A novel mechanism of autophagic cell death in dystrophic muscle regulated by P2RX7 receptor large-pore formation and HSP90

P2RX7 is an ATP-gated ion channel, which can also exhibit an open state with a considerably wider permeation. However, the functional significance of the movement of molecules through the large pore (LP) and the intracellular signaling events involved are not known. Here, analyzing the consequences of P2RX7 activation in primary myoblasts and myotubes from the Dmd^mdx mouse model of Duchenne muscular dystrophy, we found ATP-induced P2RX7-dependent autophagic flux, leading to CASP3-CASP7-independent cell death. P2RX7-evoked autophagy was triggered by LP formation but not Ca²⁺ influx or MAPK1-MAPK3 phosphorylation, 2 canonical P2RX7-evoked signals. Phosphoproteomics, protein expression inference and signaling pathway prediction analysis of P2RX7 signaling mediators pointed to HSPA2 and HSP90 proteins. Indeed, specific HSP90 inhibitors prevented LP formation, LC3-II accumulation, and cell death in myoblasts and myotubes but not in macrophages. Pharmacological blockade or genetic ablation of p2rx7 also proved protective against ATP-induced death of muscle cells, as did inhibition of autophagy with 3-MA. The functional significance of the P2RX7 LP is one of the great unknowns of purinergic signaling. Our data demonstrate a novel outcome—autophagy—and show that molecules entering through the LP can be targeted to phagophores. Moreover, we show that in muscles but not in macrophages, autophagy is needed for the formation of this LP. Given that P2RX7-dependent LP and HSP90 are critically interacting in the ATP-evoked autophagic death of dystrophic muscles, treatments targeting this axis could be of therapeutic benefit in this debilitating and incurable form of muscular dystrophy.     

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

A Comparative Quantitative Assessment of Axonal and Dendritic mRNA Transport in Maturing Hippocampal Neurons

Translation of mRNA in axons and dendrites enables a rapid supply of proteins to specific sites of localization within the neuron. Distinct mRNA-containing cargoes, including granules and mitochondrial mRNA, are transported within neuronal projections. The distributions of these cargoes appear to change during neuronal development, but details on the dynamics of mRNA transport during these transitions remain to be elucidated. For this study, we have developed imaging and image processing methods to quantify several transport parameters that can define the dynamics of RNA transport and localization. Using these methods, we characterized the transport of mitochondrial and non-mitochondrial mRNA in differentiated axons and dendrites of cultured hippocampal neurons varying in developmental maturity. Our results suggest differences in the transport profiles of mitochondrial and non-mitochondrial mRNA, and differences in transport parameters at different time points, and between axons and dendrites. Furthermore, within the non-mitochondrial mRNA pool, we observed two distinct populations that differed in their fluorescence intensity and velocity. The net axonal velocity of the brighter pool was highest at day 7 (0.002±0.001 µm/s, mean ± SEM), raising the possibility of a presynaptic requirement for mRNA during early stages of synapse formation. In contrast, the net dendritic velocity of the brighter pool increased steadily as neurons matured, with a significant difference between day 12 (0.0013±0.0006 µm/s ) and day 4 (−0.003±0.001 µm/s) suggesting a postsynaptic role for mRNAs in more mature neurons. The dim population showed similar trends, though velocities were two orders of magnitude higher than of the bright particles. This study provides a baseline for further studies on mRNA transport, and has important implications for the regulation of neuronal plasticity during neuronal development and in response to neuronal injury.     

Facultative response to a kleptoparasite by the cooperatively breeding pied babbler

In many cases of interspecific kleptoparasitism, hosts developdefensive behaviors to minimize the impact of kleptoparasites.Because vigilance and defensive behaviors are often costly,selection should favor hosts that adjust the amount of investmentneeded to minimize losses to kleptoparasitism. However, examplesof such facultative responses are rare. Here, we investigatethe response of cooperatively breeding pied babblers (Turdoidesbicolor) to the drongo (Dicrurus adsimilis), an avian kleptoparasitethat regularly follows pied babbler groups, often giving alarmcalls to alert the group to predators but also occasionallygiving false alarm calls in order to steal food items. We showthat pied babbler response to drongos varies markedly accordingto babbler group size. In small groups, where there are fewindividuals available to act as sentinels, babblers sentinelless when a drongo is present and respond strongly to drongoalarm calls. However, in large groups, where there are manyindividuals available to participate in predator vigilance,babblers sentinel more often when a drongo is present, rarelyrespond to drongo alarm calls, and aggressively displace drongos,with a consequent decline in the number of successful kleptoparasitismevents. This behavior represent a facultative response to akleptoparasite according to the costs versus benefits of toleratingtheir presence.     

Mannose-binding lectin deficiency is associated with early onset of polyarticular juvenile rheumatoid arthritis: a cohort study

Background  

Mannose-binding lectin (MBL) is an innate immune protein. The aim of our study was to determine whether genetically determined MBL deficiency is associated with susceptibility to juvenile rheumatoid arthritis (JRA) and whether MBL2 genotypes are associated with JRA severity.     

First report of metazoan parasites from the cichlid Pseudocrenilabrus philander and the cyprinid Enteromius paludinosus in a South African Ramsar wetland

Parasites of two small fish species from a Ramsar wetland in South Africa were studied in 2014–2015. The cichlid Pseudocrenilabrus philander (Weber, 1897) was parasitised by the copepod Lernaea cyprinacea Linnaeus, 1758, the monogenean Gyrodactylus thlapi Christison, Shinn & van As, 2005 and four gryporhynchid metacestode (Cyclophyllidea) species: Paradilepis scolecina (Rudolph, 1819), Paradilepis maleki Khalil, 1961, Neogryporhynchus lasiopeius Baer & Bona, 1960 and Valipora campylancristrota (Wedl, 1855). The cyprinid Enteromius paludinosus (syn. Barbus paludinosus) (Peters, 1852) was infected with the monogenean parasites Dogielius intorquens Crafford, Luus-Powell & Avenant-Oldewage, 2012, Dactylogyrus teresae Mashego, 1983, and three Dactylogyrus spp. These results represent several new locality as well as host records and further contribute information on the parasitic diversity in the Barberspan Ramsar wetland.     

IMPROVEMENT OF MICROBIAL STRAIN AND FERMENTATION PROCESS OF RAPAMYCIN BIOSYNTHESIS

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N′-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.     

Polycystic ovary syndrome in Indian women: a mass spectrometry based serum metabolomics approach

Introduction

Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrinological disorder with uncertain pathogenesis and is very common in women of reproductive age. There are few reports of utilizing metabolomics approach to understand the complex pathophysiology of PCOS. However, excluding one previous NMR-based metabolomics study, none of the study was conducted in Indian population.

Objective

The study aims to compare the serum metabolomic profile of PCOS women with controls from the Eastern region of India.

Methods

PCOS women (n?=?35) and healthy control women (n?=?30) undergoing tubal ligation were recruited for this study. Serum metabolic profiles were generated using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS). Multivariate statistical analysis was applied to spectral data obtained from both the LC-MS/MS and GC/MS.

Results

Nine metabolites were identified to be most significantly dysregulated in sera of PCOS women; however, few other identified metabolites were also altered but with lesser significance. Amongst these metabolites, riboflavin, sucrose, adenine and N-acetyl glycine, phosphoric acid and cortisol were down-regulated, whereas, thymine, cystathionine, and phenylalanine were up-regulated in PCOS when compared with controls. The observed changes in metabolite expression suggested alterations in aminoacyl-tRNA biosynthesis, metabolism of nitrogen, alanine-aspartate-glutamate, galactose, glycine-serine-threonine, and pyrimidine-purine among several metabolic pathways possibly implicated in these PCOS women.

Conclusion

The altered metabolites identified in PCOS women of Eastern Indian population, provide insight into current perceptive of the disease pathology, metabolic involvements, and may be considered as putative markers of PCOS.