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Background: A plethora of antimicrobial stewardship (AMS) programs has been initiated during the past years, focusing on hospital settings. Primary-care physicians have seldom been addressed, although the majority of antibiotic prescriptions are issued for outpatients. We sought to investigate attitudes of primary-care physicians and the impact of a customized training course.Methods: Primary-care physicians in southwest Germany were invited to a multi-part training course on AMS in the primary-care setting. Participants were asked to answer a questionnaire about their attitude and factors that hinder them from implementing AMS or enable them to perform AMS. In addition, a knowledge assessment exam at the beginning and end of the training was conducted on selected infectious diseases/syndromes.Results: In total, 36 primary-care physicians participated in the training course. The predominant age group was 51-60 years old (36%; 13/36). The majority, 23/35 (66%), indicated never having had AMS training, while 22/35 (63%) acknowledged partly implementing AMS activities in their daily routine. The primary barrier was lack of expertise, while the main motives were reducing antimicrobial resistance and optimizing patient care. The provision of guidelines was regarded as more important than feedback on their prescription behavior. Exam performance improved from the initial to the final exam on all topics.Conclusion: Customized AMS training courses are a feasible and potentially complimentary tool to address antibiotic misuse in the primary-care setting., Hintergrund: In den letzten Jahren wurde eine Vielzahl von "Antibiotic Stewardship" (ABS) Kampagnen in Krankenhäusern ins Leben gerufen. Ärzte in der Primärversorgung wurden nur selten adressiert, obwohl die Mehrzahl der Antibiotikaverordnungen bei ambulanten Patienten veranlasst wird. Wir hatten das Ziel, die Einstellung von Ärzten in der Primärversorgung zu ABS und die Auswirkungen eines maßgeschneiderten Fortbildungskurses zu untersuchen.Methoden: Ärzte in der Primärversorgung im Südwesten Deutschlands wurden zu einem mehrteiligen Fortbildungskurs über ABS in der Primärversorgung eingeladen. Die Teilnehmer wurden gebeten, einen Fragebogen zu ihrer Einstellung und zu Faktoren zu beantworten, die sie an der Durchführung von ABS hindern oder sie dazu befähigen. Darüber hinaus wurde bei der Teilnahme und beim Verlassen des Kurses ein Wissenstest zu ausgewählten Infektionssyndromen durchgeführt.Ergebnisse: Insgesamt nahmen 36 Ärzte an dem Kurs teil. Die vorherrschende Altersgruppe war die der 51- bis 60-Jährigen mit 36% (13/36). Die Mehrheit, 23/35 (66%), gab an, noch nie an einer ABS-Fortbildung teilgenommen zu haben, während 22/35 (63%) angaben, ABS-Aktivitäten teilweise in ihre tägliche Routine integriert zu haben. Das Haupthindernis war der Mangel an Fachwissen, während die Hauptmotive die Reduzierung der Antibiotikaresistenz und die Optimierung der Patientenversorgung waren. Die Bereitstellung von Leitlinien wurde als wichtiger erachtet als eine Rückmeldung über das eigene Verordnungsverhalten. Die Prüfungsleistungen im Wissenstest stiegen während des Kurses in allen Themenbereichen.Schlussfolgerung: Maßgeschneiderte ABS-Fortbildungen sind ein praktikables und potenziell ergänzendes Instrument zur Bekämpfung des übermäßigen Einsatzes von Antibiotika in der Primärversorgung.

OBJECTIVE: To compare the concordance and acceptability of saliva testing with standard-of-care oropharyngeal and bilateral deep nasal swab testing for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in children and in general practice. DESIGN: Prospective multicentre diagnostic validation study. SETTING: Royal Children's Hospital, and two general practices (cohealth, West Melbourne; Cirqit Health, Altona North) in Melbourne, July-October 2020. PARTICIPANTS: 1050 people who provided paired saliva and oropharyngeal-nasal swabs for SARS-CoV-2 testing. MAIN OUTCOME MEASURES: Numbers of cases in which SARS-CoV-2 was detected in either specimen type by real-time polymerase chain reaction; concordance of results for paired specimens; positive percent agreement (PPA) for virus detection, by specimen type. RESULTS: SARS-CoV-2 was detected in 54 of 1050 people with assessable specimens (5%), including 19 cases (35%) in which both specimens were positive. The overall PPA was 72% (95% CI, 58-84%) for saliva and 63% (95% CI, 49-76%) for oropharyngeal-nasal swabs. For the 35 positive specimens from people aged 10 years or more, PPA was 86% (95% CI, 70-95%) for saliva and 63% (95% CI, 45-79%) for oropharyngeal-nasal swabs. Adding saliva testing to standard-of-care oropharyngeal-nasal swab testing increased overall case detection by 59% (95% CI, 29-95%). Providing saliva was preferred to an oropharyngeal-nasal swab by most participants (75%), including 141 of 153 children under 10 years of age (92%). CONCLUSION: In children over 10 years of age and adults, saliva testing alone may be suitable for SARS-CoV-2 detection, while for children under 10, saliva testing may be suitable as an adjunct to oropharyngeal-nasal swab testing for increasing case detection.

Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373-374A). Further cleavage by MMPs (N341-342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.

The metalloproteinase ADAMTS-5 (A disintegrin and metalloproteinase with thrombospondin motifs) degrades aggrecan, a proteoglycan essential for cartilage structure and function. ADAMTS-5 is the major aggrecanase in mouse cartilage, and is also likely to be the major aggrecanase in humans. ADAMTS-5 is a multidomain enzyme, but the function of the C-terminal ancillary domains is poorly understood. We show that mutant ADAMTS-5 lacking the catalytic domain, but with a full suite of ancillary domains inhibits wild type ADAMTS activity, in vitro and in vivo, in a dominant-negative manner. The data suggest that mutant ADAMTS-5 binds to wild type ADAMTS-5; thus we tested the hypothesis that ADAMTS-5 associates to form oligomers. Co-elution, competition, and in situ PLA experiments using full-length and truncated recombinant ADAMTS-5 confirmed that ADAMTS-5 molecules interact, and showed that the catalytic and disintegrin-like domains support these intermolecular interactions. Cross-linking experiments revealed that recombinant ADAMTS-5 formed large, reduction-sensitive oligomers with a nominal molecular mass of ∼ 400 kDa. The oligomers were unimolecular and proteolytically active. ADAMTS-5 truncates comprising the disintegrin and/or catalytic domains were able to competitively block full-length ADAMTS-5-mediated aggrecan cleavage, measured by production of the G1-EGE(373) neoepitope. These results show that ADAMTS-5 oligomerization is required for full aggrecanase activity, and they provide evidence that blocking oligomerization inhibits ADAMTS-5 activity. The data identify the surface provided by the catalytic and disintegrin-like domains of ADAMTS-5 as a legitimate target for the design of aggrecanase inhibitors.

Many coral reef communities thriving in inshore coastal waters characterised by chronically high natural turbidity (>5 mg l-1) have adapted to low light (<200 µmol photons m-2 s-1) and high sedimentation rates (>10 mg cm-2 d-1). Yet, short (hours) acute sediment stress events driven by wind waves, dredging operations involving suction or screening, or shipping activities with vessel wake or propeller disturbance, can result in a rise in turbidity above the natural background level. Although these may not be lethal to corals given the time frame, there could be a considerable impact on photo-trophic energy production. A novel sediment delivery system was used to quantify the effects of 3 acute sediment resuspension stress events (turbidity = 100, 170, 240 mg l-1; sedimentation rates = 4, 9, 13 mg cm-2 h-1) on 3 inshore turbid water corals common in the Indo-Pacific (Merulina ampliata, Pachyseris speciosa and Platygyra sinensis). Coral photo-physiology response (respiration, net photosynthesis, and maximum quantum yield) was measured immediately after 2 h of exposure. The respiration rate increased (from 0.72-1.44 to 0.78-1.76 µmol O2 cm-2 h-1) as the severity of the acute sediment resuspension event increased, whereas the photosynthetic rate declined (from 0.25-0.41 to -0.19-0.25 µmol O2 cm-2 h-1). Merulina was the least tolerant to acute sediment resuspension, with a photosynthesis and respiration ratio (P/R ratio) of <1.0 when turbidity levels reached >170 mg l-1, while Platygyra was most tolerant (P/R > 1.0). Fluorescence yield data suggest that the rapid photo-acclimation ability of Platygyra enabled it to maintain a positive carbon budget during the experiments, illustrating species-specific responses to acute sediment stress events.

OBJECTIVE: To investigate aggrecan degradation in juvenile idiopathic arthritis (JIA). METHODS: The pattern and abundance of aggrecan fragments in synovial fluid (SF) aspirates from JIA patients were analyzed and compared with aggrecan fragments in SF from patients with other arthritides, children with knee injury, and a knee-healthy reference group. Concentrations of sulfated glycosaminoglycan (sGAG) in SF were measured by Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density-gradient centrifugation, deglycosylated, and analyzed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE, and KEEE neoepitopes or the aggrecan G3 domain. RESULTS: The concentration of sGAG in SF from patients with JIA was significantly lower compared with that in SF from patients with osteoarthritis (OA) (P < 0.001), patients with juvenile knee injury (P = 0.006), and knee-healthy controls (P = 0.022). Western blot analysis revealed KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulfate-rich region of aggrecan in patients with JIA. The pattern of aggrecan fragments in JIA patients was not identical to that in pooled OA SF, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in JIA SF, in marked contrast to levels in OA SF. CONCLUSION: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in the chondroitin sulfate-rich region. These results suggest that in JIA, unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event.

Proteolysis of the cartilage proteoglycan aggrecan is a feature of arthritis. We present a method for analyzing aggrecanolysis in in vitro cultures of 3-week-old mouse femoral head cartilage based on traditional methods developed for large animal species. Investigators can choose either a simple analysis that detects several aggrecan fragments released into culture medium only or a more comprehensive study that detects all fragments present in both the medium and the cartilage matrix. The protocol comprises (i) cartilage culture and optional cartilage extraction, (ii) a quick and simple colorimetric assay for quantitating aggrecan and (iii) neoepitope western blotting to identify specific aggrecan fragments partitioning to the medium or cartilage compartments. The crucial difference between the methods for mice and larger animals is that the proportion of aggrecan in a given sample is normalized to total aggrecan rather than to tissue wet weight. This necessary break from tradition arises because tiny volumes of liquid clinging to mouse cartilage can increase the apparent tissue wet weight, causing unacceptable errors. The protocol has broad application for the in vitro analysis of transgenic mice, particularly those with mutations that affect cartilage remodeling, arthritic disease and skeletal development. The protocol is robust, reliable and takes 7-11 d to complete.

The microstructure of keratan sulphate purified from the interglobular domain, the keratan sulphate-rich region and total aggrecan was compared using fluorophore-assisted-carbohydrate-electrophoresis. Keratan sulphate in the interglobular domain was substantially less sulphated than keratan sulphate elsewhere on aggrecan, based on the ratio of unsulphated: monosulphated disaccharides generated by endo-beta-galactosidase digestion, and the ratio of monosulphated: disulphated disaccharides generated by keratanase II digestion. The ratio of unsulphated: monosulphated: disulphated disaccharides was 1:4:5 for keratan sulphate from total aggrecan and the keratan sulphate-rich region, but only 1:0.9:0.8 for the interglobular domain. These results show that keratan sulphate in the interglobular domain of pig aggrecan has a microstructure that is distinct from keratan sulphate in the keratan sulphate-rich region.

Aim: To formulate a guideline on educational objectives and methodology for the implementation of the cross-disciplinary subject "Global Health" in German medical education, on the initiative of the German Medical Students' Association (bvmd Germany). Methods: Coordination: For this purpose, the Globalisation and Health Initiative (GandHI) of bvmd Germany established the "Global Health Curriculum" working group in May 2008. Literature review: Review and assessment of available international literature regarding Global Health in medical education. Development: Multilevel consensus development process from May 2008 to January 2009 by means of workshops, group discussions, teleconferences, and e-mail. Results: 1. Proposals: The bvmd Germany calls for i) the incorporation of the cross-disciplinary subject "Global Health" into the list of mandatory elective courses in Appendix 3 (Art. 2, Para. 8, Clause 2) of the German licensing regulations (ÄApprO 2002), which govern admission to the Second State Examination (Zweiter Abschnitt der Ärztlichen Prüfung); ii) the creation of Global Health course offerings at every medical faculty in Germany and the integration of Global Health into the mandatory elective module of respective medical curricula; iii) the modelling of the Global Health course offerings on the WHO "Health for All" framework, especially with respect to ethics; as well as iv) the adoption of modern approaches of teaching and learning, e.g., group discussions, case studies, problem-based and community-based learning. 2. Educational objectives: The formulation of 15 general educational objectives based on proposals (ii) and (iii). 3. Topics: Three clusters shall formulate specific objectives: Cluster 1, "Politics, Policies and Polity"; Cluster 2, "Individual and Population Health"; and , Zielsetzung: Formulierung einer Leitlinie zu Lernzielen und der praktischen Umsetzung eines disziplinübergreifenden Fachgebiets "Global Health" für die medizinische Ausbildung in Deutschland aus Sicht der Bundesvertretung der Medizinstudierenden (bvmd). Methodik: Expertenauswahl: Initiierung des Arbeitskreises "Global Health Curriculum" innerhalb der Globalisation and Health Initiative (GandHI) der bvmd im Mai 2008. Literaturrecherche: Nicht-systematische Recherche und Bewertung nationaler und internationaler Literatur zu Global Health in der medizinischen Ausbildung. Erstellung: Mehrstufiger Konsensbildungsprozess von Mai 2008 bis Januar 2009 durch Workshops, Arbeitstreffen, Gruppendiskussionen, Telefonkonferenzen und Email-Kommunikation, initiert und koordiniert durch den Arbeitskreis "Global Health Curriculum" der GandHI. Ergebnisse: 1. Positionen : Die bvmd plädiert für i) eine Aufnahme des disziplinübergreifenden Fachgebiets Global Health als Wahlpflichtfach in Anlage 3 (zu § 2 Abs. 8 Satz 2) der ÄApprO 2002 für die Zulassung zum Zweiten Abschnitt der Ärztlichen Prüfung, ii) die Einführung von Lehrangeboten zu Global Health an allen medizinischen Fakultäten sowie die Verankerung dieser Angebote in den medizinischen Curricula als Wahlpflichtfach, iii) eine grundsätzliche Orientierung der Lehrangebote zu Global Health an den medizinischen Fakultäten am "Health for All" Prinzip der Weltgesundheitsorganisation (WHO) als ethisch-moralische Grundlage sowie iv) eine Orientierung an modernen Lehr- und Lernmethoden wie Kleingruppenunterricht, Gruppendiskussionen, problemorientiertem Lernen, Fallbeispielen sowie community-based-learning. 2. Lernziele: Formulierung von 15 allgemeinen Lernzielen, aufbauend auf den Positionen (ii) und (iii). 3. Themenkatalog: Bestehend aus Cluster 1: "Politics, Policies and Polity", Cluster

Researchers working on environmentally relevant organisms, populations, and communities are increasingly turning to the application of OMICS technologies to answer fundamental questions about the natural world, how it changes over time, and how it is influenced by anthropogenic factors. In doing so, the need to capture meta-data that accurately describes the biological “source” material used in such experiments is growing in importance. Here, we provide an overview of the formation of the “Env” community of environmental OMICS researchers and its efforts at considering the meta-data capture needs of those working in environmental OMICS. Specifically, we discuss the development to date of the Env specification, an informal specification including descriptors related to geographic location, environment, organism relationship, and phenotype. We then describe its application to the description of environmental transcriptomic experiments and how we have used it to extend the Minimum Information About a Microarray Experiment (MIAME) data standard to create a domain-specific extension that we have termed MIAME/Env. Finally, we make an open call to the community for participation in the Env Community and its future activities.

The growth plate is a transitional region of cartilage and highly diversified chondrocytes that controls long bone formation. The composition of growth plate cartilage changes markedly from the epiphysis to the metaphysis, notably with the loss of type II collagen, concomitant with an increase in MMP-13; type X collagen; and the C-propeptide of type II collagen. In contrast, the fate of aggrecan in the growth plate is not clear: there is biosynthesis and loss of aggrecan from hypertrophic cartilage, but the mechanism of loss is unknown. All matrix metalloproteinases (MMPs) cleave aggrecan between amino acids N341 and F342 in the proteinase-sensitive interglobular domain (IGD), and MMPs in the growth plate are thought to have a role in aggrecanolysis. We have generated mice with aggrecan resistant to proteolysis by MMPs in the IGD and found that the mice develop normally with no skeletal deformities. The mutant mice do not accumulate aggrecan, and there is no significant compensatory proteolysis occurring at alternate sites in the IGD. Our studies reveal that MMP cleavage in this key region is not a predominant mechanism for removing aggrecan from growth plate cartilage.

Keratan sulfate is thought to influence the cleavage of aggrecan by metalloenzymes. We have therefore produced a recombinant substrate, substituted with keratan sulfate, suitable for the study of aggrecanolysis in vitro. Recombinant human G1-G2 was produced in primary bovine keratocytes using a vaccinia virus expression system. Following purification and digestion with specific hydrolases, fluorophore-assisted carbohydrate electrophoresis was used to confirm the presence of the monosulfated Gal-GlcNAc6S and GlcNAc6s-Gal disaccharides and the disulfated Gal6S-GlcNAc6S disaccharides of keratan sulfate. Negligible amounts of fucose or sialic acid were detected, and the level of unsulfated disaccharides was minimal. Treatment with keratanases reduced the size of the recombinant G1-G2 by approximately 5 kDa on SDS-PAGE. Treatment with N-glycosidase F also reduced the size of G1-G2 by approximately 5 kDa and substantially reduced G1-G2 immunoreactivity with monoclonal antibody 5-D-4, indicating that keratan sulfate on the recombinant protein is N-linked. Cleavage of G1-G2 by aggrecanase was markedly reduced when keratan sulfate chains were removed by treatment with keratanase, keratanase II, endo-beta-galactosidase, or N-glycosidase F. These results indicate that modification of oligosaccharides in the aggrecan interglobular domain with keratan sulfate, most likely at asparagine residue 368, potentiates aggrecanase activity in this part of the core protein.

Two major proteolytic cleavages, one at NITEGE(373)/A(374)RGSVI and the other at VDIPEN(341)/F(342)FGVGG, have been shown to occur in vivo within the interglobular domain of aggrecan. The Glu(373)-Ala(374) site is cleaved in vitro by aggrecanase-1 (ADAMTS4) and aggrecanase-2 (ADAMTS5), whereas the other site, at Asn(341)-Phe(342), is efficiently cleaved by matrix metalloproteinases (MMPs) and by cathepsin B at low pH. Accordingly, the presence of the cleavage products globular domain 1 (G1)-NITEGE(373) and G1-VDIPEN(341) in vivo has been widely interpreted as evidence for the specific involvement of ADAMTS enzymes and MMPs/cathepsin B, respectively, in aggrecan proteolysis in situ. We show here, in digests with native human aggrecan, that purified ADAMTS4 cleaves primarily at the Glu(373)-Ala(374) site, but also, albeit slowly and secondarily, at the Asn(341)-Phe(342) site. Cleavage at the Asn(341)-Phe(342) site in these incubations was due to bona fide ADAMTS4 activity (and not a contaminating MMP) because the cleavage was inhibited by TIMP-3 (a potent inhibitor of ADAMTS4), but not by TIMP-1 and TIMP-2, at concentrations that totally blocked MMP-3-mediated cleavage at this site. Digestion of recombinant human G1-G2 (wild-type and cleavage site mutants) confirmed the dual activity of ADAMTS4 and supported the idea that the enzyme cleaves primarily at the Glu(373)-Ala(374) site and secondarily generates G1-VDIPEN(341) by removal of the Phe(342)-Glu(373) peptide from G1-NITEGE(373). These results show that G1-VDIPEN(341) is a product of both MMP and ADAMTS4 activities and challenge the widely held assumption that this product represents a specific indicator of MMP- or cathepsin B-mediated aggrecan degradation.

We have expressed G1-G2 mutants with amino acid changes at the DIPEN(341) downward arrow(342)FFGVG and ITEGE(373) downward arrow(374)ARGSV cleavage sites, in order to investigate the relationship between matrix metalloproteinase (MMP) and aggrecanase activities in the interglobular domain (IGD) of aggrecan. The mutation DIPEN(341) to DIGSA(341) partially blocked cleavage by MMP-13 and MMP-8 at the MMP site, while the mutation (342)FFGVG to (342)GTRVG completely blocked cleavage at this site by MMP-1, -2, -3, -7, -8, -9, -13, -14. Each of the MMP cleavage site mutants, including a four-amino acid deletion mutant lacking residues ENFF(343), were efficiently cleaved by aggrecanase, suggesting that the primary sequence at the MMP site had no effect on aggrecanase activity in the IGD. The mutation (374)ARGSV to (374)NVYSV completely blocked cleavage at the aggrecanase site by aggrecanase, MMP-8 and atrolysin C but had no effect on the ability of MMP-8 and MMP-13 to cleave at the Asn(341) downward arrowPhe bond. Susceptibility to atrolysin C cleavage at the MMP site was conferred in the DIGSA(341) mutant but absent in the wild-type, (342)GTRVG, (374)NVYSV, and deletion mutants. To further explore the relationship between MMP and aggrecanase activities, sequential digest experiments were done in which MMP degradation products were subsequently digested with aggrecanase and vice versa. Aggrecanase-derived G1 domains with ITEGE(373) C termini were viable substrates for MMPs; however, MMP-derived G2 fragments were resistant to cleavage by aggrecanase. A 10-mer peptide FVDIPENFFG, which is a substrate analogue for the MMP cleavage site, inhibited aggrecanase cleavage at the Glu(373) downward arrowAla bond. This study demonstrates that MMPs and aggrecanase have unique substrate recognition in the IGD of aggrecan and suggests that sequences at the C terminus of the DIPEN(341) G1 domain may be important for regulating aggrecanase cleavage.