Cln5 represents a new type of cysteine-based S-depalmitoylase linked to neurodegeneration

Genetic CLN5 variants are associated with childhood neurodegeneration and Alzheimer’s disease; however, the molecular function of ceroid lipofuscinosis neuronal protein 5 (Cln5) is unknown. We solved the Cln5 crystal structure and identified a region homologous to the catalytic domain of members of the N1pC/P60 superfamily of papain-like enzymes. However, we observed no protease activity for Cln5; and instead, we discovered that Cln5 and structurally related PPPDE1 and PPPDE2 have efficient cysteine palmitoyl thioesterase (S-depalmitoylation) activity using fluorescent substrates. Mutational analysis revealed that the predicted catalytic residues histidine-166 and cysteine-280 are critical for Cln5 thioesterase activity, uncovering a new cysteine-based catalytic mechanism for S-depalmitoylation enzymes. Last, we found that Cln5-deficient neuronal progenitor cells showed reduced thioesterase activity, confirming live cell function of Cln5 in setting S-depalmitoylation levels. Our results provide new insight into the function of Cln5, emphasize the importance of S-depalmitoylation in neuronal homeostasis, and disclose a new, unexpected enzymatic function for the N1pC/P60 superfamily of proteins

Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly From Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay

Matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-TOF MS)-based direct-on-target microdroplet growth assay (DOT-MGA) was recently described as a novel method of phenotypic antimicrobial susceptibility testing (AST). Here, we developed the application of MALDI-TOF MS-based DOT-MGA for Gram-positive bacteria including AST from agar cultures and directly from positive blood cultures (BCs) using the detection of methicillin resistance as example. Consecutively collected, a total of 14 methicillin-resistant Staphylococcus aureus (MRSA) and 14 methicillin-susceptible S. aureus (MSSA) clinical isolates were included. Furthermore, a collection of MRSA challenge strains comprising different SCCmec types, mec genes, and spa types was tested. Blood samples were spiked with MRSA and MSSA and positive BC broth processed by three different methods: serial dilution of BC broth, lysis/centrifugation, and differential centrifugation. Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 μl with and without cefoxitin at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard as a quality control (QC). Spectra were acquired and evaluated using MALDI Biotyper software. First, tests were considered as valid, if the growth control achieved an identification score of ≥1.7. For valid tests, same score criterion was used for resistant isolates when incubated with cefoxitin. An identification score <1.7 after incubation with cefoxitin defined susceptible isolates. On-target protein extraction using formic acid considerably improved detection of methicillin resistance in S. aureus and DOT-MGA showed feasible results for AST from agar cultures after 4 h incubation time. Comparing the different processing methods of positive BC broth, lysis/centrifugation method with a final dilution step 10–1 of the 0.5 McFarland suspension resulted in best test performance after 4 h incubation time. Overall, 96.4% test validity, 100% sensitivity, and 100% specificity were achieved for detection of methicillin resistance in clinical isolates. All strains of the MRSA challenge collection were successfully tested as methicillin-resistant. This first study on Gram-positive organisms showed feasibility and accuracy of MALDI-TOF MS-based DOT-MGA for rapid AST of S. aureus from agar cultures and directly from positive BCs

LIMITES E POSSIBILIDADES NA PARTICIPAÇÃO DE JOGADORAS NO TIME DE FUTEBOL DO CRICIÚMA ESPORTE CLUBE: UMA ANÁLISE DE GÊNERO E DA DIVISÃO SEXUAL DO TRABALHO

A prática do futebol faz parte da cultura brasileira e está inserida, de maneira significativa, nas relações sociais. Segundo a autora Silvana Goellner (2005) o futebol se constituiu como uma profissão de hegemonia masculina, em que a participação das mulheres se deu historicamente e culturalmente num universo repleto de proibições, restrições e lutas. O presente trabalho se constitui como parte da pesquisa de mestrado em desenvolvimento e busca indagar, a partir de uma perspectiva de gênero e da divisão sexual do trabalho, quais são as limitações e possibilidades vivenciadas por mulheres que integram o time de futebol feminino do Criciúma Esporte Clube. Joan Scott (1995, p.86), ao conceituar gênero, define duas proposições: “(1) o gênero é um elemento constitutivo de relações sociais baseadas nas diferenças percebidas entre os sexos e (2) o gênero é uma forma primária de dar significado às relações de poder.” Para Danièle Kergoat (2009, p.67), “[...] a divisão sexual do trabalho tem por características a destinação prioritária dos homens à esfera produtiva e das mulheres à esfera reprodutiva [...]”. Sendo assim, a proposta da pesquisa se justifica pelo fato de acreditar que poderá fomentar ainda mais a reflexão e a análise em torno da exclusão histórica das mulheres em diversos espaços sociais, inclusive nos esportes e mais especificamente no futebol, sendo uma prática esportiva dominada por homens. Por meio da revisão da literatura, foi possível observar certa ausência de estudos sobre o tema da divisão sexual do trabalho no futebol praticado por mulheres. A metodologia será baseada na Etnografia, que prevê a observação participante como um instrumento de grande importância para o método etnográfico (OLIVEIRA, 2000). Além disso, serão realizadas entrevistas semiestruturada, grupo focal e registro em diário de campo. Os sujeitos de pesquisa serão mulheres jogadoras de futebol do Criciúma Esporte Clube, com idades entre 18 a 36 anos. As informações serão analisadas a partir de uma abordagem qualitativa e análise de conteúdo, proposta por Laurence Bardin (2011), com temas emergentes das narrativas das participantes. Para a construção do projeto, foi realizada uma revisão da literatura nas seguintes bases: Biblioteca Digital Brasileira de Dissertações e Teses, Scopus e Scielo. A partir da revisão bibliográfica, cabe ressaltar que comparando a décadas passadas, atualmente foram realizadas muitas construções e conquistas em torno da prática das mulheres no futebol. No entanto, o que se vê é uma aceitação muito mais por força de leis, que não se reflete na ampliação da infraestrutura do futebol feminino no país, ressignificação cultural e social. Ainda, conforme observado na revisão da literatura, torna-se evidente questões envolvendo as resistências das jogadoras diante do preconceito sofrido no futebol, que emerge de lutas das mulheres diante da descontinuidade de investimentos no futebol feminino, identificado ao longo da história

Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels

Background/Aims: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. Methods: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. Results: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. Conclusion: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release

Unification of M- and F- Theory Calabi-Yau Fourfold Vacua

We consider splitting type phase transitions between Calabi-Yau fourfolds. These transitions generalize previously known types of conifold transitions between threefolds. Similar to conifold configurations the singular varieties mediating the transitions between fourfolds connect moduli spaces of different dimensions, describing ground states in M- and F-theory with different numbers of massless modes as well as different numbers of cycles to wrap various p-branes around. The web of Calabi-Yau fourfolds obtained in this way contains the class of all complete intersection manifolds embedded in products of ordinary projective spaces, but extends also to weighted configurations. It follows from this that for some of the fourfold transitions vacua with vanishing superpotential are connected to ground states with nonzero superpotential.Comment: plain TeX, 22p

Conserved and species-specific molecular denominators in mammalian skeletal muscle aging

Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan (young, middle, and old) to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress (as assessed by protein carbonyl adducts and 4-hydroxynonenal) is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process

Filter(n) – Geschichte Ästhetik Praktiken

Prof. Dr. Jens Schröter, Dr. Pablo Abend und Prof. Dr. Benjamin Beil sind Herausgeber der Reihe. Die Herausgeber der einzelnen Hefte sind renommierte WissenschaftlerInnen aus dem In- und Ausland.Bei genauer Betrachtung scheint es, als sei der Filter allgegenwärtig: Von alltäglichen Gebrauchsgegenständen wie z.B. Kaffee-, Aktivkohle- und Pollenfiltern über den Moog Ladder Filter beim Moog Synthesizer, automatisierte Software-Filter im Zuge von Bewerbungsverfahren, bis hin zur 2016 im amerikanischen Präsidentschafts-Wahlkampf viel beschworenen Filter-Bubble oder eben fotografischen Filter-verfahren, wie sie derzeit prominent in einer Reihe von Apps Anwendung finden. Trotz dieser Präsenzen des Filters wurden in der Medien- und Kulturwissenschaft Operationen und Technologien des Filterns bislang weder systematisch noch in ihrer ganzen Bandbreite thematisiert. Ziel des Heftes ist es, hierzu einen Anstoß zu geben und sich mit dem Filter, aber auch mit den Praktiken des Filterns, seiner Geschichte und seinen Ästhetiken zu beschäftigen. Die Beiträge verhandeln dabei ganz unterschiedliche Beispiele: von Wahrnehmungsfiltern über Photoshop und Filtern in der Musik hin zu einer Ökonomie des Filter(n)s und dem Prozess der Verdauung

Conserved and species-specific molecular denominators in mammalian skeletal muscle aging

Aging is a complex phenomenon involving functional decline in multiple physiological systems. We undertook a comparative analysis of skeletal muscle from four different species, i.e. mice, rats, rhesus monkeys, and humans, at three different representative stages during their lifespan (young, middle, and old) to identify pathways that modulate function and healthspan. Gene expression profiling and computational analysis revealed that pathway complexity increases from mice to humans, and as mammals age, there is predominantly an upregulation of pathways in all species. Two downregulated pathways, the electron transport chain and oxidative phosphorylation, were common among all four species in response to aging. Quantitative PCR, biochemical analysis, mitochondrial DNA measurements, and electron microscopy revealed a conserved age-dependent decrease in mitochondrial content, and a reduction in oxidative phosphorylation complexes in monkeys and humans. Western blot analysis of key proteins in mitochondrial biogenesis discovered that (i) an imbalance toward mitochondrial fusion occurs in aged skeletal muscle and (ii) mitophagy is not overtly affected, presumably leading to the observed accumulation of abnormally large, damaged mitochondria with age. Select transcript expression analysis uncovered that the skeletal inflammatory profile differentially increases with age, but is most pronounced in humans, while increased oxidative stress (as assessed by protein carbonyl adducts and 4-hydroxynonenal) is common among all species. Expression studies also found that there is unique dysregulation of the nutrient sensing pathways among the different species with age. The identification of conserved pathways indicates common molecular mechanisms intrinsic to health and lifespan, whereas the recognition of species-specific pathways emphasizes the importance of human studies for devising optimal therapeutic modalities to slow the aging process.This research was funded in part by the Intramural Research Program of the NIA/NIH. E.M.M was supported by a postdoctoral grant of the Research Foundation—Flanders (FWO/12R2415N—http://www.fwo.be/en/). I.W. was supported by the Bundesministerium für Bildung und Forschung (BMBF 01GM1113B; mitoNET—Deutsches Netzwerk für mitochondriale Erkrankungen), by the Cluster of Excellence Frankfurt Macromolecular Complexes at the Goethe University Frankfurt DFG project EXC 115 and by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 815, project Z1 (Redox-Proteomics). And partly by the European Union’s Seventh Framework Programme (FP7) under Grant agreement no.. 223576 (MYOAGE) to C.F. J.M.V. was supported by the Spanish Ministerio de Economía y Competitividad (grants BFU2011-23578 and BFU2015-64630-R). We thank the personnel from the Servicio Centralizado de Apoyo a la Investigación (SCAI, University of Córdoba) for technical support.Peer reviewe