Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm−2 trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL−1 horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening

Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry

Penicillins are used universally in both human and veterinary medicine. The European Union (EU) has established maximum residue levels (MRLs) for most ß-lactam antibiotics in milk and animal tissues and included them in the National Residue Monitoring Programs. In this study, a novel method is described for the determination and confirmation of eight penicillins in porcine tissues, milk and animal feed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). To prevent degradation of penicillin residues during workup, a derivatisation procedure was developed, by which penicillins were converted to stable piperidine derivatives. Deuterated piperidine derivatives were synthesised for all relevant penicillins, enabling the use of isotope dilution for accurate quantification. Penicillin residues were derivatised in the crude extract with piperidine and isolated using solid-phase extraction. The penicillin piperidine derivatives were determined by LC–MS/MS. The method was validated at the current MRLs, which range from 25–300 µg kg−1 in muscle and kidney to 4–30 µg kg−1 in milk as well as at the target value of 100 µg kg−1 chosen for animal feed, according to the EU requirements for a quantitative confirmatory method. Accuracy ranged from 94–113% (muscle), 83–111% (kidney) and 87–103% (milk) to 88–116% (animal feed). Intra-day precision (relative standard deviation (RSD)r) ranged from 5–13% (muscle, n = 18), 4–17% (kidney, n = 7) and 5–18% (milk, n = 7) to 11–32% (animal feed, n = 18). Inter-day precision (RSDRL, n = 18) ranged from 6–23% (muscle) to 11–36% (animal feed). From the results, it was concluded that the method was fit for purpose at the target MRLs in animal tissue and target levels for animal feed

High-capacity transmission over polymer optical fiber

Abstract-Polymer optical fiber (POF) is a promising transmission medium to provide broad-band telecommunication services within the customer's premises. POF offers several attractive features for data transmission such as broad bandwidth and low cost for in-house, access, and local-area-network (LAN) applications. This paper presents a review on optical transmission systems using POF and their enabling technologies. A summary is given of experimental data links with record capacity over record transmission distances. To conclude, we discuss trends for further development and research. Index Terms-Optical communications, optical fiber, polymer optical fiber

Cardiorespiratory Fitness in Individuals Post-stroke:Reference Values and Determinants

Objective: To provide reference values of cardiorespiratory fitness for individuals post-stroke in clinical rehabilitation and to gain insight in characteristics related to cardiorespiratory fitness post stroke. Design: A retrospective cohort study. Reference equations of cardiopulmonary fitness corrected for age and sex for the fifth, 25th, 50th, 75th, and 95th percentile were constructed with quantile regression analysis. The relation between patient characteristics and cardiorespiratory fitness was determined by linear regression analyses adjusted for sex and age. Multivariate regression models of cardiorespiratory fitness were constructed. Setting: Clinical rehabilitation center. Participants: Individuals post-stroke who performed a cardiopulmonary exercise test as part of clinical rehabilitation between July 2015 and May 2021 (N=405). Main Outcome Measures: Cardiorespiratory fitness in terms of peak oxygen uptake (V˙O2peak) and oxygen uptake at ventilatory threshold (V˙O2-VT). Results: References equations for cardiorespiratory fitness stratified by sex and age were provided based on 405 individuals post-stroke. Median V˙O2peak was 17.8[range 8.4-39.6] mL/kg/min and median V˙O2-VT was 9.7[range 5.9-26.6] mL/kg/min. Cardiorespiratory fitness was lower in individuals who were older, women, using beta-blocker medication, and in individuals with a higher body mass index and lower motor ability. Conclusions: Population specific reference values of cardiorespiratory fitness for individuals post-stroke corrected for age and sex were presented. These can give individuals post-stroke and health care providers insight in their cardiorespiratory fitness compared with their peers. Furthermore, they can be used to determine the potential necessity for cardiorespiratory fitness training as part of the rehabilitation program for an individual post-stroke to enhance their fitness, functioning and health. Especially, individuals post-stroke with more mobility limitations and beta-blocker use are at a higher risk of low cardiorespiratory fitness.</p

Relative Aerobic Load of Daily Activities After Stroke

Objective: Individuals after stroke are less active, experience more fatigue, and perform activities at a slower pace than peers with no impairments. These problems might be caused by an increased aerobic energy expenditure during daily tasks and a decreased aerobic capacity after stroke. The aim of this study was to quantify relative aerobic load (ie, the ratio between aerobic energy expenditure and aerobic capacity) during daily-life activities after stroke. Methods: Seventy-nine individuals after stroke (14 in Functional Ambulation Category [FAC] 3, 25 in FAC 4, and 40 in FAC 5) and 22 peers matched for age, sex, and body mass index performed a maximal exercise test and 5 daily-life activities at a preferred pace for 5 minutes. Aerobic energy expenditure (mL O2/kg/min) and economy (mL O2/kg/unit of distance) were derived from oxygen uptake (V˙O2). Relative aerobic load was defined as aerobic energy expenditure divided by peak aerobic capacity (%V˙O2peak) and by V˙O2 at the ventilatory threshold (%V˙O2-VT) and compared in individuals after stroke and individuals with no impairments. Results: Individuals after stroke performed activities at a significantly higher relative aerobic load (39%-82% V˙O2peak) than peers with no impairments (38%-66% V˙O2peak), despite moving at a significantly slower pace. Aerobic capacity in individuals after stroke was significantly lower than that in peers with no impairments. Movement was less economical in individuals after stroke than in peers with no impairments. Conclusion: Individuals after stroke experience a high relative aerobic load during cyclic daily-life activities, despite adopting a slower movement pace than peers with no impairments. Perhaps individuals after stroke limit their movement pace to operate at sustainable relative aerobic load levels at the expense of pace and economy. Impact: Improving aerobic capacity through structured aerobic training in a rehabilitation program should be further investigated as a potential intervention to improve mobility and functioning after stroke.</p

The inclination for conscious motor control after stroke: Validating the Movement-Specific Reinvestment Scale for use in inpatient stroke patients

PURPOSE: Stroke survivors are inclined to consciously control their movements, a phenomenon termed "reinvestment". Preliminary evidence suggests reinvestment to impair patients' motor recovery. To investigate this hypothesis, an instrument is needed that can reliably assess reinvestment post-stroke. Therefore, this study aimed to validate the Movement-Specific Reinvestment Scale (MSRS) within inpatient stroke patients. METHOD: One-hundred inpatient stroke patients (<1 year post-stroke) and 100 healthy peers completed the MSRS, which was translated to Dutch for the study purpose. To assess structural validity, confirmatory factor analysis determined whether the scale measures two latent constructs, as previously reported in healthy adults. Construct validity was determined by testing whether patients had higher reinvestment than controls. Reliability analyses entailed assessment of retest reliability (ICC), internal consistency (Cronbach's alpha), and minimal detectable change. RESULTS: Both structural and construct validity of the MSRS were supported. Retest reliability and internal consistency indices were acceptable to good. The minimal detectable change was adequate on group level, but considerable on individual level. CONCLUSIONS: The MSRS is a valid and reliable tool and suitable to assess the relationship between reinvestment and motor recovery in the first months post-stroke. Eventually, this may help therapists to individualize motor learning interventions based on patients' reinvestment preferences. IMPLICATIONS FOR REHABILITATION: This study showed that the Movement-Specific Reinvestment Scale (MSRS) is a valid and reliable tool to objectify stroke patients' inclination for conscious motor control. The MSRS may be used to identify stroke patients who are strongly inclined to consciously control their movements, as this disposition may hinder their motor recovery. Eventually, the MSRS may enable clinicians to tailor motor learning interventions to stroke patients' motor control preferences

Vergelijking van een screenings- en een bevestigingsmethode voor de bepaling van [beta]-agonisten in urine

Voor de bepaling van een aantal [beta]-agonisten in urine zijn een screenings- en een bevestigingsmethode ontwikkeld. De screeningsmethode is gebaseerd op een enzym-immunologische test (EIA), waarbij antilichamen worden gebruikt voor de herkenning van clenbuterol en kruisreagerende verbindingen (metabolieten en andere [beta]-agonisten). Naast een directe screeningsmethode (minimale monstervoorbewerking; geschikt voor ca. 100 monsters per dag) is een iets meer bewerkelijke combinatie met een op immuno-affiniteitschromatografie (IAC) gebaseerde monstervoorbewerking mogelijk. De methode waarbij gebruik wordt gemaakt van gaschromatografie in combinatie met massaspectrometrie (GC-MS) en IAC als monstervoorbewerking is vanwege de grote specificiteit geschikt als bevestigingsmethode (capaciteit 10-20 monsters per dag). In het in dit rapport beschreven onderzoek worden de resultaten verkregen na analyse van 1 013 urinemonsters, met zowel de screenings- als de bevestigingsmethode, met elkaar vergeleken

Determination of zearalenone and its metabolites in endometrial cancer by coupled separation techniques

This study presents a selective method of isolation of zearalenone (ZON) and its metabolite, α-zearalenol (α-ZOL), in neoplastically changed human tissue by accelerated solvent and ultrasonic extractions using a mixture of acetonitrile/water (84/16% v/v) as the extraction solvent. Extraction effectiveness was determined through the selection of parameters (composition of the solvent mixture, temperature, pressure, number of cycles) with tissue contamination at the level of nanograms per gram. The produced acetonitrile/water extracts were purified, and analytes were enriched in columns packed with homemade molecularly imprinted polymers. Purified extracts were determined by liquid chromatography (LC) coupled with different detection systems (diode array detection - DAD and mass spectrometry - MS) involving the Ascentis RP-Amide as a stationary phase and gradient elution. The combination of UE-MISPE-LC (ultrasonic extraction - molecularly imprinted solid-phase extraction - liquid chromatography) produced high (R ≈ 95–98%) and repeatable (RSD < 3%) recovery values for ZON and α-ZOL

Valid and reliable instruments for arm-hand assessment at ICF activity level in persons with hemiplegia: a systematic review

Contains fulltext : 110141.pdf (publisher's version ) (Open Access)BACKGROUND: Loss of arm-hand performance due to a hemiparesis as a result of stroke or cerebral palsy (CP), leads to large problems in daily life of these patients. Assessment of arm-hand performance is important in both clinical practice and research. To gain more insight in e.g. effectiveness of common therapies for different patient populations with similar clinical characteristics, consensus regarding the choice and use of outcome measures is paramount. To guide this choice, an overview of available instruments is necessary. The aim of this systematic review is to identify, evaluate and categorize instruments, reported to be valid and reliable, assessing arm-hand performance at the ICF activity level in patients with stroke or cerebral palsy. METHODS: A systematic literature search was performed to identify articles containing instruments assessing arm-hand skilled performance in patients with stroke or cerebral palsy. Instruments were identified and divided into the categories capacity, perceived performance and actual performance. A second search was performed to obtain information on their content and psychometrics. RESULTS: Regarding capacity, perceived performance and actual performance, 18, 9 and 3 instruments were included respectively. Only 3 of all included instruments were used and tested in both patient populations. The content of the instruments differed widely regarding the ICF levels measured, assessment of the amount of use versus the quality of use, the inclusion of unimanual and/or bimanual tasks and the inclusion of basic and/or extended tasks. CONCLUSIONS: Although many instruments assess capacity and perceived performance, a dearth exists of instruments assessing actual performance. In addition, instruments appropriate for more than one patient population are sparse. For actual performance, new instruments have to be developed, with specific focus on the usability in different patient populations and the assessment of quality of use as well as amount of use. Also, consensus about the choice and use of instruments within and across populations is needed