Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Contribution of direct electron transfer mechanisms to overall electron transfer in microbial fuel cells utilising <Emphasis Type="Italic">Shewanella oneidensis</Emphasis> as biocatalyst

Objectives

To investigate the contribution of direct electron transfer mechanisms to electricity production in microbial fuel cells by physically retaining Shewanella oneidensis cells close to or away from the anode electrode.

Results

A maximum power output of 114 ± 6 mWm^?2 was obtained when cells were retained close to the anode using a dialysis membrane. This was 3.5 times more than when the cells were separated away from the anode. Without the membrane the maximum power output was 129 ± 6 mWm^?2. The direct mechanisms of electron transfer contributed significantly to overall electron transfer from S. oneidensis to electrodes, a result that was corroborated by another experiment where S. oneidensis cells were entrapped in alginate gels.

Conclusion

S. oneidensis transfers electrons primarily by direct electron transfer as opposed to mediated electron transfer.

     

Degradation of carbazole,dibenzothiophene and polyaromatic hydrocarbons by recombinant <Emphasis Type="Italic">Rhodococcus</Emphasis> sp.

Objectives

With the view of designing a single biocatalyst for biorefining, carbazole dioxygenase was cloned from Pseudomonas sp. and expressed in Rhodococcus sp.

Results

The recombinant, IGTS8, degraded both carbazole and dibenzothiophene at 400 mg/l in 24 h. Maximum carbazole degradation was in 1:1 (v/v) hexadecane/aqueous phase. Anthracene, phenanthrene, pyrene, fluoranthene and fluorine were also degraded without affecting the aliphatic component.

Conclusions

Recombinant Rhodococcus sp. IGTS8 can function as a single biocatalyst for removing major contaminants of fossil fuels viz. dibenzothiophene, carbazole and polyaromatic compounds.

     

Expression,purification and characterization of a thermostable leucine dehydrogenase from the halophilic thermophile <Emphasis Type="Italic">Laceyella sacchari</Emphasis>

Objective

A potential thermotolerant l-leucine dehydrogenase from Laceyella sacchari (Ls-LeuDH) was over-expressed in E. coli, purified and characterized.

Results

Ls-LeuDH had excellent thermostability with a specific activity of 183 U/mg at pH 10.5 and 25 °C. It retained a high activity in 200 mM carbonate buffer from pH 9.5 to 11. The optimal temperature for Ls-LeuDH was 60 °C.

Conclusion

It is the first time that a thermostable and highly active LeuDH originating from L. sacchari has been characterized. It may be useful for medical and pharmaceutical applications.

     

Inactivation of virginiamycin by <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis>

Objective

To test the inactivation of the antibiotic, virginiamycin, by laccase-induced culture supernatants of Aureobasidium pullulans.

Results

Fourteen strains of A. pullulans from phylogenetic clade 7 were tested for laccase production. Three laccase-producing strains from this group and three previously identified strains from clade 5 were compared for inactivation of virginiamycin. Laccase-induced culture supernatants from clade 7 strains were more effective at inactivation of virginiamycin, particularly at 50 °C. Clade 7 strain NRRL Y-2567 inactivated 6 µg virginiamycin/ml within 24 h. HPLC analyses indicated that virginiamycin was degraded by A. pullulans.

Conclusions

A. pullulans has the potential for the bioremediation of virginiamycin-contaminated materials, such as distiller’s dry grains with solubles (DDGS) animal feed produced from corn-based fuel ethanol production.

     

Medical students and physical education students as CPR instructors: an appropriate solution to the CPR-instructor shortage in secondary schools?

Background

Integrating cardiopulmonary resuscitation (CPR) training in secondary schools will increase the number of potential CPR providers. However, currently too few certified instructors are available for this purpose. Training medical students and physical education student teachers to become CPR instructors could decrease this shortage.

Aim

Examine whether medical students and physical education student teachers can provide CPR training for secondary school pupils as well as (i.?e., non-inferior to) registered nurses.

Methods

A total of 144 secondary school pupils were randomly assigned to CPR training by a registered nurse (n = 12), a  medical student (n = 17) or a physical education student teacher (n = 15). CPR performance was assessed after training and after eight weeks in a simulated cardiac arrest scenario on a resuscitation manikin, using manikin software and video recordings.

Results

No significant differences were found between the groups on the overall Cardiff Test scores and the correctness of the CPR techniques during the post-training and retention test. All pupils showed sufficient CPR competence, even after eight weeks.

Conclusion

Training by medical students or physical education student teachers is non-inferior to training by a registered nurse, suggesting that school teachers, student teachers and medical students can be recruited for CPR training in secondary schools.

     

Increased riboflavin production by knockout of 6-phosphofructokinase I and blocking the Entner–Doudoroff pathway in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To construct an Escherichia coli strain capable of producing riboflavin with high titer and yield.

Results

A low copy number plasmid pLS01 containing a riboflavin operon under the control of a constitutive promoter was constructed and introduced into Escherichia coli MG1655. Subsequently, the pfkA, edd and ead genes were disrupted, and the resulting strain LS02T produced 667 mg riboflavin/l in MSY medium supplied with 10 g glucose/l in flask cultivation. In a fed-batch process, riboflavin production of the strain reached 10.4 g/l with a yield of 56.8 mg riboflavin/g glucose.

Conclusion

To our knowledge, this is the first report of engineered E. coli strains that can produce more than 10 g riboflavin/l in fed-batch cultivation, indicating that E. coli has potential for riboflavin production.

     

Genotyping and Persistence of <Emphasis Type="Italic">Candida albicans</Emphasis> from Pregnant Women with Vulvovaginal Candidiasis

Objective

To study Candida albicans genotypes using RAPD and their susceptibility to fluconazole in healthy pregnant women and in vulvovaginal candidiasis (VVC) patients after topical treatment with clotrimazole.

Methods

Vaginal swabs were collected at t = 0 and t = 1 (1 month later) in pregnant women (control group, n = 33), and before (t = 0), at 1 month (t = 1) and at 2 months (t = 2) after clotrimazole treatment in pregnant women with VVC.

Results

Candida albicans was isolated in 30% of healthy pregnant women and 80% of patients with VVC. A high genetic heterogeneity was observed in C. albicans genotypes between individuals. In patients with VVC, topical antifungal treatment with clotrimazole was clinically effective, but only in a 62% C. albicans was eradicated. In patients in which C. albicans was not eradicated, this microorganism persisted for 1 or 2 months after the antifungal treatment. The persistent colonies were not associated with a specific genotype, but they were associated with higher MICs in comparison with colonies isolated from the control group.

Conclusions

Therapy with topical clotrimazole, despite a good clinical outcome, could not eradicate completely C. albicans allowing the persistence of genotypes, with higher MICs to fluconazole. More studies with higher number of patients are needed to validate this preliminary finding.

     

Left ventricular outflow tract velocity time integral outperforms ejection fraction and Doppler-derived cardiac output for predicting outcomes in a select advanced heart failure cohort

Background

Left ventricular outflow tract velocity time integral (LVOT VTI) is a measure of cardiac systolic function and cardiac output. Heart failure patients with low cardiac output are known to have poor cardiovascular outcomes. Thus, extremely low LVOT VTI may predict heart failure patients at highest risk for mortality.

Methods

Patients with heart failure and extremely low LVOT VTI were identified from a single-center database. Baseline characteristics and heart failure related clinical outcomes (death, LVAD) were obtained at 12 months. Correlation between clinical endpoints and the following variables were analyzed: ejection fraction (EF), pulmonary artery systolic pressure (PASP), NYHA class, renal function, Doppler cardiac output (CO), and LVOT VTI.

Results

Study cohort consisted of 100 patients. At the 12-month follow up period, 30 events (28 deaths, 2 LVADs) were identified. Occurrence of death and LVAD implantation was statistically associated with a lower LVOT VTI (p = 0.039) but not EF (p = 0.169) or CO (p = 0.217). In multivariate analysis, LVOT VTI (p = 0.003) remained statistically significant, other significant variables were age (p = 0.033) and PASP (p = 0.022). Survival analysis by LVOT VTI tertile demonstrated an unadjusted hazard ratio of 4.755 (CI 1.576-14.348, p = 0.006) for combined LVAD and mortality at one year.

Conclusions

Extremely low LVOT VTI strongly predicts adverse outcomes and identifies patients who may benefit most from advanced heart failure therapies.

     

Purging behaviors relate to impaired subjective sleep quality in female patients with anorexia nervosa: a prospective observational study

Background

We examined how purging behaviors relate to subjective sleep quality and sleep patterns and how symptoms of disordered eating behaviors relate to global sleep quality in female patients with anorexia nervosa (AN).

Methods

Participants were new consecutive female inpatients with a primary diagnosis of AN admitted to the Department of Psychosomatic Medicine at Kohnodai Hospital between June 26 and December 25, 2015. We recorded patients’ habitual eating behaviors, laxative overuse, or uretic misuse, and administered the Japanese versions of the Pittsburgh Sleep Quality Index (PSQI-J) and Center for Epidemiologic Studies Depression Scale. Raw PSQI-J data were used to determine sleep patterns (sleep-onset time, wake-up time, and sleep duration). To examine how purging behaviors related to sleep quality, we compared variables between AN restricting type (ANr) and AN binge-eating/purging type (ANbp). Spearman’s rank correlation analysis was used to examine which potential factors influence global PSQI-J score.

Results

Participants were 20 patients, of whom 12 had ANbp. Two ANr patients (25%) had global PSQI-J scores greater than 5, compared to 9 ANbp patients (75%; P < 0.05). Circadian rhythm disruption and abnormal sleep duration were significantly greater in ANbp patients than in ANr patients (P < 0.05). Global PSQI-J was significantly correlated with a diagnosis of ANbp (ρ = 0.525; P < 0.05), vomiting (ρ = 0.561; P < 0.05), and duration of illness (ρ = 0.536; P < 0.05).

Conclusions

ANbp patients had worse global sleep quality and greater disrupted sleep than did ANr patients. This suggests that treatments focusing on sleep would be useful, especially for ANbp patients. Furthermore, vomiting and duration of illness should be considered essential factors related to impaired global sleep quality.

Trial registration

Not applicable.

     

Rapid evolution of hyaluronan synthase to improve hyaluronan production and molecular mass in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Objectives

To improve the production and molecular mass of the glycosaminoglycan hyaluronan (HA) in Bacillus subtilis by engineering hyaluronan synthase (HAS) from Streptococcus zooepidemicus.

Results

By mutating regions within HAS intracellular domains, five positive variants exhibiting higher HA production (from 1.22 to 2.24 g l^?1) and molecular mass values (from 1.20 to 1.36 × 10⁶ Da) were constructed and characterized. Overexpression of the V5 variant and the genes tuaD and glmU increased HA production and molecular mass to 2.8 g l^?1 and 2.4 × 10⁶ Da, respectively.

Conclusions

This study provides a novel strategy for improving HA production and its molecular mass.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Expression of a high sweetness and heat-resistant mutant of sweet-tasting protein,monellin, in <Emphasis Type="Italic">Pichia pastoris</Emphasis> with a constitutive GAPDH promoter and modified <Emphasis Type="Italic">N</Emphasis>-terminus

Objectives

To improve the stability and sweetness of the sweet-tasting protein, monellin, by using site-directed mutagenesis and a Pichia pastoris expression system with a GAPDH constitutive promoter.

Results

Both wild-type and E2 N mutant of single-chain monellin gene were cloned into the PGAPZαA vector and expressed in Pichia pastoris. The majority of the secreted recombinant protein, at 0.15 g/l supernatant, was monellin. This was purified by Sephadex G50 chromatography. The sweetness threshold of wild-type and E2 N were 30 μg/ml and 20 μg/ml, respectively. Compared with the proteins expressed in Escherichia coli, the thermostability of both proteins was improved. The N-terminal sequence is determinative for the sweetness of the proteins expressed in yeast strains.

Conclusions

Site-directed mutagenesis, modification of the N-terminus of monellin, and without the need of methanol induction in P. pastoris expression system, indicate the possibility for large-scale production of this sweet-tasting protein.

     

Growth and antioxidant production of <Emphasis Type="Italic">Spirulina</Emphasis> in different NaCl concentrations

Objective

To evaluate the quantity of Spirulina cultured in seawater, salt-tolerant strains were screened out and their growth and antioxidant accumulation were studied in different salt concentrations

Results

Salt tolerance of five Spirulina strains were investigated with modified Zarrouk medium (with 200–800 mM NaCl). All strains grew well with 400 mM NaCl; their growth rates were almost same as in the control medium. Spirulina strains FACHB-843 (SP843) and FACHB-972 (SP972) had the highest salt tolerance their growth rates in 600 mM NaCl were nearly same as the control. Both strains produced more carotene, phycocyanin, polysaccharides, proline and betaine in 400–600 mM NaCl than the control. Salt stress also induced them to produce higher activities of superoxide dismutase and peroxidase. Total antioxidant capacities of SP843 and SP972 peaked at 600 and 400 mM NaCl, respectively.

Conclusion

Spirulina strains cultured with seawater accumulate more bioactive substances and will have a higher nutritive value.

     

PEGylation of cytochrome <Emphasis Type="Italic">c</Emphasis> at the level of lysine residues mediated by a microbial <Emphasis Type="Italic">trans</Emphasis>glutaminase

Objectives

To establish a method for microbial transglutaminase (mTG)-mediated PEGylation of proteins at the level of lysine (Lys) residues.

Results

Carboxybenzyl-glutaminyl–glycinyl-methoxypolyethylene glycol (CBZ-QG-mPEG) was prepared by introducing carboxybenzyl-glutaminyl-glycine (CBZ-QG) to mPEG amine. The analysis by Fourier transform infrared spectroscopy and SDS-PAGE showed that CBZ-QG-mPEG was successfully synthesized and can be recognized by mTG as an acyl donor to modify therapeutic protein, cytochrome c (cyt c). Finally, under an optimized condition (cyt c 0.5 mg/ml, CBZ-QG-mPEG 11.25 mg/ml, mTG 0.5 mg/ml, 37 °C, 2 h), the PEGylation yield reached 76.5 %.

Conclusions

This is the first study regarding the PEGylation of protein at the level of Lys residues catalyzed by mTG. The novel method could be employed to immobilize active proteins and modify therapeutic proteins.

     

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Introducing transcatheter aortic valve implantation with a new generation prosthesis: Institutional learning curve and effects on acute outcomes

Objectives

We present our single-centre experience with the direct flow medical (DFM) trans-catheter aortic valve implantation (TAVI) prosthesis addressing the impact of learning curve upon outcomes.

Background

The DFM has been recently introduced for TAVI. The prosthesis presents original design and implantation features.

Methods

Patients were divided into three groups according to the chronological implantation sequence that reflected technical skills acquisition of the entire team.

Results

Group I included the first 20 patients (early learning phase), group II the second 20 patients (proctoring to other members of the team), and group III the following 93 patients (technique consolidation). Differences in baseline and procedural variables were analysed. Nonparametric correlation and linear regression were used to identify changes according to institutional cumulative experience. There was a significant correlation between catheterisation time and institutional experience (rho = ?0.4; p < 0.0001) confirmed at linear regression (beta = ?0.2; p = 0.001; CI: ?0.3?–??0.08). Moreover, there was lower rate of valve retrieval in group III (15% vs. 20% vs. 10%; p = 0.5). No intra-procedural mortality was reported and improved early safety (at 30 days) was observed (80% vs. 85% vs. 87.1; p = 0.7). At hospital discharge, valve haemodynamic performance was satisfactory with only mild regurgitation in 10% (I), 20% (II), and 9.7% (III) (p = 0.8).

Conclusions

DFM adequate sizing and implantation can be achieved after the early learning phases. A significant reduction in catheterisation time is reported after the first 20 patients. Results remain satisfactory during the proctoring and technical consolidation phase.

     

High intensity smoking cessation interventions: Cardiac patients of low socioeconomic status and low intention to quit profit most

Background

Without assistance, smokers being admitted to the hospital for coronary heart disease often return to regular smoking within a year.

Objective

This study assessed the 12-month effectiveness of a telephone and a face-to-face counselling intervention on smoking abstinence among cardiac patients. Differential effects for subgroups varying in their socioeconomic status and intention to quit smoking were also studied.

Methods

A randomised controlled trial was used. During hospital stay, smokers hospitalised for coronary heart disease were assigned to usual care (n = 245), telephone counselling (n = 223) or face-to-face counselling (n = 157). Eligible patients were allocated to an intervention counselling group and received nicotine patches. After 12 months, self-reported continued abstinence was assessed and biochemically verified in quitters. Effects on smoking abstinence were tested using multilevel logistic regression analyses applying the intention-to-treat approach.

Results

Compared with usual care, differential effects of telephone and face-to-face counselling on continued abstinence were found in patients with a low socioeconomic status and in patients with a low quit intention. For these patients, telephone counselling increased the likelihood of abstinence threefold (OR = 3.10, 95?% CI 1.32–7.31, p = 0.01), whereas face-to-face counselling increased this likelihood fivefold (OR = 5.30, 95?% CI 2.13–13.17, p < 0.001). Considering the total sample, the interventions did not result in stronger effects than usual care.

Conclusion

Post-discharge telephone and face-to-face counselling interventions increased smoking abstinence rates at 12 months compared with usual care among cardiac patients of low socioeconomic status and low quit intentions. The present study indicates that patients of high socioeconomic status and high quit motivation require different cessation approaches.

     

Metabolic profiling identifies trehalose as an abundant and diurnally fluctuating metabolite in the microalga <Emphasis Type="Italic">Ostreococcus tauri</Emphasis>

Introduction

The picoeukaryotic alga Ostreococcus tauri (Chlorophyta) belongs to the widespread group of marine prasinophytes. Despite its ecological importance, little is known about the metabolism of this alga.

Objectives

In this work, changes in the metabolome were quantified when O. tauri was grown under alternating cycles of 12 h light and 12 h darkness.

Methods

Algal metabolism was analyzed by gas chromatography-mass spectrometry. Using fluorescence-activated cell sorting, the bacteria associated with O. tauri were depleted to below 0.1% of total cells at the time of metabolic profiling.

Results

Of 111 metabolites quantified over light–dark cycles, 20 (18%) showed clear diurnal variations. The strongest fluctuations were found for trehalose. With an intracellular concentration of 1.6 mM in the dark, this disaccharide was six times more abundant at night than during the day. This fluctuation pattern of trehalose may be a consequence of starch degradation or of the synchronized cell cycle. On the other hand, maltose (and also sucrose) was below the detection limit (~10 μM). Accumulation of glycine in the light is in agreement with the presence of a classical glycolate pathway of photorespiration. We also provide evidence for the presence of fatty acid methyl and ethyl esters in O. tauri.

Conclusions

This study shows how the metabolism of O. tauri adapts to day and night and gives new insights into the configuration of the carbon metabolism. In addition, several less common metabolites were identified.