Imaging in vivo neuronal transport in genetic model organisms using microfluidic devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.     

Plasma peptidome profiling of acute hepatitis E patients by MALDI-TOF/TOF

Background  

Hepatitis E is endemic to resource-poor regions, where it manifests as sporadic cases and large waterborne outbreaks. The disease severity ranges from acute self-limited hepatitis with low mortality to fulminant hepatic failure with high mortality. It is believed that the host response plays an important role in determining the progression and outcome of this disease. We profiled the plasma peptidome from hepatitis E patients to discover suitable biomarkers and understand disease pathogenesis.     

The Effects of Salinity on Nitrogen Fixation and Trehalose Metabolism in Mycorrhizal Cajanus cajan (L.) Millsp. Plants

Arbuscular mycorrhizal (AM) fungi exist widely in natural ecosystems as well as in salt-affected soils and are considered suitable candidates for bio-amelioration of saline soils. Plants respond to salinity by accumulating sugars and other low-molecular-weight compatible solutes. One such compound is trehalose, which has been found to play an important role as a stress protectant. The aim of the present investigation was to study interactions between an AM fungus and salinity stress on growth, nitrogen fixation, and trehalose metabolism in Cajanus cajan (L.) Millsp. (pigeonpea). Two genotypes [Sel 85N (salt-tolerant) and ICP 13997 (salt-sensitive)] were subjected to saline treatments with and without mycorrhizal inoculations. Salinity reduced plant biomass (shoot and root) in both genotypes and resulted in a decline in shoot-to-root ratio (SRR); however, a smaller decline was observed in Sel 85N than in ICP 13997. AM colonization was reduced with increasing salinity levels but mycorrhizal responsiveness (MR) increased. Genotypic variability in nitrogen fixation and trehalose metabolism in response to salinity and mycorrhization was observed. An increment in nodule number was accompanied by a reduction in dry mass. Subsequently, nodular activity (leghemoglobin, acetylene-reduction activity [ARA], nitrogen content) was reduced under soil salinity, which was more profound in ICP 13997 than in Sel 85N. The symbiotic association with Glomus mosseae led to significant improvement in plant dry mass and nitrogen-fixing potential of nodules under salt stress. Salinity led to an increase in trehalose-6-P synthetase (TPS) and trehalose-6-P phosphatase (TPP) activities resulting in increased trehalose content in nodules, which was accompanied by inhibition of trehalose catabolism (trehalase activity). AM plants had lower trehalase activity under saline and nonsaline conditions. Thus, a symbiotic relationship between plant roots and G. mosseae might have resulted in salinity tolerance in a genotype-dependent manner.     

Molecular modeling and computational simulation of the photosystem-II reaction center to address isoproturon resistance in Phalaris minor

Isoproturon is the only herbicide that can control Phalaris minor, a competitive weed of wheat that developed resistance in 1992. Resistance against isoproturon was reported to be due to a mutation in the psbA gene that encodes the isoproturon-binding D1 protein. Previously in our laboratory, a triazole derivative of isoproturon (TDI) was synthesized and found to be active against both susceptible and resistant biotypes at 0.5 kg/ha but has shown poor specificity. In the present study, both susceptible D1((S)), resistant D1((R)) and D2 proteins of the PS-II reaction center of P. minor have been modeled and simulated, selecting the crystal structure of PS-II from Thermosynechococcus elongatus (2AXT.pdb) as template. Loop regions were refined, and the complete reaction center D1/D2 was simulated with GROMACS in lipid (1-palmitoyl-2-oleoylglycero-3-phosphoglycerol, POPG) environment along with ligands and cofactor. Both S and R models were energy minimized using steepest decent equilibrated with isotropic pressure coupling and temperature coupling using a Berendsen protocol, and subjected to 1,000 ps of MD simulation. As a result of MD simulation, the best model obtained in lipid environment had five chlorophylls, two plastoquinones, two phenophytins and a bicarbonate ion along with cofactor Fe and oxygen evolving center (OEC). The triazole derivative of isoproturon was used as lead molecule for docking. The best worked out conformation of TDI was chosen for receptor-based de novo ligand design. In silico designed molecules were screened and, as a result, only those molecules that show higher docking and binding energies in comparison to isoproturon and its triazole derivative were proposed for synthesis in order to get more potent, non-resistant and more selective TDI analogs.     

The HDAC9-associated risk locus promotes coronary artery disease by governing TWIST1

Genome wide association studies (GWAS) have identified thousands of single nucleotide polymorphisms (SNPs) associated with the risk of common disorders. However, since the large majority of these risk SNPs reside outside gene-coding regions, GWAS generally provide no information about causal mechanisms regarding the specific gene(s) that are affected or the tissue(s) in which these candidate gene(s) exert their effect. The ‘gold standard’ method for understanding causal genes and their mechanisms of action are laborious basic science studies often involving sophisticated knockin or knockout mouse lines, however, these types of studies are impractical as a high-throughput means to understand the many risk variants that cause complex diseases like coronary artery disease (CAD). As a solution, we developed a streamlined, data-driven informatics pipeline to gain mechanistic insights on complex genetic loci. The pipeline begins by understanding the SNPs in a given locus in terms of their relative location and linkage disequilibrium relationships, and then identifies nearby expression quantitative trait loci (eQTLs) to determine their relative independence and the likely tissues that mediate their disease-causal effects. The pipeline then seeks to understand associations with other disease-relevant genes, disease sub-phenotypes, potential causality (Mendelian randomization), and the regulatory and functional involvement of these genes in gene regulatory co-expression networks (GRNs). Here, we applied this pipeline to understand a cluster of SNPs associated with CAD within and immediately adjacent to the gene encoding HDAC9. Our pipeline demonstrated, and validated, that this locus is causal for CAD by modulation of TWIST1 expression levels in the arterial wall, and by also governing a GRN related to metabolic function in skeletal muscle. Our results reconciled numerous prior studies, and also provided clear evidence that this locus does not govern HDAC9 expression, structure or function. This pipeline should be considered as a powerful and efficient way to understand GWAS risk loci in a manner that better reflects the highly complex nature of genetic risk associated with common disorders.     

A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for <Emphasis Type="Italic">Streptococcus</Emphasis>

The use of rrs (16S rRNA) gene is widely regarded as the “gold standard” for identifying bacteria and determining their phylogenetic relationships. Nevertheless, multiple copies of this gene in a genome is likely to give an overestimation of the bacterial diversity. In each of the 50 Streptococcus genomes (16 species, 50 strains), 4–7 copies of rrs are present. The nucleotide sequences of these rrs genes show high similarity within and among genomes, which did not allow unambiguous identification. A genome-wide search revealed the presence of 27 gene sequences common to all the Streptococcus species. Digestion of these 27 gene sequences with 10 type II restriction endonucleases (REs) showed that unique RE digestion in purH gene is sufficient for clear cut identification of 30 genomes belonging to 16 species. Additional gene-RE combinations allowed identification of another 15 strains belonging to S. pneumoniae, S. pyogenes, and S. suis. For the rest 5 strains, a combination of 2 genes was required for identifying them. The proposed strategy is likely to prove helpful in proper detection of pathogens like Streptococcus.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0561-5) contains supplementary material, which is available to authorized users.     

Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate

An alkalophilic bacterial isolate identified as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 degrees C. The Km (for casein) was 11 mg/ml and Vmax was 380-microg tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 degrees C with a pH and temperature optimum at 8.4 and 40 degrees C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production.     

Mitochondrial Complex III-generated Oxidants Activate ASK1 and JNK to Induce Alveolar Epithelial Cell Death following Exposure to Particulate Matter Air Pollution

We have previously reported that airborne particulate matter air pollution (PM) activates the intrinsic apoptotic pathway in alveolar epithelial cells through a pathway that requires the mitochondrial generation of reactive oxygen species (ROS) and the activation of p53. We sought to examine the source of mitochondrial oxidant production and the molecular links between ROS generation and the activation of p53 in response to PM exposure. Using a mitochondrially targeted ratiometric sensor (Ro-GFP) in cells lacking mitochondrial DNA (ρ⁰ cells) and cells stably expressing a small hairpin RNA directed against the Rieske iron-sulfur protein, we show that site III of the mitochondrial electron transport chain is primarily responsible for fine PM (PM_2.5)-induced oxidant production. In alveolar epithelial cells, the overexpression of SOD1 prevented the PM_2.5-induced ROS generation from the mitochondria and prevented cell death. Infection of mice with an adenovirus encoding SOD1 prevented the PM_2.5-induced death of alveolar epithelial cells and the associated increase in alveolar-capillary permeability. Treatment with PM_2.5 resulted in the ROS-mediated activation of the oxidant-sensitive kinase ASK1 and its downstream kinase JNK. Murine embryonic fibroblasts from ASK1 knock-out mice, alveolar epithelial cells transfected with dominant negative constructs against ASK1, and pharmacologic inhibition of JNK with SP600125 (25 μm) prevented the PM_2.5-induced phosphorylation of p53 and cell death. We conclude that particulate matter air pollution induces the generation of ROS primarily from site III of the mitochondrial electron transport chain and that these ROS activate the intrinsic apoptotic pathway through ASK1, JNK, and p53.Epidemiologic studies have consistently demonstrated a strong link between the daily levels of particulate matter air pollution <2.5 μm in diameter (PM_2.5)³ and PM <10 μmin diameter (PM₁₀) and cardiopulmonary morbidity and mortality (1–3). In humans, exposure to PM₁₀ has been associated with an increase in mortality from ischemic cardiovascular events including stroke and myocardial infarction, an acceleration in the age-related decline in lung function in normal adults, impairment in normal lung development in children, exacerbations of asthma in children and adults, accelerated atherosclerosis in women, increased rates of lung cancer, and the development of myocardial ischemia in men with stable coronary artery disease (4–10). The intracellular generation of reactive oxygen species (ROS) has emerged as a common mechanism by which particulates might initiate signaling pathways that end in these diverse pathologic conditions (11). We have reported that the PM-induced generation of ROS requires a functional electron transport chain, suggesting that PM might induce the inadvertent transfer of electrons from one or more sites in the electron transport chain to molecular oxygen (12).One of the mechanisms by which exposure to PM can contribute to alveolar epithelial dysfunction, lung injury and inflammation, and lung cancer is by activating the intrinsic apoptotic pathway to induce cell death (11, 12). We have reported that this process requires the activation of p53; however, the molecular events linking the generation of ROS by the mitochondrial electron transport chain with the activation of p53 are not known (12). In this paper, we show that exposure of alveolar epithelial cells to PM_2.5 induces the generation of ROS from site III of the mitochondrial electron transport chain. These mitochondrially derived oxidants activate the mitogen-activated signaling kinase kinase kinase (MAPKKK) apoptosis signaling kinase 1 (ASK1), which activates the c-Jun N-terminal kinase (JNK) signaling pathway. The activation of JNK is required for the phosphorylation of p53 and the subsequent cell death. Inhibition of mitochondrial oxidant production in mouse lungs prevents PM_2.5-induced cell death and the associated PM_2.5-induced increase in the permeability of the alveolar-capillary barrier.     

BH3 Peptides Induce Mitochondrial Fission and Cell Death Independent of BAX/BAK

BH3 only proteins trigger cell death by interacting with pro- and anti-apoptotic members of the BCL-2 family of proteins. Here we report that BH3 peptides corresponding to the death domain of BH3-only proteins, which bind all the pro-survival BCL-2 family proteins, induce cell death in the absence of BAX and BAK. The BH3 peptides did not cause the release of cytochrome c from isolated mitochondria or from mitochondria in cells. However, the BH3 peptides did cause a decrease in mitochondrial membrane potential but did not induce the opening of the mitochondrial permeability transition pore. Interestingly, the BH3 peptides induced mitochondria to undergo fission in the absence of BAX and BAK. The binding of BCL-X_L with dynamin-related protein 1 (DRP1), a GTPase known to regulate mitochondrial fission, increased in the presence of BH3 peptides. These results suggest that pro-survival BCL-2 proteins regulate mitochondrial fission and cell death in the absence of BAX and BAK.