Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection.

NOD2 receptor and the cytosolic protein kinase RIPK2 regulate NF-κB and MAP kinase signaling during bacterial infections, but the role of this immune axis during viral infections has not been addressed. We demonstrate that Nod2(-/-) and Ripk2(-/-) mice are hypersusceptible to infection with influenza A virus. Ripk2(-/-) cells exhibited defective autophagy of mitochondria (mitophagy), leading to enhanced mitochondrial production of superoxide and accumulation of damaged mitochondria, which resulted in greater activation of the NLRP3 inflammasome and production of IL-18. RIPK2 regulated mitophagy in a kinase-dependent manner by phosphorylating the mitophagy inducer ULK1. Accordingly, Ulk1(-/-) cells exhibited enhanced mitochondrial production of superoxide and activation of caspase-1. These results demonstrate a role for NOD2-RIPK2 signaling in protection against virally triggered immunopathology by negatively regulating activation of the NLRP3 inflammasome and production of IL-18 via ULK1-dependent mitophagy.

Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects.

We have prepared mouse fur extensively 13C,15N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13C double quantum-single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and ß-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.

Preparation of highly and generally enriched mammalian tissues for solid state NMR.

An appreciable level of isotope labelling is essential for future NMR structure elucidation of mammalian biomaterials, which are either poorly expressed, or unexpressable, using micro-organisms. We present a detailed protocol for high level (13)C enrichment even in slow turnover murine biomaterials (fur keratin), using a customized diet supplemented with commercial labelled algal hydrolysate and formulated as a gel to minimize wastage, which female mice consumed during pregnancy and lactation. This procedure produced approximately eightfold higher fur keratin labelling in pups, exposed in utero and throughout life to label, than in adults exposed for the same period, showing both the effectiveness, and necessity, of this approach.

Knowledge and Use of, and Attitudes toward, Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Practice: A Survey of Ontario Physiotherapists.

Purpose: To investigate Ontario physiotherapists' knowledge and use of, and attitudes toward, non-steroidal anti-inflammatory drugs (NSAIDs) to identify whether there is a need for physiotherapists to receive education specific to NSAIDs. Method: An existing survey instrument was modified and tested by five Ontario physiotherapists. The final version was distributed electronically to approximately 4,400 Ontario Physiotherapy Association members as a self-administered online questionnaire. Results: A total of 294 physiotherapists responded to the survey (response rate=6.7%). Respondents demonstrated variability in their knowledge of NSAID contraindications, side effects, and drug interactions. Most respondents (62.6%) were incorrect or unsure about where and how to obtain most NSAIDs, and most demonstrated incorrect or uncertain knowledge of the relevant legislation. Despite this lack of knowledge, 50% of respondents recommend NSAIDs to their patients. Conclusions: Many Ontario physiotherapists who participated in this survey recommend NSAIDs to their patients despite having a variable understanding of the legislation and medication-related factors. A lack of thorough knowledge of risks and contraindications has implications for patient safety. Physiotherapists who incorporate medications into their practice should access comprehensive information on appropriate NSAID use and should inform themselves about legislative restrictions to ensure that associated treatment is provided in a manner that is evidence based, safe, and in keeping with regulatory boundaries.

Objectif : enquêter sur les connaissances et l'usage de médicaments anti-inflammatoires non stéroïdiens par les physiothérapeutes de l'Ontario, ainsi que leurs opinions à l'égard de ceux-ci, afin de déterminer si les physiothérapeutes devraient recevoir une formation spécifique sur les anti-inflammatoires. Méthodes : un instrument de sondage existant a été modifié et mis à l'essai par cinq physiothérapeutes ontariens. La version finale a été distribuée électroniquement à environ 4 400 membres de la Ontario Physiotherapy Association. Résultats : 294 physiothérapeutes ont répondu au sondage (taux de réponse=6,7%). Un niveau de connaissance variable a été observé chez les répondants quant aux contre-indications, aux effets secondaires et aux interactions médicamenteuses des anti-inflammatoires. La majorité des répondants (62,6%) avaient une connaissance erronée ou incertaine du mode d'obtention de la plupart des anti-inflammatoires, et la plupart avaient une connaissance erronée ou incertaine des lois régissant leur usage. Malgré ce manque de connaissance, 50% des répondants recommandent des anti-inflammatoires à leurs patients. Conclusions : les physiothérapeutes ontariens qui ont participé au sondage sont nombreux à recommander des anti-inflammatoires à leurs patients malgré un niveau de connaissance variable des lois applicables et de certains facteurs liés aux médicaments. Un manque de connaissance des risques et des contre-indications a des répercussions sur la sécurité des patients. Les physiothérapeutes qui intègrent la médicamentation dans leur pratique devraient se renseigner en profondeur sur l'usage des anti-inflammatoires et les restrictions législatives afin de s'assurer que le traitement associé est dispensé en toute sécurité et conformément aux données probantes et aux lois.

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15. The resulting spectra allowed us to refine an in vitro model of developing bone and to probe its detailed structure. The identification of an unexpected molecule, poly(adenosine diphosphate ribose), that may be implicated in calcification of the bone matrix, illustrates the analytical power of this approach.