TRPM8 is required for cold sensation in mice

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling; however, it is unclear whether either ion channel is required for thermosensation in vivo. We show that mice lacking TRPM8 have severe behavioral deficits in response to cold stimuli. In thermotaxis assays of temperature gradient and two-temperature choice assays, TRPM8-deficient mice exhibit strikingly reduced avoidance of cold temperatures. TRPM8-deficient mice also lack behavioral response to cold-inducing icilin application and display an attenuated response to acetone, an unpleasant cold stimulus. However, TRPM8-deficient mice have normal nociceptive-like responses to subzero centigrade temperatures, suggesting the presence of at least one additional noxious cold receptor. Finally, we show that TRPM8 mediates the analgesic effect of moderate cooling after administration of formalin, a painful stimulus. Therefore, depending on context, TRPM8 contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia.     

Aphid-repellent pheromone E-β-farnesene is generated in transgenic Arabidopsis thaliana over-expressing farnesyl diphosphate synthase2

Background and Aims Plant-synthesized sesquiterpenes play a pivotal role in chemotactic interactions with insects. Biosynthesis of functionally diverse sesquiterpenes is dependent on the availability of a pool of the precursor farnesyldiphosphate (FDP). In Arabidopsis thaliana, FPS2, encoding cytosolic farnesyldiphosphate synthase, is implicated in the synthesis of cytosolic FDP, but it is not known whether enhanced levels of FDP have a commensurate effect on sesquiterpene-mediated defence responses. This study examined transgenic arabidopsis plants generated to over-express FPS2 in order to determine if any effects could be observed in the response of aphids, Myzus persicae.Methods Transgenic arabidopsis plants were generated to over-express FPS2 to produce FPS2 in either the cytosol or the chloroplasts. Morphochemical analyses of the transgenic plants were carried out to detremine growth responses of roots and shoots, and for GC-MS profiling of sesquiterpenes. Aphid response to hyrdo-distillate extracts and head-space volatiles from transgenic plants was assessed using a bioassay.Key Results Either over-expression of FPS2 in the cytosol or targetting of its translated product to chlorplasts resulted in stimulatory growth responses of transgenic arabidopsis at early and late developmental stages. GC-MS analysis of hydro-distillate extracts from aerial parts of the plants revealed biosynthesis of several novel sesquiterpenes, including E-β-farnesene, an alarm pheromone of aphids. Both entrapped volatiles and hydro-distillate extracts of the transgenic leaves triggered agitation in aphids, which was related to both time and dose of exposure.Conclusions Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in arabidopsis led to synthesis of several novel sesquiterpenes, including E-β-farnesene, and induced alarm responses in M. persicae. The results suggest a potential for engineering aphid-resistant strains of arabidopsis.     

Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-μm fragments, compared to 100% of >2-μm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.     

Seasonal variation in in vitro immune activity of milk leukocytes in elite and non-elite crossbred cows of Indian sub-tropical semi-arid climate

Immunity of mammary gland in terms of in vitro activity of milk leukocytes has been evaluated during hot-humid, summer, and winter season in elite (n = 10) and non-elite (n = 10) crossbred cows. Milk samples were collected from all the cows throughout the year at 15-day interval. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were evaluated microscopically. Milk neutrophils, macrophages, and lymphocytes were isolated and cultured in vitro. In vitro PI of milk neutrophils and macrophages was evaluated by colorimetric NBT (nitro-blue tetrazolium) reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (thiazolyl blue tetrazolium bromide) assay. Milk SCC was found to be significantly (p < 0.01) higher in elite cows compared to non-elite cows irrespective of season. There was significant (p < 0.05) increase in milk SCC during hot-humid season compared to winter season in both the group of the cows. There was no significant difference between group and season in terms of DLC. In vitro phagocytic index of elite cows was significantly (p < 0.01) higher than non-elite cows. The phagocytic index was significantly (p < 0.01) decreased in summer and hot-humid season compared to winter season in both the group of animals. Macrophages isolated from elite cows having significantly (p < 0.01) lower phagocytic index than non-elite cows which significantly (p < 0.01) decreased during summer and hot-humid season compared to winter. In vitro milk lymphocyte proliferative response was significantly (p < 0.01) lower in elite cows. Activity of B-lymphocytes decreased significantly (p < 0.01) during summer and hot-humid season than winter, but activity of T-lymphocytes remains unaltered during different seasons. In conclusion, the mammary immunity in terms of in vitro activity of milk leukocytes is compromised during summer and hot-humid season in elite crossbred cows; therefore, better care and management should be taken in high-yielding cows during summer and hot-humid season to minimize intramammary infections.     

The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub

Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-Troglitazone-induced Apoptosis in Prostate Cancer Cells Involve AMP-activated Protein Kinase

Prostate cancer (PCa) is one of the most frequently diagnosed cancers in men with limited treatment options for the hormone-resistant forms. Development of novel therapeutic options is critically needed to target advanced forms. Here we demonstrate that combinatorial treatment with the thiazolidinedione troglitazone (TZD) and TNF-related apoptosis-inducing ligand (TRAIL) can induce significant apoptosis in various PCa cells independent of androgen receptor status. Because TZD is known to activate AMP-activated protein kinase (AMPK), we determined whether AMPK is a molecular target mediating this apoptotic cascade by utilizing PCa cell lines stably overexpressing AMPKα1 dominant negative (C4-2-DN) or empty vector (C4-2-EV). Our results indicated a significantly higher degree of apoptosis with TRAIL-TZD combination in C4-2-EV cells compared with C4-2-DN cells. Similarly, results from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a larger reduction of viability of C4-2-EV cells compared with C4-2-DN cells when treated with TRAIL-TZD, thus suggesting that C4-2-DN cells were more apoptosis-resistant. Additionally, siRNA-mediated knockdown of endogenous AMPKα1 expression showed a reduction of TRAIL-TZD-induced apoptosis, further confirming the participation of AMPK in mediating this apoptosis. Apoptosis induction by this combinatorial treatment was also associated with a cleavage of β-catenin that was inhibited in both C4-2-DN cells and those cells in which AMPKα1 was knocked down. In addition, time course studies showed an increase in pACC^S79 (AMPK target) levels coinciding with the time of apoptosis. These studies indicate the involvement of AMPK in TRAIL-TZD-mediated apoptosis and β-catenin cleavage and suggest the possibility of utilizing AMPK as a therapeutic target in apoptosis-resistant prostate cancer.     

Zinc Inhibits Hedgehog Autoprocessing: LINKING ZINC DEFICIENCY WITH HEDGEHOG ACTIVATION*

Zinc is an essential trace element with wide-ranging biological functions, whereas the Hedgehog (Hh) signaling pathway plays crucial roles in both development and disease. Here we show that there is a mechanistic link between zinc and Hh signaling. The upstream activator of Hh signaling, the Hh ligand, originates from Hh autoprocessing, which converts the Hh precursor protein to the Hh ligand. In an in vitro Hh autoprocessing assay we show that zinc inhibits Hh autoprocessing with a K_i of 2 μm. We then demonstrate that zinc inhibits Hh autoprocessing in a cellular environment with experiments in primary rat astrocyte culture. Solution NMR reveals that zinc binds the active site residues of the Hh autoprocessing domain to inhibit autoprocessing, and isothermal titration calorimetry provided the thermodynamics of the binding. In normal physiology, zinc likely acts as a negative regulator of Hh autoprocessing and inhibits the generation of Hh ligand and Hh signaling. In many diseases, zinc deficiency and elevated level of Hh ligand co-exist, including prostate cancer, lung cancer, ovarian cancer, and autism. Our data suggest a causal relationship between zinc deficiency and the overproduction of Hh ligand.     

The biological basis of degenerative disc disease: proteomic and biomechanical analysis of the canine intervertebral disc

IntroductionIn the present study, we sought to quantify and contrast the secretome and biomechanical properties of the non-chondrodystrophic (NCD) and chondrodystrophic (CD) canine intervertebral disc (IVD) nucleus pulposus (NP).MethodsWe used iTRAQ proteomic methods to quantify the secretome of both CD and NCD NP. Differential levels of proteins detected were further verified using immunohistochemistry, Western blotting, and proteoglycan extraction in order to evaluate the integrity of the small leucine-rich proteoglycans (SLRPs) decorin and biglycan. Additionally, we used robotic biomechanical testing to evaluate the biomechanical properties of spinal motion segments from both CD and NCD canines.ResultsWe detected differential levels of decorin, biglycan, and fibronectin, as well as of other important extracellular matrix (ECM)-related proteins, such as fibromodulin and HAPLN1 in the IVD NP obtained from CD canines compared with NCD canines. The core proteins of the vital SLRPs decorin and biglycan were fragmented in CD NP but were intact in the NP of the NCD animals. CD and NCD vertebral motion segments demonstrated significant differences, with the CD segments having less stiffness and a more varied range of motion.ConclusionsThe CD NP recapitulates key elements of human degenerative disc disease. Our data suggest that at least some of the compromised biomechanical properties of the degenerative disc arise from fibrocartilaginous metaplasia of the NP secondary to fragmentation of SLRP core proteins and associated degenerative changes affecting the ECM. This study demonstrates that the degenerative changes that naturally occur within the CD NP make this animal a valuable animal model with which to study IVD degeneration and potential biological therapeutics.

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