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This study aimed to examine the relationship between teachers' perceived psychological distance and structural distance from management and teachers' affective organisational commitment (AOC) and organisational citizenship behaviour (OCB). Teachers' trust in management was expected to mediate these relationships. Furthermore, the adequacy and openness of communication and participation in decision making were expected to reduce psychological distance. At 10 Dutch schools for vocational education and training, 884 teachers completed a questionnaire. The data were analysed using structural equation modelling. Teachers' psychological distance had a negative effect on trust in management (supervisor and higher management) and AOC. Trust in management had a positive effect on AOC, and trust in the supervisor had a positive effect on OCB. Structural distance did not influence teacher outcomes. Communication and participation in decision making reduced teachers' psychological distance from management. The discussion focuses on the implications of the findings to improve the effectiveness of schools.

In this study we examined the role of trust as a mediator in social exchange between teachers and their school, particularly between perceived procedural justice and perceived organizational support, on the one hand, and teachers' affective organizational commitment and organizational citizenship behaviour, on the other hand. A model was developed that distinguished between trust in three agents: team members, the supervisor and higher management. The model was tested in the context of Dutch schools for vocational education and training on a sample of 845 teachers. Data was collected using questionnaires and was analysed with structural equation modelling. Results show that trust was a predictor for desirable teacher outcomes and a mediator in social exchange. In particular trust in team members was related to affective organizational commitment and organizational citizenship behaviour. Trust in the supervisor and trust in higher management were neither related to organizational commitment nor to organizational citizenship behaviour. Perceived organizational support was related to teachers' trust in all trust targets. It also had a direct effect on organizational commitment. Procedural justice influenced trust in the supervisor and trust in higher management. The authors discuss the results in light of previous findings and the context of the study.

Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1−/− mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function. [ABSTRACT FROM AUTHOR]

The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by a reverse Michael addition. The overall fold and the constitution of the active site of the enzyme completely differ from the well-characterised chalcone isomerase of plants. For (+)-taxifolin, CHI catalyses the intramolecular ring contraction to alphitonin. In this study, Fwe perform crystal structure analyses of CHI and its active site mutant His33Ala in the presence of the substrate taxifolin at 2.15 and 2.8 Å resolution, respectively. The inactive enzyme binds the substrate (+)-taxifolin as well defined, whereas the electron density maps of the native CHI show a superposition of substrate, product alphitonin, and most probably also the reaction intermediate taxifolin chalcone. Evidently, His33 mediates the stereospecific acid-base reaction by abstracting a proton from the flavonoid scaffold. The stereospecificity of the product is discussed. [ABSTRACT FROM AUTHOR]

The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel `hairpin-like' architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the `hairpin-like' hypothesis. [ABSTRACT FROM AUTHOR]

This study aimed to examine the relationship between teachers’ perceived psychological distance and structural distance from management and teachers’ affective organisational commitment (AOC) and organisational citizenship behaviour (OCB). Teachers’ trust in management was expected to mediate these relationships. Furthermore, the adequacy and openness of communication and participation in decision making were expected to reduce psychological distance. At 10 Dutch schools for vocational education and training, 884 teachers completed a questionnaire. The data were analysed using structural equation modelling. Teachers’ psychological distance had a negative effect on trust in management (supervisor and higher management) and AOC. Trust in management had a positive effect on AOC, and trust in the supervisor had a positive effect on OCB. Structural distance did not influence teacher outcomes. Communication and participation in decision making reduced teachers’ psychological distance from management. The discussion focuses on the implications of the findings to improve the effectiveness of schools. [ABSTRACT FROM PUBLISHER]

Purpose. Granulocyte transfusions have been used to treat immune cell dysfunction in sepsis. A granulocyte bioreactor for the extracorporeal treatment of sepsis was tested in a prospective clinical study focusing on the dosage of norepinephrine in patients and influence on dynamic and cell based liver tests during extracorporeal therapies. Methods and Patients. Ten patients with severe sepsis were treated twice within 72 h with the system containing granulocytes from healthy donors. Survival, physiologic parameters, extended hemodynamic measurement, and the indocyanine green plasma disappearance rate (PDR) were monitored. Plasma of patients before and after extracorporeal treatments were tested with a cell based biosensor for analysis of hepatotoxicity. Results. The observed mortality rate was 50% during stay in hospital. During the treatments, the norepinephrine-dosage could be significantly reduced while mean arterial pressure was stable. In the cell based analysis of hepatotoxicity, the viability and function of sensor-cells increased significantly during extracorporeal treatment in all patients and the PDR-values increased significantly between day 1 and day 7 only in survivors. Conclusion. The extracorporeal treatment with donor granulocytes showed promising effects on dosage of norepinephrine in patients, liver cell function, and viability in a cell based biosensor. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

In this study we examined the role of trust as a mediator in social exchange between teachers and their school, particularly between perceived procedural justice and perceived organizational support, on the one hand, and teachers’ affective organizational commitment and organizational citizenship behaviour, on the other hand. A model was developed that distinguished between trust in three agents: team members, the supervisor and higher management. The model was tested in the context of Dutch schools for vocational education and training on a sample of 845 teachers. Data was collected using questionnaires and was analysed with structural equation modelling. Results show that trust was a predictor for desirable teacher outcomes and a mediator in social exchange. In particular trust in team members was related to affective organizational commitment and organizational citizenship behaviour. Trust in the supervisor and trust in higher management were neither related to organizational commitment nor to organizational citizenship behaviour. Perceived organizational support was related to teachers’ trust in all trust targets. It also had a direct effect on organizational commitment. Procedural justice influenced trust in the supervisor and trust in higher management. The authors discuss the results in light of previous findings and the context of the study. [ABSTRACT FROM AUTHOR]

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2 S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2 S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific 1H/2H isotope exchange observed by 1H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1. [ABSTRACT FROM AUTHOR]

The importance of amine transaminases for producing optically pure chiral precursors for pharmaceuticals and chemicals has substantially increased in recent years. The X-ray crystal structure of the (R) -selective amine transaminase from the fungus Aspergillus fumigatus was solved by S-SAD phasing to 1.84 Å resolution. The refined structure at 1.27 A resolution provides detailed knowledge about the molecular basis of substrate recognition and conversion to facilitate protein-engineering approaches. The protein forms a homodimer and belongs to fold class IV of the pyridoxal-5'-phosphate-dependent enzymes. Both subunits contribute residues to form two active sites. The structure of the holoenzyme shows the catalytically important cofactor pyridoxal-5'-phosphate bound as an internal aldimine with the catalytically responsible amino-acid residue Lys179, as well as in its free form. A long N-terminal helix is an important feature for the stability of this fungal (R)-selective amine transaminase, but is missing in branched-chain amino-acid aminotransferases and d-amino-acid aminotransferases. [ABSTRACT FROM AUTHOR]

Flavonoide stellen eine große Gruppe pflanzlicher Sekundärmetabolite dar und haben eine Vielzahl unterschiedlicher biologischer Eigenschaften. Aufgrund dessen stehen sie im Interesse vieler Wissenschaftler, da sie zu vielen industriell interessanten Verbindungen führen können. Das anaerobe Darmbakterium Eubacterium ramulus kann über einen bislang unbekannten Abbauweg diese Flavonoide verstoffwechseln. Über eine Vollgenom ‐ Sequenzierung wurden in dieser Arbeit die entscheidenden Enzyme, eine Chalconisomerase (CHI) und eine Enoatreduktase (ERED), identifiziert, kloniert und funktionell sogar unter aeroben Bedingungen exprimiert. Mittels Biotransformation des Flavanons zum Dihydrochalcon konnte der Flavonoid ‐ Abbauweg bewiesen werden. Der beide Enzyme exprimierende E. ‐ coli ‐ Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden. Dies unterstreicht die Anwendbarkeit des in dieser Arbeit entwickelten biokatalytischen Systems. Der biokatalytisch ‐ metabolische Weg für die Umsetzung von Flavonoiden aus Eubacterium ramulus wurde identifiziert. Eine Chalconisomerase und eine Enoatreduktase wurden erfolgreich kloniert und in E. coli exprimiert und unter aeroben Bedingungen eingesetzt, obwohl E. ramulus ein strikt anaerobes Bakterium ist. Der beide Enzyme exprimierende E. ‐ coli ‐ Stamm kann für die Umsetzung verschiedener Flavanone eingesetzt werden. [ABSTRACT FROM AUTHOR]

Flavonoids are a large group of plant secondary metabolites with a variety of biological properties and are therefore of interest to many scientists, as they can lead to industrially interesting intermediates. The anaerobic gut bacterium Eubacterium ramulus can catabolize flavonoids, but until now, the pathway has not been experimentally confirmed. In the present work, a chalcone isomerase (CHI) and an enoate reductase (ERED) could be identified through whole genome sequencing and gene motif search. These two enzymes were successfully cloned and expressed in Escherichia coli in their active form, even under aerobic conditions. The catabolic pathway of E. ramulus was confirmed by biotransformations of flavanones into dihydrochalcones. The engineered E. coli strain that expresses both enzymes was used for the conversion of several flavanones, underlining the applicability of this biocatalytic cascade reaction. [ABSTRACT FROM AUTHOR]

In an extracorporeal combination therapy, the impact of different replacement fluids on survival was tested in a bacterial sepsis model in pigs. In an animal study 19 pigs, weighing 7.5-11.1 kg, were included. All groups received an intravenous lethal dose of live Staphylococcus aureus over 1 h. The animals were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration for 4 h. The extracorporeal circuit was pre-filled with 400 mL replacement fluid. In the P0 group 100% hydroxyethyl starch 130/0.4 was used as replacement fluid; in the P30 group 30% pig plasma and 70% hydroxyethyl starch; and in the P100 group 100% pig plasma. The observation time was 7 days. All animals of the group P100 survived, while all animals of group P0 and five out of seven animals of the P30 group died during the observation time. Extracorporeal therapy consisting of online centrifugation and plasma filtration with 100% pig plasma as replacement fluid significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

The impact on survival of a combination of plasma separation by centrifugation and subsequent plasma filtration was tested in a bacterial sepsis model in pigs. In this animal study 19 pigs were included. Groups II and III received an intravenous lethal dose of live Staphylococcus aureus over 1 h; group I received saline (non-septic control-NC). Groups I and II were treated by an extracorporeal circuit consisting of online centrifugation and subsequent plasma filtration (group II: treated group-TG) for 4 h; group III had no specific treatment (septic control, SC). The observation time was 7 days. All animals of group I (NC) and group II (TG) survived, while all animals of group III (SC) died during the observation time. Extracorporeal therapy with online centrifugation and plasma filtration significantly improved survival in a pig model of sepsis. Further studies with this approach are encouraged. [ABSTRACT FROM AUTHOR]

The ( R)-selective amine transaminase from Aspergillus fumigatus was expressed in Escherichia coli and purified to homogeneity. Bright yellow crystals appeared while storing the concentrated solution in the refrigerator and belonged to space group C2221. X-ray diffraction data were collected to 1.27 Å resolution, as well as an anomalous data set to 1.84 Å resolution that was suitable for S-SAD phasing. [ABSTRACT FROM AUTHOR]

Membrane proteins are traditionally extracted and purified in detergent for biochemical and structural characterisation. This process is often costly and laborious, and the stripping away of potentially stabilising lipids from the membrane protein of interest can have detrimental effects on protein integrity. Recently, styrene-maleic acid (SMA) co -polymers have offered a solution to this problem by extracting membrane proteins directly from their native membrane, while retaining their naturally associated lipids in the form of stable SMA lipid particles (SMALPs). However, the inherent nature and heterogeneity of the polymer renders their use challenging for some downstream applications – particularly mass spectrometry (MS). While advances in cryo-electron microscopy (cryo-EM) have enhanced our understanding of membrane protein:lipid interactions in both SMALPs and detergent, the resolution obtained with this technique is often insufficient to accurately identify closely associated lipids within the transmembrane annulus. Native-MS has the power to fill this knowledge gap, but the SMA polymer itself remains largely incompatible with this technique. To increase sample homogeneity and allow characterisation of membrane protein:lipid complexes by native-MS, we have developed a novel SMA-exchange method; whereby the membrane protein of interest is first solubilised and purified in SMA, then transferred into amphipols or detergents. This allows the membrane protein and endogenously associated lipids extracted by SMA co -polymer to be identified and examined by MS, thereby complementing results obtained by cryo-EM and creating a better understanding of how the lipid bilayer directly affects membrane protein structure and function. • First reported exchange protocol for transferring membrane proteins solubilised in SMALPs, into detergent or amphipols. • Purification of protein:lipid complexes without detergent for mass spectrometry and subsequent lipid identification. • Cost effective membrane protein purification requiring only minimal amounts of detergents in the exchange process. [ABSTRACT FROM AUTHOR]

Summary Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. Highlights • Crystal structure of the kinesin-1 KLC2 TPR domain bound to the cargo JIP3 • JIP3 binding site is located on KLC TPR1 and is conserved from sponges to humans • Molecular mechanism by which ARF6 regulates kinesin-1 recruitment by JIP3/4 revealed • A unified framework explaining how unrelated cargoes activate kinesin-1 Cockburn et al. present the structure of the cargo binding TPR domain of kinesin light chain bound to the cargo molecule JIP3. From this they propose a mechanism by which multiple, unrelated cellular cargo molecules can "hotwire" the kinesin-1 molecule into motility. [ABSTRACT FROM AUTHOR]

Size-exclusion chromatography coupled to multiangle laser light scattering (SEC-MALLS) is the perfect method to determine the oligomeric state of membrane proteins as this method works in solution and is totally independent from prior assumptions such as detergent-to-protein ratio or the shape of the protein. In a relatively short time (ca. 30 min), the molecular mass and quality of a membrane protein preparation can be determined. Here, I provide a detailed protocol on how to perform a SEC-MALLS run and show exemplary chromatograms and their analysis.

Nucleoside transporters (NTs) play critical biological roles in humans, and to understand the molecular mechanism of nucleoside transport requires high-resolution structural information. However, the main bottleneck for structural analysis of NTs is the production of pure, stable, and high quality native protein for crystallization trials. Here we report a novel membrane protein expression and purification strategy, including construction of a high-yield membrane protein expression vector, and a new and fast purification protocol for NTs. The advantages of this strategy are the improved time efficiency, leading to high quality, active, stable membrane proteins, and the efficient use of reagents and consumables. Our strategy might serve as a useful point of reference for investigating NTs and other membrane proteins by clarifying the technical points of vector construction and improvements of membrane protein expression and purification.

Introduction: Women often develop malignant mesothelioma (MM) without occupational asbestos exposure. Northern Jutland has a high prevalence of MM due to previously high occupational exposures to asbestos. The aim of this study was to elucidate a possible domestic exposure to asbestos through first-degree relatives in women who develop MM., Material and Methods: This was a retrospective study in women with MM of the pleura. A total of 30 women were diagnosed with and treated for MM in Northern Jutland from 1996 to 2012. In all, 24 women were included. Demographic data, subtype of MM, time from first hospital contact to diagnosis, survival and information on occupational and domestic exposure to asbestos were obtained from hospital records., Results: A total of 12.5% of the study population were primarily exposed to asbestos. 46% had domestic exposure to asbestos through their husbands or sons. The median age of the study population was 66.5 years. In all, 75% suffered from the epitheloid subtype, 12.5% from the biphasic and 8.4% from the sarcomatoid subtype. Time from first hospital contact to diagnosis was one month and the median survival time was 12 months. The 1- and 5- year-survival were 58% and 0%, respectively., Conclusion: Nearly 50% of the women affected by MM have been domestically exposed to asbestos through first-degree relatives., Funding: not relevant., Trial Registration: not relevant.