caprylic acid to the culture at mid-growth phase was more suitable for 9-HHIA and 10-HHIA production due to less cell growth inhibition by caprylic acid. DO-stat batch fermentation with 3% glucose and 14·4 g l-1 of caprylic acid in a 1·5 l jar fermenter resulted in the production titres of 9-HHIA and 10-HHIA being 0·48 and 1·54 g l-1 respectively after 10 days of cultivation. CONCLUSIONS: Addition of caprylic acid to the culture of A. niger S17-5 enhances 9-HHIA and 10-HHIA production. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that 9-HHIA and 10-HHIA are synthesized with octanoyl-CoA derived from caprylic acid, and that the supply of octanoyl-CoA is a rate-limiting step in 9-HHIA and 10-HHIA production. To the best of our knowledge, this is the first report regarding the fermentation of naturally occurring itaconic acid derivatives in a jar fermenter.

Stability analysis of silver nanoparticle suspensions by cyclic voltammetry.

We report the application of cyclic voltammetry and absorption spectroscopy to the characterization and study of the stability of silver colloids in water. The samples are prepared via chemical reduction and the reactions are catalyzed by irradiation with white light. The electrochemical response is related to the characteristic sample surface plasmon resonance (SPR) in the UV-visible absorption spectra. Cyclic voltammetry shows a characteristic reduction peak whose position is specific to each analyzed sample. Optical analysis of a colloid precursor during a 12 h time span, under low-power white-light irradiation, shows that nanoparticles undergo change in size and surface state (absorption bands splitting and inversion) to attain the "stable" colloidal form. While the absorption spectrum bands of the precursor return almost periodically to similar positions, the cyclic voltammogram characteristic reduction peak is displaced as a function of time. Finally, we follow the SPR changes of one "stable" colloid being subjected to electrolysis, heating, and sunlight irradiation, for environmental remediation purposes. Sunlight exposure produces the most significant SPR intensity drop, but the electrochemical technique shows itself promising as well.

PolyTag: A peptide tag that affords scaffold-less covalent protein assembly catalyzed by microbial transglutaminase.

Assembling proteins in close vicinity to each other provides an opportunity to gain unique function because collaborative and even synergistic functionalities can be expected in an assembled form. There have been a variety of strategies to synthesize functional protein assemblies but site-specific covalent assembly of monomeric protein units without impairing their intrinsic function remains challenging. Herein we report a powerful strategy to design protein assemblies by using microbial transglutaminase (MTG). A serendipitous discovery of self-crosslinking of enhanced green fluorescent protein (EGFP) fused with StrepTag I at the C-terminus revealed that EGFP was assembled through the crosslinking of the Lys (K) residue in the C-terminus of EGFP and the Gln (Q) residue in StrepTag I (AWRHPQFGG). Site-directed mutagenesis of the residues next to the K and Q yielded EGFP assemblies with higher molecular weights. An optimized peptide tag comprised of both K and Q residues (HKRWRHYQRGG) enabled the assembly of different types of proteins of interest (POI) when it was fused to either the N- or C-terminus. The peptide tag that enabled the self-polymerization of the functional POI without a scaffold was designated as a 'PolyTag'.

Non-uniformity smoothing of direct-driven fuel target implosion by phase control in heavy ion inertial fusion.

We have proposed a dynamic smoothing method based on a phase control to smooth plasma non-uniformities in perturbed plasma systems. In this paper, the dynamic smoothing method is applied to a spherical direct-driven fuel target implosion in heavy ion inertial confinement fusion. We found that the wobbling motion of each heavy ion beam (HIB) axis induces a phase-controlled HIBs energy deposition, and consequently the phase-controlled implosion acceleration is realized, so that the HIBs irradiation non-uniformity is successfully smoothed. HIB accelerators provide a well-established performance to oscillate a HIB axis at a high frequency. In inertial confinement fusion, a fuel implosion uniformity is essentially significant for achieving the DT fuel compression and for releasing the fusion energy, and the non-uniformity of the implosion acceleration should be less than a few %. The results in this paper demonstrate that the wobbling HIBs would provide an improvement in the fuel target implosion uniformity.

Propensity score-matched comparison of non-anatomical resection and radiofrequency ablation for hepatocellular carcinoma in patients with up to three tumours, each measuring up to 3 cm in diameter.

BACKGROUND: Non-anatomical liver resection (NAR) and radiofrequency ablation (RFA) are treatment options for early-stage hepatocellular carcinoma (HCC). The aim was to compare the outcomes of NAR and RFA for HCC in patients with three or fewer tumour nodules, each measuring not more than 3 cm in maximum diameter. METHODS: Eligible patients undergoing NAR or RFA with curative intent between September 2002 and December 2014 were identified. A propensity score-matching analysis was performed to reduce bias, and outcomes in these patients were analysed. RESULTS: From a total of 199 patients, 1:1 propensity score matching identified 70 matched pairs. Patients having NAR had a longer hospital stay (median 10 days versus 4 days for those who had RFA; P < 0·001) and a higher morbidity rate (24 versus 10 per cent respectively; P = 0·042). Patients who had NAR had slightly better recurrence-free survival but this failed to reach statistical significance in univariable analysis (P = 0·064). There was no significant difference in overall survival between the two groups (P = 0·475). RFA was identified as an independent risk factor for recurrence-free survival (hazard ratio (HR) 1·57; P = 0·041) in multivariable analysis. Local recurrence was significantly more common in patients receiving RFA (23 versus 1 per cent; P < 0·001). CONCLUSION: RFA was an independent risk factor for shorter recurrence-free survival, with a significantly higher local recurrence rate than NAR. Despite these differences, overall survival was not affected.

Calibrated Gastrojejunostomy in Gastric Bypass: It Is a Myth.

BACKGROUND: Many surgeons prefer a narrow gastrojejunostomy in gastric bypass believing that this provides greater weight loss. The question is whether the anastomosis remains at its initial diameter, whether it narrows or widens over time. This study tests the hypothesis that the initial diameter of the anastomosis varies over time. MATERIALS AND METHODS: One hundred obese had undergone surgery. Group A with anastomosis 1.5 cm wide and group B with anastomosis 3 cm wide. One-year follow-up was done for 83 patients. The size of the anastomosis diameter was evaluated using a balloon for cardia dilation. RESULTS: Comparing the results of surgery with the values presented 1 year after the procedure, (i) BMI decreased from 43.3 ± 6.6 to 28.7 ± 7.0 kg/m2 for group A and 44.6 ± 5.9 to 29.1 ± 6.3 kg/m2 for group B, (ii) the total weight loss was 30.6 ± 4.7% for group A and 31.9 ± 5.2% for group B, and (iii) the initial 1.5-cm anastomosis in group A measured 2.3 ± 1.0 cm while the initial 3-cm anastomosis in group B measured 2.7 ± 1.0 cm 1 year after surgery. No statistically significant difference was found for any of the variables evaluated before and after the surgical procedure. CONCLUSION: Calibrated anastomosis has no utility in increasing weight loss in gastric bypass.

Familial Congenital Methemoglobinemia in Pomeranian Dogs Caused by a Missense Variant in the NADH-Cytochrome B5 Reductase Gene.

BACKGROUND: In veterinary medicine, congenital methemoglobinemia associated with nicotinamide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5R) deficiency is rare. It has been reported in several breeds of dogs, but little information is available about its etiology. OBJECTIVES: To analyze the NADH-cytochrome b5 reductase gene, CYB5R3, in a Pomeranian dog family with methemoglobinemia suspected to be caused by congenital b5R deficiency. ANIMALS: Three Pomeranian dogs from a family with methemoglobinemia were analyzed. Five healthy beagles and 5 nonrelated Pomeranian dogs without methemoglobinemia were used as controls. METHODS: Methemoglobin concentration, b5R activity, and reduced glutathione (GSH) concentration were measured, and a turbidity index was used to evaluate Heinz body formation. The CYB5R3 genes of the affected dog and healthy dogs were analyzed by direct sequencing. RESULTS: Methemoglobin concentrations in erythrocytes of the affected dogs were remarkably higher than those of the control dogs. The b5R activity of the affected dogs was notably lower than that of the control dogs. DNA sequencing indicated that this Pomeranian family carried a CYB5R3 gene missense variant (ATC→CTC at codon 194) that resulted in the replacement of isoleucine (Ile) by leucine (Leu). CONCLUSIONS AND CLINICAL IMPORTANCE: This dog family had familial congenital methemoglobinemia caused by b5R deficiency, which resulted from a nonsynonymous variant in the CYB5R3 gene. This variation (c.580A>C) led to an amino acid substitution (p.Ile194Leu), and Ile194 was located in the proximal region of the NADH-binding motif. Our data suggested that this variant in the canine CYB5R3 gene would affect function of the b5R in erythrocytes.

A Double-blind, Placebo-controlled, Multicenter, Prospective, Randomized Study of Beraprost Sodium Treatment for Cats with Chronic Kidney Disease.

BACKGROUND: Chronic kidney disease (CKD) is a common progressive and irreversible disease in cats. The efficacy and safety of beraprost sodium (BPS) in cats with CKD have not been evaluated. HYPOTHESIS/OBJECTIVES: To evaluate the efficacy and safety of BPS in the treatment of cats with CKD, as compared to placebo. ANIMALS: Seventy-four client-owned cats with naturally occurring CKD. METHODS: Double-blind, placebo-controlled, multicenter, prospective, randomized trial. The cats received BPS (55 µg/cat) or a placebo PO q12 h for 180 days. The primary endpoint was prospectively defined as a change in the serum creatinine (sCr), serum phosphorus-to-calcium ratio or urine specific gravity (USG). RESULTS: The sCr increased significantly (P = 0.0030) in the placebo group (mean ± SD: 2.8 ± 0.7 to 3.2 ± 1.3 mg/dL) but not in the BPS group (2.4 ± 0.7 to 2.5 ± 0.7 mg/dL). The difference between the groups at day 180 was significant (0.8 mg/dL, 95% CI: 0.2 to 1.3 mg/dL, P = 0.0071). The serum phosphorus-to-calcium ratio was significantly (P = 0.0037) increased in the placebo group (0.46 ± 0.10 to 0.52 ± 0.21 mg/dL) but not in the BPS group (0.50 ± 0.08 to 0.51 ± 0.11 mg/dL). There was no significant change in the USG in either group. An adverse event judged as being treatment-related included vomiting that occurred in 1 case in the placebo group. No clinically relevant change was observed in the CBC and other blood chemistry tests. CONCLUSIONS AND CLINICAL IMPORTANCE: Beraprost sodium treatment was well tolerated and safe in cats with CKD. BPS inhibited the reduction in renal filtration function as measured by sCr increase.

Novel biallelic mutations in the PNPT1 gene encoding a mitochondrial-RNA-import protein PNPase cause delayed myelination.

Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.

Expression of uncoupling protein 1 in bovine muscle cells.

Uncoupling protein 1 (Ucp1) is predominantly expressed in brown/beige adipocytes in mammals. Although myogenic cells have been suggested to commit to a brown adipocyte lineage through the induction of Prdm16 expression, Prdm16 is also expressed in skeletal muscle. Thus, we examined expression of Ucp1 in bovine myogenic cells. Considering that Ucp1 is a principle molecule that induces energy expenditure in brown/beige adipocytes, expression of Ucp1 is not preferable in beef cattle because of potential decrease in energy (fattening) efficiency. The RT-PCR analyses revealed the expression of Ucp1 in the skeletal muscle of cattle; expression levels were markedly lower than those in the brown fat of calves. Immunohistochemical analyses showed that Ucp1 surrounded muscle fibers, but not adipocytes residing in skeletal muscle. Myosatellite cells cultured in myogenic medium showed an increase in the expression levels of myogenic regulatory factors ( < 0.05), while those in cells cultured in adipogenic medium were decreased ( < 0.05). The Ucp1 expression was also detected in myosatellite cells; expression levels were greater in cells after myogenic culture for 12 d than in those after myogenic culture for 6 d ( < 0.05) and were decreased when cells were cultured in adipogenic medium ( < 0.05). The Prdm16 expression was not affected by culture conditions, suggesting that the expression of Ucp1 is not regulated by that of Prdm16. The results of the present study provide an insight into the unexpected expression of Ucp1 in bovine skeletal muscle, which suggests the necessity for further studies on Ucp1-mediated energy expenditure in bovine skeletal muscle.

Determination of a localized surface plasmon resonance mode of Cu7S4 nanodisks by plasmon coupling.

Plasmon properties such as peak position, extinction cross-section and local electric field intensity are strongly dependent on excited, localized surface plasmon resonance (LSPR) modes. In non-spherical copper chalcogenide nanoparticles, assignment of the LSPR peaks to the corresponding oscillation modes has been controversial and requires experimental verification. We determined the in-plane LSPR mode of roxbyite Cu7S4 nanodisks from the plasmon coupling effect of nanodisks in solution. Compared with individual Cu7S4 nanodisks, self-assembled Cu7S4 nanodisk arrays in chloroform exhibited a blue-shifted LSPR peak with weaker optical density. This strongly suggests that the singular LSPR peak in the near-infrared region mainly originates from the in-plane oscillation mode. In addition, we demonstrate that the same LSPR peak can be readily tuned by controlling the number of disks in the array.

Analysis of factors for poor activated partial thromboplastin time control after dabigatran administration.

In this study, we compared patients whose activated partial thromboplastin time (APTT) was prolonged excessively with those whose APTT was controlled within the normal range after dabigatran administration. We analyzed the factors for the APTT prolongation. We divided the patients into two groups: those whose APTTs prolonged to more than 65 s and those whose APTTs were less than 65 s after dabigatran administration. There were 130 patients from March 2011 to July 2013, and we analyzed the background features and laboratory data of these patients. Results showed that there were no significant differences in the patients' background and laboratory data except for the high-density lipoprotein cholesterol (HDL-C) level. However, details of the relationship between the APTT prolongation and the HDL-C level are currently unknown. We hypothesize that the reason for the APTT prolongation is the variability in such parameters as the time of blood drawing, internal time of dabigatran, individual variability, and blood concentration. Therefore, we consider that these parameters need to be carefully evaluated even if APTT does not show prolongation.

Identification of a new disease cluster of pemphigus vulgaris with autoimmune thyroid disease, rheumatoid arthritis and type I diabetes.

BACKGROUND: Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering skin disease. It is known that individuals with autoimmune diseases such as PV, as well as their family members, are at increased risk of developing other autoimmune diseases. However, it is unknown whether there are specific autoimmune diseases that cluster with PV. OBJECTIVES: To investigate the frequency of coexisting autoimmune diseases in patients with PV and their relatives, to determine the prevalence of specific autoimmune diseases in patients with PV vs. the general population and to identify statistically significant clinical clusters linking PV with other autoimmune disorders. METHODS: We performed a cross-sectional study and meta-analysis of patient data from our own patient database (n = 230), an anonymous online survey conducted by our laboratory (n = 171) and the International Pemphigus & Pemphigoid Foundation registry (n = 393). RESULTS: We found that the prevalences of autoimmune thyroid disease (AITD), rheumatoid arthritis and type 1 diabetes were significantly increased in patients with PV compared with the general population. These diseases were also among the most frequent in family members of patients with PV, in addition to systemic lupus erythematosus (SLE). Descriptive cluster analysis using basic principle components methods revealed that PV forms a distinct cluster with AITD, rheumatoid arthritis and type 1 diabetes, and another cluster with SLE, AITD and rheumatoid arthritis. CONCLUSIONS: PV belongs to an established autoimmune disease cluster that includes AITD, rheumatoid arthritis and type 1 diabetes. Our data suggest the possibility of common genetic elements across clinically distinct diseases that might underlie autoimmune susceptibility.

Persistent organic pollutants in liver of Brazilian sharpnose shark (Rhizoprionodon lalandii) from southeastern coast of Brazil.

In the present study, persistent organic pollutants (POPs) were determined in 14 livers from specimens of the Brazilian sharpnose shark (Rhizoprionodon lalandii), which is an important economic resource for small-scale fisheries on the southeastern coast of Brazil. The following concentrations (lipid weight) of POPs were found: ∑PCBs: 1019±267 ng g(-1); ∑DDTs: 111±40 ng g(-1) and ∑PBDEs: 10.4±4.78. PCB 153 made the greatest contribution to ∑PCB (21.4%), followed by PCB 138 (14.6%) and PCB 180 (9.94%). Among chlorinated pesticides, only the p,p'-DDE and p,p'-DDD isomers had concentrations above the detection limit. Moreover, levels above the detection limit were found only for PBDE congeners 47 and 100 (BDEs 47>99). On average, BDE 47 accounted for 88% of the total PBDE load. The feeding habits of the Brazilian sharpnose shark close to the Brazilian coastline are likely the most important difference regarding the accumulation of POPs in comparison to oceanic species that feed in deeper waters. Thus, this species may be used to evaluate the pollution of coastal areas as well as human exposure to contaminants, as the Brazilian sharpnose shark is a frequently used for human consumption. Further studies in other areas of Brazil and involving other species are needed to clarify the mechanisms and potential impact of POPs, which can affect the biology of different organisms and cause population declines.

Effectiveness of brief suicide management training programme for medical residents in Japan: a cluster randomized controlled trial.

Aims. To evaluate the effectiveness of a brief suicide management training programme for Japanese medical residents compared with the usual lecture on suicidality. Methods. In this multi-center, clustered randomized controlled trial, the intervention group attended a structured suicide management programme and the control group, the usual lecture on depression and suicidality. The primary outcome was the difference in residents' cumulative competency score to manage suicidal persons from baseline (T0) to 1 month after the intervention (T2), determined using the Suicide Intervention Response Inventory (SIRI-1) score, at individual level. Results. Analysis of 114 residents (intervention group n = 65, control group n = 49) assigned to two clusters in each group revealed no change in SIRI-1 score from T0 to T2 or immediately after the intervention (T1) between the two groups. As a secondary analysis, discrepancy in judgement between the participants and Japanese suicidologists was examined immediately after the intervention in the adjusted model, with a mean difference in score of 9.98 (95% confidence interval: 4.39-15.56; p = 0.001). Conclusions. The structured programme was not proven to improve competency in suicide management when measured by the SIRI-1 score. Further elaboration of the programme and valid measurement of its outcome would be needed to show the program's effectiveness.

Herd prevalence of Enterobacteriaceae producing CTX-M-type and CMY-2 ß-lactamases among Japanese dairy farms.

AIMS: To determine the herd prevalence of Enterobacteriaceae producing CTX-M-type extended-spectrum ß-lactamases (ESBLs) among 381 dairy farms in Japan. METHODS AND RESULTS: Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 µg ml(-1)). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX-M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX-M-15 (n = 7), CTX-M-2 (n = 12), CTX-M-14 (n = 3), CMY-2 (n = 2) or CTX-M-15/2/14 and CMY-2 (n = 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (n = 23) and three genera of Enterobacteriaceae (n = 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed-field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX-M-15 or CTX-M-27 was not detected. CONCLUSIONS: Three clusters of CTX-M (CTX-M-15, CTX-M-2, CTX-M-14) had spread among Japanese dairy farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the prevalence of multidrug-resistant CTX-M-15-producing E. coli among Japanese dairy farms.