Reliability and validity of the modified shuttle test-paeds to measure cardiorespiratory fitness in children.

BACKGROUND: The Modified Shuttle Test-Paeds (Paeds), a recently developed 10-meter Shuttle run test for aerobic capacity in children. This study aims to investigate the construct validity (known-group and convergent validity) and test-retest reliability of the recently developed test for cardiorespiratory fitness, the Modified Shuttle Test-Paeds (Paeds). METHODS: A total of 144 participants (6-12 y) were tested on the Paeds test, and 84 children were tested on the 20-meter Shuttle Run test (20 m-SRT) to assess construct validity. To evaluate test-retest reliability, 46 children were tested twice on the Paeds. RESULTS: No sex differences were found, but there was an age effect. A strong correlation was found between Paeds and the 20 m-SRT (rs=0.78, p < 0.001). The test-retest reliability was good (ICC 0.84; 95% CI 0.74-0.91). CONCLUSION: Paeds appears to be a reliable and valid tool for estimating cardiorespiratory fitness in typically developing children aged 6-12 years and has the advantages of being shorter, needing less space, not requiring pacing and being self-motivational. More studies are needed to assess whether children reach an aerobic steady state in three minutes and how much of the results of the Paeds test can be explained by the agility component of the task (turning and grasping or aiming a bean bag). For clinical use, psychometric properties should be studied in various patient groups (e.g., ADHD, DCD, and children with intellectual disabilities).

Climate footprint assessment of plastic waste pyrolysis and impacts on the Danish waste management system.

Increased plastic recycling is necessary to reduce environmental impacts related to manufacturing and end-of-life of plastic products, however, mechanical recycling (MR) - currently the most widespread recycling option for plastic waste - is limited by quality requirements for inputs and reduced quality of outputs. In this study, pyrolysis of plastic waste is assessed against MR, municipal solid waste incineration (MSWI) and fuel substitution through climate footprint assessment (CFA) based on primary data from pyrolysis of plastic waste sourced from Danish waste producers. Results of the CFA are scaled to the Danish plastic waste resource in an impact assessment of current Danish plastic waste management, and scenarios are constructed to assess reductions through utilization of pyrolysis. Results of the CFA show highest benefits utilizing pyrolysis for monomer recovery (-1400 and -4800 kg CO2e per ton polystyrene (PS) and polymethyl methacrylate (PMMA), respectively) and MR for single polymer polyolefins (-1000 kg CO2e per ton PE). The two management options perform similarly with mixed plastic waste (200 kg CO2e per ton plastic waste). MSWI has the highest impact (1600-2200 kg CO2e per ton plastic waste) and should be avoided when alternatives are available. Scaling the results of the CFA to the full Danish plastic waste resource reveals an impact of 0.79 Mt CO2e in year 2020 of current plastic waste management. Utilizing pyrolysis to manage MR residues reduces the system impact by 15%. Greater reductions are possible through increased separation of plastic from residual waste. The best performance is achieved through a combination of MR and pyrolysis.

Animal production, animal health and food safety: Gaps and challenges in the chilean industry.

This paper summarizes the gaps and challenges related to animal production, health, and food safety as discussed by a panel at the 1st International Symposium of Food Safety (ISFS) in Santiago, Chile, in December 2016. Participating representatives of academia, industry, and government and statements from the audience confirmed that food safety is essential for increasing food security. First, panelists identified the need for a science-based regulatory framework to implement effective regulations. Second, they highlighted the importance of a risk analysis framework to quantify the risk of the potential for antimicrobial resistance associated with the use of antimicrobials, and the need of studies to evaluate foodborne prevention/control strategies. Third, the challenges of filling the gaps between industry and academia were addressed, including examples of successful collaboration, opportunities, and weakness identified by industry. Finally, challenges in animal food production included issues related to changing consumer preferences, animal welfare, the use of antimicrobials, and sustainable animal production. The symposium provided a regional platform to share experiences from the implementation of methods and approaches for food safety. The roundtable successfully explored the future science and technology challenges that are of strategic importance for Chile and the region in animal health and food safety.

Serum neurofilament light chain, contactin-1 and complement activation in anti-MAG IgM paraprotein-related peripheral neuropathy.

INTRODUCTION: In anti-myelin-associated glycoprotein IgM paraprotein-related peripheral neuropathy (anti-MAG PN), there is a lack of reliable biomarkers to select patients eligible for therapy and for evaluating treatment effects, both in routine practice and in clinical trials. Neurofilament light chain (NfL) and contactin-1 (CNTN1) can serve as markers of axonal and paranodal damage. Complement activation is involved in the pathogenesis in anti-MAG PN. We, therefore, hypothesized that serum NfL, CNTN1, C3b/c and C4b/c may function as biomarkers of disease activity in anti-MAG PN. METHODS: In this prospective cohort study, we included 24 treatment-naïve patients with anti-MAG PN (mean age 69 years, 57% male) that had IgM paraproteinemia, a high IgM MAG-antibody, and clinical diagnosis of anti-MAG PN by a neurologist specialized in peripheral nerve disorders. We measured serum NfL, CNTN1, C3b/c and C4b/c, reference values were based on healthy controls. As controls, 10 treatment-naïve patients with IgM Monoclonal gammopathy of undetermined significance (MGUS) or Waldenström's Macroglobulinemia (mean age 69 years, 60% male) without signs of neuropathy were included (non-PN). RESULTS: NfL, CNTN1 levels in serum were mostly normal in anti-MAG PN patients and comparable to non-PN patients. C3b/c and C4b/c levels were normal in anti-MAG PN patients. CONCLUSION: Our results do not support serum NfL, CNTN1, and C3b/c and C4b/c as potential biomarkers in anti-MAG PN, although we cannot exclude that subgroups or subtle abnormalities could be found in a much larger cohort with longitudinal follow-up.

Asparaginase inhibits the lectin pathway of complement activation.

Oncological treatment has been associated with an increased risk of infection, most often related to therapy-induced pancytopenia. However, limited research has been conducted on the effect of oncological therapy on the complement system, being part of the non-cellular innate immune system. This became the rationale for an observational clinical study (C2012) in which we have investigated the prevalence of transient complement defects. Once we had observed such defects, a correlation of the complement defects to specific clinical parameters or to specific therapeutic regimens was investigated. A prominent defect observed in C2012 was the inhibition of the lectin pathway (LP) of complement activation during the treatment of acute lymphoblastic leukemia (ALL), which we could directly associate to the use of asparaginase (ASNase). Ex-vivo experiments confirmed a direct dose-dependent inhibitory effect of ASNase on the LP functionality.

Soil feedback of exotic savanna grass relates to pathogen absence and mycorrhizal selectivity.

Enemy release of exotic plants from soil pathogens has been tested by examining plant-soil feedback effects in repetitive growth cycles. However, positive soil feedback may also be due to enhanced benefit from the local arbuscular mycorrhizal fungi (AMF). Few studies actually have tested pathogen effects, and none of them did so in arid savannas. In the Kalahari savanna in Botswana, we compared the soil feedback of the exotic grass Cenchrus biflorus with that of two dominant native grasses, Eragrostis lehmanniana and Aristida meridionalis. The exotic grass had neutral to positive soil feedback, whereas both native grasses showed neutral to negative feedback effects. Isolation and testing of root-inhabiting fungi of E. lehmanniana yielded two host-specific pathogens that did not influence the exotic C. biflorus or the other native grass, A. meridionalis. None of the grasses was affected by the fungi that were isolated from the roots of the exotic C. biflorus. We isolated and compared the AMF community of the native and exotic grasses by polymerase chain reaction-denaturing gradient gel elecrophoresis (PCR-DGGE), targeting AMF 18S rRNA. We used roots from monospecific field stands and from plants grown in pots with mixtures of soils from the monospecific field stands. Three-quarters of the root samples of the exotic grass had two nearly identical sequences, showing 99% similarity with Glomus versiforme. The two native grasses were also associated with distinct bands, but each of these bands occurred in only a fraction of the root samples. The native grasses contained a higher diversity of AMF bands than the exotic grass. Canonical correspondence analyses of the AMF band patterns revealed almost as much difference between the native and exotic grasses as between the native grasses. In conclusion, our results support the hypothesis that release from soil-borne enemies may facilitate local abundance of exotic plants, and we provide the first evidence that these processes may occur in arid savanna ecosystems. Pathogenicity tests implicated the involvement of soil pathogens in the soil feedback responses, and further studies should reveal the functional consequences of the observed high infection with a low diversity of AMF in the roots of exotic plants.

Characterization of cysteine-degrading and H2S-releasing enzymes of higher plants - from the field to the test tube and back.

Due to the clean air acts and subsequent reduction of emission of gaseous sulfur compounds sulfur deficiency became one of the major nutrient disorders in Northern Europe. Typical sulfur deficiency symptoms can be diagnosed. Especially plants of the Cruciferae family are more susceptible against pathogen attack. Sulfur fertilization can in part recover or even increase resistance against pathogens in comparison to sulfur-deficient plants. The term sulfur-induced resistance (SIR) was introduced, however, the molecular basis for SIR is largely unknown. There are several sulfur-containing compounds in plants which might be involved in SIR, such as high levels of thiols, glucosinolates, cysteine-rich proteins, phytoalexins, elemental sulfur, or H2S. Probably more than one strategy is used by plants. Species- or even variety-dependent differences in the development of SIR are probably used. Our research focussed mainly on the release of H2S as defence strategy. In field experiments using different BRASSICA NAPUS genotypes it was shown that the genetic differences among BRASSICA genotypes lead to differences in sulfur content and L-cysteine desulfhydrase activity. Another field experiment demonstrated that sulfur supply and infection with PYRENOPEZIZA BRASSICA influenced L-cysteine desulfhydrase activity in BRASSICA NAPUS. Cysteine-degrading enzymes such as cysteine desulfhydrases are hypothesized to be involved in H2S release. Several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially analyzed from the model plant ARABIDOPSIS THALIANA. However, it cannot be excluded that H2S is also released in a partial back reaction of O-acetyl-L-serine(thiol)lyase or enzymes not yet characterized. For the exact determination of the H2S concentration in the cell a H2S-specific microsensor was used the first time for plant cells. The transfer of the results obtained for application back on BRASSICA was initiated.

Biosynthesis, processing and secretion of mucus glycoprotein in the rat stomach.

For the study of the biosynthesis, processing and secretion of mucus glycoproteins in rat gastric mucous cells, antibodies were raised against purified gastric mucus glycoproteins and against deglycosylated gastric mucus glycoproteins. Indirect immunofluorescence analysis of gastric mucosa sections revealed that both antibodies specifically labelled the mucus glycoprotein-synthesizing cells in the gastric mucosa. Stomach segments were pulse-labelled with [35S]cysteine and chased for various times. The radioactively labelled (glyco)proteins were quantitatively immunoprecipitated and analyzed by SDS-polyacrylamide gel electrophoresis. Less than 3% of the total radioactivity incorporated in protein was found to be present in mucus glycoproteins. Antibodies raised against native mucus glycoproteins recognized only high-molecular-weight mucus glycoproteins, while the antibodies against deglycosylated glycoproteins also bound to probable precursor forms. The synthesis of mature mucus glycoproteins (Mr greater than 300 000) required about 90 min. After 3 h of chase, only a small portion of the pulse-labelled mucus glycoproteins had been secreted; the majority of the radioactive glycoproteins at that time was still associated with the tissue. Immature (glyco)proteins were not secreted into the medium.

Gp96/GRP94 is a putative high density lipoprotein-binding protein in liver.

We have previously shown that three high density lipoproteins (HDL)-binding proteins in liver, of 90, 110 and 180 kDa, are structurally related. In this study, these proteins are identified as gp96/GRP94. This protein is known to occur as a homodimer and has a dual subcellular localization: it is both an endoplasmic reticulum resident protein, where it is supposed to act as a chaperonin, and a plasma membrane protein, whose significance is unknown. In ultrastructural studies the plasma membrane localization of the homodimeric form was verified. The 90-kDa protein was abundantly present at the membranes of the endosomal/lysosomal vesicles as well as at the apical hepatocyte membranes, comprising the bile canaliculi. The monomeric protein is scarcely present at the basolateral membrane of the hepatocytes, but could be demonstrated in coated pits, suggesting involvement in receptor-mediated endocytosis. Labeling of the endoplasmic reticulum was virtually absent. Gp96/GRP94 was transiently expressed in COS-1 cells. However, the expressed protein was exclusively localized in the endoplasmic reticulum. Transfection with constructs in which the C-terminal KDEL sequence had been deleted, resulted in plasma membrane localized expression of protein, but only in an extremely low percentage of cells. In order to evaluate the HDL-binding capacities of this protein, stably transfected cells were generated, using several cell types. It appeared to be difficult to obtain a prolonged high level expression of gp96. In these cases, however, a marked increase of HDL-binding activity compared with the control cells could be observed.

Regulation of chloride transport in cultured normal and cystic fibrosis keratinocytes.

Cultured normal (N) cystic fibrosis (CF) keratinocytes were evaluated for their Cl(-)-transport properties by patch-clamp-, Ussing chamber- and isotopic efflux-measurements. Special attention was paid to a 32 pS outwardly rectifying Cl- channel which has been reported to be activated upon activation of cAMP-dependent pathways in N, but not in CF cells. This depolarization-induced Cl- channel was found with a similar incidence in N and CF apical keratinocyte membranes. However, activation of this channel in excised patches by protein kinase (PK)-A or PK-C was not successful in either N or CF keratinocytes. Forskolin was not able to activate Cl- channels in N and CF cell-attached patches. The Ca(2+)-ionophore A23187 activated in cell-attached patches a linear 17 pS Cl- channel in both N and CF cells. This channel inactivated upon excision. No relationship between the cell-attached 17 pS and the excised 32 pS channel could be demonstrated. Returning to the measurement of Cl- transport at the macroscopic level, we found that a drastic rise in intracellular cAMP induced by forskolin did in N as well as CF cells not result in a change in the short-circuit current (Isc) or the fractional efflux rates of 36Cl- and 125I-. In contrast, addition of A23187 resulted in an increase of the Isc and in the isotopic anion efflux rates in N and CF cells. We conclude that Cl(-)-transport in cultured human keratinocytes can be activated by Ca2+, but not by cAMP-dependent pathways.

Fibroblast function and the maintenance of the aortic-valve matrix.

The long-term behaviour of the aortic-valve allograft seems to be dependent on the maintenance of its matrix. Protein and collagen synthesis was studied in rat aortic-valves. Quantitative and qualitative methods proved the production of at least two protein pools. One protein pool is localised intracellularily with a 'turn-over' time of about 2 weeks, and a second pool is localised extracellularly with a 'turn-over' time of at least 8 weeks. The latter protein pool mainly consists of collagen.

Cell survival in canine aortic heart valves stored in nutrient medium.

For transplantation of viable aortic valves a period of preservation will generally be needed. To maintain cell viability during this preservation period valves can be stored in nutrient medium after sterilisation in an antibiotic solution. To obtain quantitative data about the survival of aortic valve fibroblasts after preservation, we determined the number of viable fibroblasts in canine aortic valves after several weeks of preservation. The results shows that after storage of 1 week in nutrient medium cell survival is about 80%, after 2 weeks cell survival has declined substantially, while after 3 weeks survival is already unacceptably low. Storage for periods over 4 weeks results in almost completely non-viable aortic valves. These results show that only valves preserved for 1 to 2 weeks in nutrient medium can be considered as viable aortic valves.

Telemetric EEG and video monitoring in epilepsy.

We report a clinical evaluation of EEG and video monitoring. In 181 consecutive records, the most common clinical indications were differentiation of epileptic and nonepileptic events (99), seizure recordings for locating a possible focus (23), seizure frequency determination (32), and investigation of possible trigger factors (19). Overall, useful information was obtained in 72% and the clinical question was answered in 67% of records. Prolonged EEG and video monitoring is therefore an important diagnostic aid in patients with proven or suspected epilepsy.

Production of monoclonal antibodies against inactivated alpha 1-antitrypsin. Cross-reactivity with complexed alpha 1-antitrypsin and application in an assay to determine inactivated and complexed alpha 1-antitrypsin in biological fluids.

15 different monoclonal antibodies (mcAbs) have been raised against the cleaved (inactive) form of the serpin alpha 1-antitrypsin (AT). In initial experiments these mcAbs were analysed for their ability to bind the native and the cleaved form of this inhibitor: eight of the 15 mcAbs appeared to react predominantly with cleaved AT. Additional experiments with mixtures of purified native AT, AT complexed to neutrophilic elastase and inactivated AT revealed that all mAbs that preferentially reacted with inactivated AT also bound to complexed AT. Using two of the mcAbs against inactivated AT a quantitative and sensitive sandwich-type radioimmunoassay was developed to determine levels of proteolytically inactivated AT in biological fluids. With this assay increased levels of inactivated AT were found in synovial fluid from patients with rheumatoid arthritis corresponding to about 2.4% (range 0.3-11%) of total AT. Approximately 10% of this inactivated AT appeared to consist of AT complexed to neutrophil elastase. The mcAbs described here further illustrate the structural resemblance between the complexed and cleaved forms of AT. In addition, these mcAbs appear to be useful tools for the study of AT in human disease.

Production and characterization of monoclonal antibodies raised against recombinant human granzymes A and B and showing cross reactions with the natural proteins.

The human serine proteases granzymes A and B are expressed in cytoplasmic granules of activated cytotoxic T lymphocytes and natural killer cells. Recombinant granzyme A and granzyme B proteins were produced in bacteria, purified and then used to raise specific mouse monoclonal antibodies. Seven monoclonal antibodies (mAb) were raised against granzyme A, which all recognized the same or overlapping epitopes. They reacted specifically in an immunoblot of interleukin-2 (IL-2) stimulated PBMNC with a disulfide-linked homodimer of 43 kDa consisting of 28 kDa subunits. Seven mAb against granzyme B were obtained, which could be divided into two groups, each recognizing a different epitope. On an immunoblot, all mAb reacted with a monomer of 33 kDa protein. By immunohistochemistry, these mAb could be used to detect granzymes A and B expression in activated CTL and NK cells. The availability of these mAb may facilitate studies on the role of human cytotoxic cells in various immune reactions and may contribute to a better understanding of the role of granzymes A and B in the cytotoxic response in vivo.

Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo.

It has been shown that the most important inhibitor of plasmin is alpha 2-antiplasmin, however, other protease inhibitors are able to inhibit this proteolytic enzyme as well. The contribution of the various protease inhibitors to the inhibition of plasmin in vivo has never been quantitatively assessed. To assess the relative contribution of the different protease inhibitors on the inhibition of plasmin we developed a series of sensitive immunoassays for the detection of complexes between plasmin and the protease inhibitors alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin and C1-inhibitor, utilizing monoclonal antibodies that are specifically directed against complexed protease inhibitors and a monoclonal antibody against plasmin. It was confirmed that alpha 2-antiplasmin is the most important inhibitor of plasmin in vivo, however, complexes of plasmin with alpha 2-macroglobulin, antithrombin III, alpha 1-antitrypsin- and C1-inhibitor were also detected. Particularly during activation of fibrinolysis complexes between plasmin and inhibitors other than alpha 2-antiplasmin were detected. It was observed that during different situations the inhibition profile of plasmin was not constant e.g. in patients with diffuse intravascular coagulation plasma levels of plasmin-alpha 1-antitrypsin and plasmin-C1-inhibitor were increased whereas in plasma from patients who were treated with thrombolytic agents complexes of plasmin with alpha 2-macroglobulin and with antithrombin III were significantly elevated. In conclusion, we confirmed the important role of alpha 2-antiplasmin in the inhibition of plasmin, however, in situations in which fibrinolysis is activated other protease inhibitors also account for the inhibition of plasmin in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathologic explanation for postoperative obstipation in Hirschsprung's disease revealed with monoclonal antibody staining.

Until now, no pathologic explanation could be found for the postoperative obstipation occurring in some patients with intestinal aganglionosis. Twenty-two of 108 infants treated for intestinal aganglionosis suffered from postoperative obstipation. Resected material from these 22 patients and from 17 control subjects was investigated with monoclonal anti-neurofilament antibody staining. An abnormal staining pattern was revealed in 18 of the constipated patients. Consequently, this new immunohistochemical staining technic has revealed a hitherto unsuspected cause for postoperative obstipation in aganglionosis. The monoclonal antibody may provide early warning of such postoperative constipation.

Hippocampal EEG and motor activity in the cat: the role of eye movements and body acceleration.

In cat the relation between various behaviours and the spectral properties of the hippocampal EEG was investigated. Both EEG and behaviour were quantified and results were evaluated statistically. Significant relationships were found between the properties of the hippocampal EEG and motor acts (walking, sitting, eating, stepping and eye movements). These results were compared with those obtained in dog under similar experimental circumstances. Species differences were found particularly regarding the fact that in the cat a dissociation between frequency and amplitude parameters was obtained for some behaviours; this may explain why appreciable differences in the visual interpretation of EEG records of different species are often reported. A simple model of the modulation of hippocampal EEG by brainstem inputs is presented. Particular attention is paid to species differences regarding the anatomy and physiology of the pathways involved in this modulation. It is concluded that in cat a strong relation exists between the modulation of spectral properties of hippocampal EEG and vestibular inputs and/or eye movements. The effects of body acceleration on hippocampal EEG are put in evidence and related to theories of hippocampal function.

Sister chromatid exchanges, hyperdiploidy and chromosomal rearrangements studied in cells from melanoma-prone individuals belonging to families with the dysplastic nevus syndrome.

Cytogenetic investigations were performed on 25 individuals belonging to six melanoma-prone families with multiple melanocytic lesions (the dysplastic nevus syndrome, DNS). Patients having DNS with or without a history of melanoma were compared with clinically normal relatives and unrelated normal controls. The results indicate normal frequencies of hyperdiploidy and spontaneous sister chromatid exchanges in the fibroblasts of all individuals studied. Karyotypic analyses were carried out on the members of one family. The patients with DNS had a normal constitutional karyotype. In lymphocytes or fibroblasts from five patients, however, increased frequencies of cells with random chromosomal rearrangements were observed. These abnormalities, mainly translocations and inversions, were not found in two of the patients' spouses and in six clinically normal relatives. In the fibroblast cultures considerable clonal selection of cytogenetically abnormal cells occurred.

The distribution and characterization of HNK-1 antigens in the developing avian heart.

The heart originates from splanchnic mesoderm and to a lesser extent from neural crest cells. The HNK-1 monoclonal antibody is a marker for early migrating neural crest cells, but reacts also with structures which are not derived from the neural crest. We investigated whether heart structures are HNK-1 positive before neural crest cells colonize these target tissues. To that end, we determined the HNK-1 antigen expression in the developing avian heart on immunohistochemical sections and on Western blots. The HNK-1 immunoreactivity in the developing chick heart is compared with data from literature on the localization of neural crest cells in chick/quail chimeras. Structures with neural crest contribution, including parts of the early outflow tract and the related endocardial cushions, the primordia of the semilunar valve leaflets and the aorticopulmonary septum were HNK-1 positive. Furthermore, other structures were HNK-1 positive, such as the atrioventricular cushions, the wall of the sinus venosus at stage HH 15 through 21, parts of the endocardium at E3, parts of the myocardium at E6, and the extracellular matrix in the myocardial base of the semilunar valves at E14. HNK-1 expression was particularly observed in morphologically dynamic regions such as the developing valves, the outflow tract cushion, the developing conduction system and the autonomic nervous system of the heart. We observed that atrioventricular endocardial cushions are HNK-1 positive. We conclude that: a HNK-1 immunoreactivity does not always coincide with the presence of neural crest cells or their derivatives; (2) the outflow tract cushions and atrioventricular endocardial cushions are HNK-1 positive before neural crest cells are expected (stage HH 19) to enter the endocardial cushions of the outflow tract; (3) the observed spatio-temporal HNK-1 patterns observed in the developing heart correspond with various HNK-1 antigens. Apart from a constant pattern of HNK-1 antigens during development, stage-dependent HNK-1 antigens were also found.