Regular enzyme recovery enhances cellulase production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis> in fed-batch culture

Objective

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.

Results

Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l^?1.

Conclusion

Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Recent advances in human sweat metabolomics for lung cancer screening

Introduction

Lung cancer is the leading cause of cancer related mortality owing to the advanced stage it is usually detected because the available diagnostic tests are expensive and invasive; therefore, they cannot be used for general screening.

Objectives

To increase robustness of previous biomarker panels—based on metabolites in sweat samples—proposed by the authors, new samples were collected within different intervals (4 months and 2 years), analyzed at different times (2012 and 2014, respectively) by different analysts to discriminate between LC patients and smokers at risk factor.

Methods

Sweat analysis was carried out by LC–MS/MS with minimum sample preparation and the generated analytical data were then integrated to minimize variability in statistical analysis.

Results

Panels with capability to discriminate LC patients from smokers at risk factor were obtained taken into account the variability between both cohorts as a consequence of the different intervals for samples collection, the times at which the analyses were carried out and the influence of the analyst. Two panels of metabolites using the PanelomiX tool allow reducing false negatives (95 % specificity) and false positives (95 % sensitivity). The first panel (96.9 % specificity and 83.8 % sensitivity) is composed by monoglyceride MG(22:2), muconic, suberic and urocanic acids, and a tetrahexose; the second panel (81.2 % specificity and 97.3 % sensitivity) is composed by the monoglyceride MG(22:2), muconic, nonanedioic and urocanic acids, and a tetrahexose.

Conclusion

The study has allowed obtaining a prediction model more robust than that obtained in the previous study from the authors.

     

Effecten van geriatrische COPD-revalidatie op ziekenhuisopnames en inspanningstolerantie: een retrospectief observationele studie

Introduction

Frail COPD patients are frequently not accepted for regular pulmonary rehabilitation programs due to low physical condition and functional limitations. Rehabilitation programs in nursing homes for geriatric patients with COPD have been developed. The effects of such programs are largely unknown.

Aims

To assess the course of COPD-related hospital admissions and exercise tolerance in a cohort of frail COPD patients participating in geriatric COPD rehabilitation.

Methods

Retrospective observational study with a follow up of 12 months after discharge from rehabilitation. COPD related hospital admission days were measured in the year before and after participating rehabilitation. Exercise tolerance was measured by the six minute walk test (6MWT) at admission and at discharge from rehabilitation.

Results

Fifty-eight participants accomplished the rehabilitation program. Twelve patients died in the first year after discharge. The median number of hospital admission days in the year before participating rehabilitation was 21 (IQR 10–33). The first year after discharge this was decreased to a median of 6 (IQR 0–12). The 6MWT increased from 194 (SD 85) meters at admission to 274 (SD 95) meters at discharge (mean difference 80 m, SD 72; p < 0.05).

Conclusions

Geriatric COPD rehabilitation in a nursing home setting seems to reduce hospital admissions in frail COPD patients and to increase exercise tolerance.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

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.

     

Trends in service time of pacemakers in the Netherlands: a long-term nationwide follow-up study

Aims

After decades of experience and strongly improved technology, service time of pacemaker generators is expected to increase. To test this hypothesis, we conducted a retrospective review of a large cohort of patients with a pacemaker.

Methods

We reviewed data collected between 1984 and 2006 in the first national Dutch pacemaker registry. This registry covered 96% of all generators implanted. We analysed the time of and reason for explantation of pacemaker generators. A 7-year follow-up interval after first implantation and following replacements was used to analyse changes over time.

Results

During 22 years of data collection, nearly 97,000 first pacemaker generators were implanted. A total of 27,937 (22.4%) generators were explanted within a mean of 6.3 (standard deviation 3.3) years. Reasons for approximately 60% of these explantations were ‘end of life’ of the pacemaker generator or elective system change. Complications or failures such as infections and recalls accounted for approximately 20% of the explantations. For the remaining 20%, the reasons for explantation had not been registered.

Conclusion

Despite progress in technology, a substantial proportion of pacemaker generators is explanted before its expected service time, with one in five generators being replaced due to technical failures, infections or other complications. Furthermore, the time interval between pacemaker implantation and explantation due to normal ‘end of life’ (battery EOL) decreased. Infections continue to rank highly as a cause for pacing system replacement, despite all current preventive measures.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Metabolomic biomarkers and novel dietary factors associated with gestational diabetes in China

Introduction

Gestational diabetes mellitus (GDM) is impaired glucose tolerance first recognised during pregnancy; its development is associated with many adverse outcomes. Mechanisms of GDM development are not fully elucidated and few studies have used Chinese participants.

Objectives

The aim of this study was to investigate the maternal metabolome associated with GDM in a Chinese population, and explore the relationship with maternal diet.

Methods

Ninety-three participants were recruited at 26–28 weeks’ gestation from Chongqing, China. Maternal urine, serum, and hair metabolomes were analysed using gas and liquid chromatography–mass spectrometry. Dietary intake was assessed using a 96-item food frequency questionnaire.

Results

Of the 1064 metabolites identified, 73 were significantly different between cases and controls (P?<?0.05), but only 2-aminobutyric acid had both a p- and q-value?<?0.05. A “snack-based-dietary-pattern” was associated with an increased likelihood of GDM (odds ratio 2·1; 95% confidence interval 1.1–3.9). The association remained significant after adjustment for calorie intake but not food volume.

Conclusion

This study provides a comprehensive characterization of the maternal metabolome. The snack-based dietary pattern associated with GDM suggests that timing and frequency of consumption are important factors in the relationship between maternal diet and GDM.

     

Variety specific relationships between effects of rhizobacteria on root exudation,growth and nutrient uptake of soybean

Aims

It has been increasingly recognized that only distal lower order roots turn over actively within the <2 mm fine root system of trees. This study aimed to estimate fine root production and turnover rate based on lower order fine roots and their relations to soil variables in mangroves.

Methods

We conducted sequential coring in five natural mangrove forests at Dongzhai Bay, China. Annual fine root production and turnover rate were calculated based on the seasonal variations of the biomass and necromass of lower order roots or the whole fine root system.

Results

Annual fine root production and turnover rate ranged between 571 and 2838 g m^?2 and 1.46–5.96 yr^?1, respectively, estimated with lower order roots, and they were increased by 0–30 % and reduced by 13–48 %, respectively, estimated with the whole fine root system. Annual fine root production was 1–3.5 times higher than aboveground litter production and was positively related to soil carbon, nitrogen and phosphorus concentrations. Fine root turnover rate was negatively related to soil salinity.

Conclusions

Mangrove fine root turnover plays a more important role than aboveground litter production in soil C accumulation. Sites with higher soil nutrients and lower salinity favor fine root production and turnover, and thus favor soil C accumulation.

     

Chilling slows anaerobic metabolism to improve anoxia tolerance of insects

Background

Insects are renowned for their ability to survive anoxia. Anoxia tolerance may be enhanced during chilling through metabolic suppression.

Aims

Here, the metabolomic response of insects to anoxia, both with and without chilling, for different durations (12–36 h) was examined to assess the potential cross-tolerance mechanisms.

Results

Chilling during anoxia (cold anoxia) significantly improved survival relative to anoxia at warmer temperatures. Reduced intermediate metabolites and increased lactic acid, indicating a switch to anaerobic metabolism, were characteristic of larvae in anoxia.

Conclusions

Anoxia tolerance was correlated survival improvements after cold anoxia were correlated with a reduction in anaerobic metabolism.

     

The elimination half-life of crystalloid fluid is shorter in female than in male volunteers: a retrospective population kinetic analysis

Background

A recent review article suggests that elimination of infused crystalloid fluid might occur faster in females than in males. To study this question, a population kinetic analysis was performed to compare the turnover of buffered Ringer’s solution when infused at different rates in males and females.

Methods

Data were retrieved from seven series of experiments where 44 intravenous infusions of Ringer’s acetate had been given to female volunteers and 67 to male volunteers. Frequent measurements of the blood hemoglobin and the urinary excretion were used as input in a kinetic two-volume model with micro-constants and covariates, using a nonlinear mixed effects software. The key outcome measure was the rate of irreversible elimination of infused fluid, which was expressed as the half-life, obtained as the excreted urine divided by the modeled plasma volume expansion over time.

Results

The half-life amounted to 24 min (95 % confidence interval, 21–27) in the females and 38 min (33–42) in the males. The urinary excretion differed somewhat less than suggested by these figures during the experimental period (3–4 h) because the plasma volume became less expanded in the females. This was due to that fluid that had been distributed peripheral tissues (the interstitium) returned slightly more slowly to the central fluid space (the plasma) in the females. Gender did not serve as a statistically significant covariate to other rate constants in the kinetic model.

Conclusions

The half-life of infused Ringer’s acetate was 60 % longer in healthy male volunteers than in female volunteers.

     

Noninvasive characterization of metabolites secreted in culture media by bovine embryos during in vitro production

Introduction

Secreted molecules could be correlated with the potential of embryonic development. The development of new technologies, such as mass spectrometry (MS), has enabled analyzes in culture medium to favor the determination of embryos viability in order to improve embryo selection.

Objectives

To perform a non-invasive characterization of the secretome of in vitro produced embryos with different kinetics of cleavage and in different stages of development to obtain specific patterns based on embryonic phenotype through MALDI–TOF–MS.

Methods

Bovine embryos were produced in vitro by standard protocols. The zygotes were transferred to individual culture medium and divided into two groups: Fast [4 cells-22 hours past the beginning of culture (hpc)] and Slow (2 cells-22 hpc). Culture media drops were collected at 22, 96 and 168 hpc. Analysis of embryonic secretome was made by MALDI–TOF–MS after extractions of the metabolites. Spectra were acquired in positive ionization mode. Univariate (Fold-change) and multivariate (Partial Least Squares Discriminants Analysis) analyses were performed by the online software Metaboanalyst.

Results

It was demonstrated that embryos with different kinetics have different spectrometric profiles during embryonic development. Moreover, secreted molecules in each developmental stage are differentially represented in embryos with different kinetics, and are related to specific pathways such as lipid and amino acids metabolism and cell proliferation.

Conclusion

We propose that the analysis of culture media by MALDI–TOF–MS can be used for qualitative characterization of bovine embryos, allowing the identification of key molecules during in vitro culture.

     

Daily variation and effect of dietary folate on urinary pteridines

Introduction

Urinary pteridines are putative molecular biomarkers for noninvasive cancer screening and prognostication. Central to their translational biomarker development is the need to understand the sources and extent of their non-epidemiological variation.

Objectives

This study was designed to characterize the two primary sources of urinary pteridine variance: daily variation and the effect of dietary folate.

Methods

Daily variation was studied by collecting urine specimens (n = 81) three times daily for 3 days. The effect of dietary folate was investigated in a treatment study in which urine specimens (n = 168) were collected daily during a control week and a treatment week during which participants received dietary folate supplements. Measurements of six urinary pteridines were made using high-performance liquid chromatography–tandem mass spectrometry. Coefficients of variation were calculated to characterize daily variance between and within subjects, while nearest neighbor non-parametric analyses were used to identify diurnal patterns and measure dietary folate effects.

Results

Daily variance was approximately 35 % RSD for both within-day and between-day periods for most pteridines. Diurnal patterns in response to circadian rhythms were similarly observed for urinary pteridines. Folate supplementation was shown to alter urinary pteridine profiles in a pathway dependent manner, suggesting that dietary folate may regulate endogenous neopterin and biopterin biosynthesis.

Conclusions

Urinary pteridine levels were found to be responsive to both daily variation and folate supplementation. These findings provide new insights into pteridine biosynthesis and regulation as well as useful information for the design of future clinical translational research.

     

High level soluble expression and one-step purification of IBDV VP2 protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To improve the expression of soluble IBDV VP2 protein by using different tagged vectors in Escherichia coli.

Results

Fusion tags, Grifin, MBP, SUMO, thioredoxin, γ-crystallin, ArsC and PpiB, enhanced the expression and solubility of VP2 protein. The fusion proteins were purified by Ni–NTA chromatography, MBP-VP2 showed the highest purity about 90 %. After removing the MBP tag, VP2 self-assembled into virus-like particles, ~25 nm diam. Results from AGP suggested the recombinant IBDV VP2 protein identified by reference serum like IBDV.

Conclusion

All the seven tags enhanced the expression and solubility of IBDV VP2 protein. The recombinant protein self-assembly into virus like particles and possess antigenicity as reference IBDV.

     

Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD)

Introduction

Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC–MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters.

Objective

Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using design of experiments (DoE).

Methods

We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD). Multivariate statistical analysis guided our decision process in the method optimizations.

Results

LC–MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5 % (p < 0.0001) over initial conditions with a 13.3 % increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8 and 57.3 %, with median metabolite response increases of 106.1 and 10.3 % (p < 0.0001 and p < 0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8 % response increase (p < 0.0001) over initial conditions.

Conclusions

The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.

     

Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank

Background

Population-based biorepositories are important resources, but sample handling can affect data quality.

Objective

Identify metabolites of value for clinical investigations despite extended postcollection freezing delays, using protocols representing a California mid-term pregnancy biobank.

Methods

Blood collected from non-pregnant healthy female volunteers (n?=?20) underwent three handling protocols after 30 min clotting at room temperature: (1) ideal—samples frozen (??80 °C) within 2 h of collection; (2) delayed freezing—samples held at room temperature for 3 days, then 4 °C for 9 days, the median times for biobank samples, and then frozen; (3) delayed freezing with freeze–thaw—the delayed freezing protocol with a freeze–thaw cycle simulating retrieved sample sub-aliquoting. Mass spectrometry-based untargeted metabolomic analyses of primary metabolism and complex lipids and targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference.

Results

Sixty-two percent of 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze–thaw effects were assay-specific with lipids being most stable.

Conclusions

Despite extended post-collection freezing delays characteristic of some biobanks of opportunistically collected clinical samples, numerous metabolomic compounds had both stable levels and good concordance.

     

Dexamethasone downregulates SIRT1 and IL6 and upregulates EDN1 genes in stem cells derived from gingivae via the AGE/RAGE pathway

Objectives

To evaluate the effects of dexamethasone on the aging of mesenchymal stem cells from human gingiva using next-generation sequencing.

Results

Four mRNAs were upregulated and 12 were downregulated when the results of dexamethasone at 24 h were compared with the control at 24 h. Expressions of SIRT1 and IL6 were decreased in dexamethasone at 24 h but expression of EDN1 was increased.

Conclusions

Application of dexamethasone reduced the expression of SIRT1 and IL6 but enhanced the expression of EDN1 of stem cells.

     

Exploration of serum biomarkers for predicting the response to Inchinkoto (ICKT), a Japanese traditional herbal medicine

Introduction

In patients with obstructive jaundice, biliary drainage sometimes fails to result in improvement. A pharmaceutical-grade choleretic herbal medicine, Inchinkoto (ICKT), has been proposed to exert auxiliary effects on biliary drainage; however, its effects are variable among patients.

Objectives

The aim of this study is to explore serum biomarkers that are associated with pharmaceutical efficacy of ICKT.

Methods

Obstructive jaundice patients who underwent external biliary decompression were enrolled (n?=?37). ICKT was given orally 3 times a day at daily dose of 7.5 g. Serum and bile samples were collected before, 3 h after, and 24 h after ICKT administration. The concentrations of total bilirubin, direct bilirubin, and total bile acid in bile specimens were measured. Metabolites in serum samples were comprehensively profiled using LC–MS/MS and GC–MS/MS. Pharmacokinetic analysis of major ICKT components was also performed.

Results

ICKT administration significantly decreased serum ALT and increased bile volume after 24 h. The serum concentrations of ICKT components were not well correlated with the efficacy of ICKT. However, the ratio of 2-hydroxyisobutyric acid to arachidonic acid and the ratio of glutaric acid to niacinamide, exhibited good performance as biomarkers for the efficacy of ICKT on bile flow and ALT, respectively. Additionally, comprehensive correlation analysis revealed that serum glucuronic acid was highly correlated with serum total bilirubin, suggesting that this metabolite may be deeply involved in the pathogenesis of jaundice.

Conclusions

The present study indicates that ICKT is efficacious and provides candidates for predicting ICKT efficacy. Further validation studies are warranted.

     

Outpatient treatment with the wearable cardioverter defibrillator: clinical experience in two Dutch centres

Introduction

The latest European Society of Cardiology Guidelines recommend consideration of a wearable cardioverter-defibrillator (WCD) for patients with a poor left ventricular ejection fraction (LVEF) who are at risk of sudden arrhythmic death but are not eligible for an implantable defibrillator. For these patients a WCD can be an alternative to long-term hospitalisation.

Purpose

To evaluate the use of WCD therapy in these patient groups in two Dutch centres.

Methods

All consecutive patients treated with the WCD between 2009 and 2016 were included from two centres in the Netherlands. Data on events and compliance were collected retrospectively through home monitoring systems and adjudicated by the investigators.

Results

A total of 79 patients were treated with a WCD. Common indications were newly diagnosed cardiomyopathy without optimal medical treatment in 46 patients (58.2%) and bridge to implantable cardioverter-defibrillator (ICD) implant in 33 patients (41.8%). Bridge to implant indications consisted of contraindications for immediate implantation such as infections (e.?g. previous device-related infections) and radiotherapy. Compliance was over 97% per day (median 23.3?h, 22.6–23.7), during a median of 79 days (50.0–109.8.0). Two patients (2.6%) received an appropriate shock (annual rate 13.6%), there was 1 (1.3%) inappropriate shock (annual rate 6.7%). In 24 patients (52.2%) without optimal medical treatment, the LVEF was sufficiently improved and ICD implant was avoided. Eight (10.1%) patients did not receive an ICD. In 45 patients an ICD was implanted (57.0%).

Conclusion

WCD therapy provides a safe and effective treatment in outpatient setting for patients at high risk for sudden cardiac death and reduces the number of ICDs implanted.