Application of fluorescent indicators to analyse intracellular calcium and morphology in filamentous fungi

A novel staining and quantification method to investigate changes in intracellular calcium levels [Ca²⁺]_i and morphology in filamentous fungus is presented. Using a simple protocol, two fluorescent dyes, Fluo-4-AM and Cell trace calcein red-orange-AM were loaded into the filamentous fungus Penicillium chrysogenum. The present study investigates the applicability of using Ca²⁺-sensitive dye to quantify and image [Ca²⁺]_i in P. chrysogenum cultures chosen for its potential as an experimental system to study Ca²⁺ signalling in elicited cultures. The dye loading was optimised and investigated at different pH loading conditions. It was observed that the fluorophore was taken up throughout the hyphae, retaining cell membrane integrity and no dye compartmentalisation within organelles was observed. From the fluorescent plate-reader studies a significant rise (p < 0.001) in the relative fluorescence levels corresponding to [Ca²⁺]_i levels in the hyphae was observed when challenged with an elicitor (mannan oligosaccharide, 150 mg L^?1) which was dependent upon extracellular calcium. Concurrently a novel application of dye-loaded hyphae for morphological analysis was also examined using the imaging software Filament Tracer (Bitplane). Essential quantitative mycelial information including the length and diameter of the segments and number of branch points was obtained using this application based on the three-dimensional data.     

A new anti-depressive strategy for the elderly: ablation of FKBP5/FKBP51

The gene FKBP5 codes for FKBP51, a co-chaperone protein of the Hsp90 complex that increases with age. Through its association with Hsp90, FKBP51 regulates the glucocorticoid receptor (GR). Single nucleotide polymorphisms (SNPs) in the FKBP5 gene associate with increased recurrence of depressive episodes, increased susceptibility to post-traumatic stress disorder, bipolar disorder, attempt of suicide, and major depressive disorder in HIV patients. Variation in one of these SNPs correlates with increased levels of FKBP51. FKBP51 is also increased in HIV patients. Moreover, increases in FKBP51 in the amygdala produce an anxiety phenotype in mice. Therefore, we tested the behavioral consequences of FKBP5 deletion in aged mice. Similar to that of naïve animals treated with classical antidepressants FKBP5−/− mice showed antidepressant behavior without affecting cognition and other basic motor functions. Reduced corticosterone levels following stress accompanied these observed effects on depression. Age-dependent anxiety was also modulated by FKBP5 deletion. Therefore, drug discovery efforts focused on depleting FKBP51 levels may yield novel antidepressant therapies.     

Inosine-containing RNA is a novel innate immune recognition element and reduces RSV infection

During viral infections, single- and double-stranded RNA (ssRNA and dsRNA) are recognized by the host and induce innate immune responses. The cellular enzyme ADAR-1 (adenosine deaminase acting on RNA-1) activation in virally infected cells leads to presence of inosine-containing RNA (Ino-RNA). Here we report that ss-Ino-RNA is a novel viral recognition element. We synthesized unmodified ssRNA and ssRNA that had 6% to16% inosine residues. The results showed that in primary human cells, or in mice, 10% ss-Ino-RNA rapidly and potently induced a significant increase in inflammatory cytokines, such as interferon (IFN)-β (35 fold), tumor necrosis factor (TNF)-α (9.7 fold), and interleukin (IL)-6 (11.3 fold) (p<0.01). Flow cytometry data revealed a corresponding 4-fold increase in influx of neutrophils into the lungs by ss-Ino-RNA treatment. In our in vitro experiments, treatment of epithelial cells with ss-Ino-RNA reduced replication of respiratory syncytial virus (RSV). Interestingly, RNA structural analysis showed that ss-Ino-RNA had increased formation of secondary structures. Our data further revealed that extracellular ss-Ino-RNA was taken up by scavenger receptor class-A (SR-A) which activated downstream MAP Kinase pathways through Toll-like receptor 3 (TLR3) and dsRNA-activated protein kinase (PKR). Our data suggests that ss-Ino-RNA is an as yet undescribed virus-associated innate immune stimulus.     

Mutations in Drosophila myosin rod cause defects in myofibril assembly

The roles of myosin during muscle contraction are well studied, but how different domains of this protein are involved in myofibril assembly in vivo is far less understood. The indirect flight muscles (IFMs) of Drosophila melanogaster provide a good model for understanding muscle development and function in vivo. We show that two missense mutations in the rod region of the myosin heavy-chain gene, Mhc, give rise to IFM defects and abnormal myofibrils. These defects likely result from thick filament abnormalities that manifest during early sarcomere development or later by hypercontraction. The thick filament defects are accompanied by marked reduction in accumulation of flightin, a myosin binding protein, and its phosphorylated forms, which are required to stabilise thick filaments. We investigated with purified rod fragments whether the mutations affect the coiled-coil structure, rod aggregate size or rod stability. No significant changes in these parameters were detected, except for rod thermodynamic stability in one mutation. Molecular dynamics simulations suggest that these mutations may produce localised rod instabilities. We conclude that the aberrant myofibrils are a result of thick filament defects, but that these in vivo effects cannot be detected in vitro using the biophysical techniques employed. The in vivo investigation of these mutant phenotypes in IFM development and function provides a useful platform for studying myosin rod and thick filament formation generically, with application to the aetiology of human myosin rod myopathies.     

Time-dependent predominance of nonhomologous DNA end-joining pathways during embryonic development in mice

Repair of DNA double-strand breaks (DSBs) is crucial for maintaining genomic integrity during the successful development of a fertilized egg into a whole organism. To date, the mechanism of DSB repair in postimplantation embryos has been largely unknown. In the present study, using a cell-free repair system derived from the different embryonic stages of mice, we find that canonical nonhomologous end joining (NHEJ), one of the major DSB repair pathways in mammals, is predominant at 14.5 day of embryonic development. Interestingly, all four types of DSBs tested were repaired by ligase IV/XRCC4 and Ku-dependent classical NHEJ. Characterization of end-joined junctions and expression studies further showed evidences for canonical NHEJ. Strikingly, in contrast to the above, we observed noncanonical end joining accompanied by DSB resection, dependent on microhomology and ligase III in 18.5-day embryos. Interestingly, we observed an elevated expression of CtIP, MRE11, and NBS1 at this stage, suggesting that it could act as a switch between classical end joining and microhomology-mediated end joining at later stages of embryonic development. Thus, our results establish for the first time the existence of both canonical and alternative NHEJ pathways during the postimplantation stages of mammalian embryonic development.     

Construction of amperometric uric acid biosensor based on uricase immobilized on PBNPs/cMWCNT/PANI/Au composite

A chitosan-glutaraldehyde crosslinked uricase was immobilized onto Prussian blue nanoparticles (PBNPs) absorbed onto carboxylated multiwalled carbon nanotube (c-MWCNT) and polyaniline (PANI) layer, electrochemically deposited on the surface of Au electrode. The nanohybrid-uricase electrode was characterized by scanning electron microscopic (SEM), Fourier transform infrared spectroscopy (FTIR) and cyclic voltammetry. An amperometric uric acid biosensor was fabricated using uricase/c-MWCNT/PBNPs/Au electrode as working electrode, Ag/AgCl as standard and Pt wire as auxiliary electrode connected through a potentiostat. The biosensor showed optimum response within 4 s at pH 7.5 and 40 °C, when operated at 0.4 V vs. Ag/AgCl. The linear working range for uric acid was 0.005-0.8 mM, with a detection limit of 5 μM. The sensor was evaluated with 96% recovery of added uric acid in sera and 4.6 and 5.4% within and between batch of coefficient of variation respectively and a good correlation (r = 0.99) with standard enzymic colorimetric method. This sensor measured uric acid in real serum samples. The sensor lost only 37% of its initial activity after its 400 uses over a period of 7 months, when stored at 4 °C.     

Identification of SSR Markers for Salt-tolerance in Rice Variety CSR10 by Selective Genotyping

A population of 171 F3 genotypes derived from a cross between CSR10 (salt tolerant, indica) and Taraori Basmati (HBC19) was evaluated for various salt-tolerance attributes at vegetative stage using a hydroponic culture system. Substantial variation was observed in F₃ population for relative growth rate (range 0.065–0.187), Na-K ratio (0.023–0.376) and visual injury symptoms (score 1–9). The mean individual score of CSR10 × HBC19 F₃ plants ranged from 1.7 to 9.0 with mean value of 5.07. Seven of the F₃ plants showed transgressive segregation for salt tolerance. F₃ individuals at both extremes of the response distribution were selected and genotyped using 30 SSR markers displaying polymorphism between the two parental genotypes. As many as 18/30 SSR markers showed distorted segregation ratios among the 30 selected salt-tolerant and salt-sensitive CSR10 × HBC19 F3 plants. Linear regression analysis showed significant association of three markers (RM162 mapped on chromosome 6, and RM209 and RM287 on chromosome 11) with relative growth rate and two markers (RM212 on chromosome 1 and RM206 on chromosome 11) with Na-K ratio explaining 31.3% and 25.6% of phenotypic variation, respectively.