Identification of CNS Injury-Related microRNAs as Novel Toll-Like Receptor 7/8 Signaling Activators by Small RNA Sequencing

Toll-like receptors (TLRs) belong to pattern recognition receptors, which respond to danger signals such as pathogen-associated molecular patterns or damage-associated molecular patterns. Upon TLR activation in microglia, the major immune cells in the brain, distinct signaling cascades trigger the production of inflammatory molecules, being a critical feature in neuroinflammation and neurodegenerative processes. Recently, individual microRNAs (miRNAs) were shown to act as endogenous TLR ligands. Here, we conducted systematic screening for miRNAs as potential TLR7/8 ligands by small RNA sequencing of apoptotic neurons and their corresponding supernatants. Several miRNA species were identified in both supernatants and injured neurons, and 83.3% of the media-enriched miRNAs activated murine and/or human TLR7/8 expressed in HEK293-derived TLR reporter cells. Among the detected extracellular miRNAs, distinct miRNAs such as miR-340-3p and miR-132-5p induced cytokine and chemokine release from microglia and triggered neurotoxicity in vitro. Taken together, our systematic study establishes miRNAs released from injured neurons as new TLR7/8 activators, which contribute to inflammatory and neurodegenerative responses in the central nervous system (CNS)

Problem-Based Learning in Mathematics

A teacher of mathematics has a great opportunity. If he fills his allotted time with drilling his students in routine operations, he kills their interest, hampers their intellectual development, and misuses his opportunity. But if he challenges the curiosity of his students by setting them problems proportionate to their knowledge and helps them to solve their problems with stimulating questions, he may give them a taste for, and some independent means of, independent thinking

Recent Deformation in the Bottom Sediments of Western and Southeastern Lake Ontario and its Association with Major Structures and Seismicity

Geophysical surveys, undertaken in the Toronto-Burlington corridor of western Lake Ontario and in the Rochester Basin of southeastern Lake Ontario, revealed the presence of features affecting the young lake-bottom sediments. In the western part of the lake, they include inferred pop-ups in bedrock, and plumose structures, dark linear patterns, and linear belts of circular to elliptical signatures in the modern mud. In southeastern Lake Ontario the glacial and post-glacial sediments display vertical separations of on the order of 10-15 m. Pop-ups are tectonically-induced structures. The features in the modern mud commonly parallel the orientation of P-stresses measured in Paleozoic rocks nearby and, along with the pop-ups, are spatially related to an aeromagnetic lineament. Furthermore, all of these features occur within a seismically active belt. The vertical displacements of the layered glacial and post-glacial sediments, within the Rochester Basin, are located along the southern margin of the postulated WSW extension of the seismically active St. Lawrence rift system and are interpreted to be due to faulting. The geologically young age of the sediments affected by the various deformational features, along with the characteristics of the features themselves, suggest that the lake-bottom sediments surveyed in this study may have recorded the effects of neotectonic processes.Des levés géophysiques effectués dans le corridor Toronto-Burlington, dans l'ouest du lac Ontario, a révélé la présence d'éléments qui altèrent les jeunes sédiments lacustres du fond. Dans la partie ouest du lac, dans la roche en place, il s'agit de structures de soulèvement (pop-ups) et, dans les boues récentes, de structures plumeuses, de réseaux de traits sombres et de zones linéaires de tracés circulaires à elliptiques. Dans la partie sud-est du lac Ontario, il y a dans les sédiments glaciaires et postglaciaires des rejets verticaux de l'ordre de 10 à 15 m. Les structures de soulèvement sont d'origine tectonique. Les formes dans les boues récentes, généralement parallèles à l'orientation des contraintes de compression mesurées dans les roches paléozoïques voisines, sont spatialement reliés, comme les structures de soulèvement, à un linéament aéromagnétique. De plus, toutes ces formes se trouvent dans une zone sismique active. Les rejets verticaux dans les sédiments glaciaires et postglaciaires stratifiés, à l'intérieur du basssin de Rochester, sont localisés le long de la bordure sud du prolongement présumé WSW du système actif du rift du Saint-Laurent et sont probablement attribuables à la formation de failles. La jeunesse des sédiments altérés par les déformations et les caractéristiques des déformations mêmes laissent croire que ces sédiments ont probablement enregistré les effets de processus néotectoniques.Geophysikalische Vermessungen, die im Toronto Burlington-Korridor des westlichen Ontariosees und im Rochester-Becken des sùdôstlichen Ontariosees durchgefùhrt wurden, deckten die Anwesenheit von Elementen auf, welche auf die jungen Seegrundsedimente einwirken. Im westlichen Teil des Sees bestehen sie aus Hebungen im anste-henden Gestein und im modernen Schlamm aus federartigen Strukturen, dunklen linearen Mustern und linearen Gùrteln mit kreisformigen bis ellipsenfôrmigen Umrissen. Im sùdôstlichen Ontariosee gibt es in den glazialen und postglazialen Sedimenten verti-kale Verwùrfe der GrôRenordnung von 10-15 m. Die Hebungen sind tektonischen Ursprungs. Die Formen im modernen Schlamm liegen im allgemeinen paralell zu der Orientierung der in den benachbarten palàozoischen Felsen gemessenen P-Stresse und sind zusammen mit den Hebungen ràumlich mit einem aeromagnetischen Lineament verbunden. AuRerdem treten aile dièse Formen innerhalb eines seismisch aktiven Gùrtels auf. Die vertikalen Verstellungen der geschichteten glazialen und postglazialen Sedimente innerhalb des Rochester-Beckens werden entlang des sùdlichen Rands der angenommenen WSW-Verlàngerung des seismisch aktiven Sankt-Lorenz-Spaltensystems lokalisiert. Das geologisch relativ junge Alter der durch die verschiedenen Verformungen Iàf3t vermu-ten, daB die in dieser Studie gemessenen Seegrundsedimente moglicherweise die Wirkungen neotektonischer Prozesse aufgezeichnet haben

Interaction of Circadian Clock Proteins CRY1 and PER2 Is Modulated by Zinc Binding and Disulfide Bond Formation

SummaryPeriod (PER) proteins are essential components of the mammalian circadian clock. They form complexes with cryptochromes (CRY), which negatively regulate CLOCK/BMAL1-dependent transactivation of clock and clock-controlled genes. To define the roles of mammalian CRY/PER complexes in the circadian clock, we have determined the crystal structure of a complex comprising the photolyase homology region of mouse CRY1 (mCRY1) and a C-terminal mouse PER2 (mPER2) fragment. mPER2 winds around the helical mCRY1 domain covering the binding sites of FBXL3 and CLOCK/BMAL1, but not the FAD binding pocket. Our structure revealed an unexpected zinc ion in one interface, which stabilizes mCRY1-mPER2 interactions in vivo. We provide evidence that mCRY1/mPER2 complex formation is modulated by an interplay of zinc binding and mCRY1 disulfide bond formation, which may be influenced by the redox state of the cell. Our studies may allow for the development of circadian and metabolic modulators

Syntheses, analytical and pharmacological characterizations of the “legal high” 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo) and analogues

New psychoactive substances (NPS) are commonly referred to as “research chemicals”, “designer drugs” or “legal highs.” One NPS class is represented by dissociative anesthetics, which include analogues of the arylcyclohexylamine phencyclidine (PCP), ketamine, and diphenidine. A recent addition to the NPS market was 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo), a morpholine analogue of 3-MeO-PCP. Although suspected to have dissociative effects in users, information about its pharmacological profile is not available. From clinical and forensic perspectives, detailed analytical data are needed for identification, especially when facing the presence of positional isomers, as these are frequently unavailable commercially. This study presents the analytical and pharmacological characterization of 3-MeO-PCMo along with five additional analogues including the 2- and 4-MeO- isomers, 3,4-methylenedioxy-PCMo (3,4-MD-PCMo), 3-Me-PCMo and PCMo. All six arylcyclohexylmorpholines were synthesized and characterized by chromatographic, mass spectrometric and spectroscopic techniques. The three positional isomers could be differentiated and the identity of 3-MeO-PCMo obtained from an internet vendor was verified. All six compounds were also evaluated for affinity at 46 central nervous system receptors including the N-methyl-D-aspartate receptor (NMDAR), an important target for dissociative anesthetics such as PCP and ketamine. In vitro binding studies using [3H]-MK-801 in rat forebrain preparations revealed moderate affinity for NMDAR in the rank order of 3-Me > 3-MeO >PCMo > 3,4-MD > 2-MeO > 4-MeO-PCMo. 3-MeO-PCMo was found to have moderate affinity for NMDAR comparable to that of ketamine, and had an approximate 12-fold lower affinity than PCP. These results support the anecdotal reports of dissociative effects from 3-MeO-PCMo in humans

The circadian clock regulates rhythmic erythropoietin expression in the murine kidney

Generation of circadian rhythms is cell-autonomous and relies on a transcription/translation feedback loop controlled by a family of circadian clock transcription factor activators including CLOCK, BMAL1 and repressors such as CRY1 and CRY2. The aim of the present study was to examine both the molecular mechanism and the hemopoietic implication of circadian erythropoietin expression. Mutant mice with homozygous deletion of the core circadian clock genes cryptochromes 1 and 2 (Cry-null) were used to elucidate circadian erythropoietin regulation. Wild-type control mice exhibited a significant difference in kidney erythropoietin mRNA expression between circadian times 06 and 18. In parallel, a significantly higher number of erythropoietin-producing cells in the kidney (by RNAscope®) and significantly higher levels of circulating erythropoietin protein (by ELISA) were detected at circadian time 18. Such changes were abolished in Cry-null mice and were independent from oxygen tension, oxygen saturation, or expression of hypoxia-inducible factor 2 alpha, indicating that circadian erythropoietin expression is transcriptionally regulated by CRY1 and CRY2. Reporter gene assays showed that the CLOCK/BMAL1 heterodimer activated an E-box element in the 5' erythropoietin promoter. RNAscope® in situ hybridization confirmed the presence of Bmal1 in erythropoietin-producing cells of the kidney. In Cry-null mice, a significantly reduced number of reticulocytes was found while erythrocyte numbers and hematocrit were unchanged. Thus, circadian erythropoietin regulation in the normoxic adult murine kidney is transcriptionally controlled by master circadian activators CLOCK/BMAL1, and repressors CRY1/CRY2. These findings may have implications for kidney physiology and disease, laboratory diagnostics, and anemia therapy

Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity

In all organisms with circadian clocks, post-translational modifications of clock proteins control the dynamics of circadian rhythms, with phosphorylation playing a dominant role. All major clock proteins are highly phosphorylated, and many kinases have been described to be responsible. In contrast, it is largely unclear whether and to what extent their counterparts, the phosphatases, play an equally crucial role. To investigate this, we performed a systematic RNAi screen in human cells and identified protein phosphatase 4 (PPP4) with its regulatory subunit PPP4R2 as critical components of the circadian system in both mammals an

Some discretizations of geometric evolution equations and the Ricci iteration on the space of Kahler metrics, I

In this article and in its sequel we propose the study of certain discretizations of geometric evolution equations as an approach to the study of the existence problem of some elliptic partial differential equations of a geometric nature as well as a means to obtain interesting dynamics on certain infinite-dimensional spaces. We illustrate the fruitfulness of this approach in the context of the Ricci flow, as well as another flow, in Kahler geometry. We introduce and study dynamical systems related to the Ricci operator on the space of Kahler metrics that arise as discretizations of these flows. We pose some problems regarding their dynamics. We point out a number of applications to well-studied objects in Kahler and conformal geometry such as constant scalar curvature metrics, Kahler-Ricci solitons, Nadel-type multiplier ideal sheaves, balanced metrics, the Moser-Trudinger-Onofri inequality, energy functionals and the geometry and structure of the space of Kahler metrics. E.g., we obtain a new sharp inequality strengthening the classical Moser-Trudinger-Onofri inequality on the two-sphere

Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-κB (NF-κB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8 mice), which is a deubiquitinating enzyme that is integral to NF-κB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occurs in this strain. The B lymphocytes themselves exhibit prolonged survival and manifest a variety of signaling disarrangements that do not occur in mice with a complete deletion of CYLD. Although both the full-length and the mutant CYLD are able to interact with Bcl-3, a predominant nuclear accumulation of Bcl-3 occurs in the CYLD mutant B cells. More dramatic, however, is the accumulation of the NF-κB proteins p100 and RelB in CYLDex7/8 B cells, which, presumably in combination with nuclear Bcl-3, results in increased levels of Bcl-2 expression. These findings suggest that CYLD can both positively and negatively regulate signal transduction and homeostasis of B cells in vivo, depending on the expression of CYLD splice variants