Plant regeneration through direct and indirect organogenesis,phyto-molecular profiles,antioxidant properties and swertiamarin production in elicitated cell suspension cultures of Swertia minor (Griseb.) Knobl

Plant Cell, Tissue and Organ Culture (PCTOC) - The present study reports an optimized protocol for high frequency in vitro plant propagation through direct and indirect organogenesis, phytochemical...     

Activation of the pyrin inflammasome by intracellular Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP-pyrin and ASC (YFP-ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.     

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.     

Effect of chlortetracycline supplementation during prebreeding and early gestation on age at puberty, ovulation rate, embryo survival and fetal development in gilts

A total of 166 crossbred gilts weighing approximately 87 +/- 1 kg was limit-fed (2.5 kg/d) a corn-soybean meal gestation diet containing either 0 or 220 ppm of chlortetracycline (CTC) from 157 +/- 1 d of age until 15 d after breeding. These gilts were slaughtered at 31 +/- 1 or 71 d +/- 1 d of gestation for evaluation of reproductive performance. Age (190 +/- 3 d vs 195 +/- 3 d) and body weights (106 +/- 2 kg vs 106 +/- 2 kg) at puberty were similar for control and CTC-fed gilts, respectively. Although not significant (P > 0.05), ovulation rate was higher in CTC-fed than in control gilts as assessed at both 31 d (14.2 +/- 0.7 vs 12.9 +/- 0.9, P = 0.31) and 71 d (13.9 +/- 0.6 vs 12.4 +/- 0.5, P = 0.10) of gestation. There was an increase (P = 0.04) in the number of live embryos for CTC-fed gilts at 31 d (12.1 +/- 0.7 vs 9.7 +/- 0.7) but not at 71 d (10.0 +/- 1.1 vs 9.6 +/- 1.0) of gestation. The mean uterine length, placental length, placental weight, fetal length, fetal weight, and allantoic fluid volumes were similar between the control and CTC-fed gilts. Results indicated that feeding CTC during prebreeding and early gestation did not influence the proportion or age of gilts at puberty. However, CTC feeding may have influenced a trend to-ward an increased ovulation rate and increased number of live embryos in gilts.     

Apoptosis-associated speck-like protein (ASC) controls Legionella pneumophila infection in human monocytes

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.     

Scale-up of an alkaline protease from Bacillus pumilus MTCC 7514 utilizing fish meal as a sole source of nutrients

Fish meal grades SL1 and SL2 from Sardine (Sardinella longiceps) and NJ from Pink Perch (Nemipterus japonicas) were evaluated as a sole source of carbon and nitrogen in the medium for alkaline protease production by Bacillus pumilus MTCC 7514. The analysis of the fish meal suggests that the carbon and nitrogen contents in fish meal are sufficient to justify its choice as replacement for other nutrients. Protease production increased significantly (4,914 U/ml) in medium containing only fish meal, compared with the basal medium (2,646 U/ml). However, the elimination of inorganic salts from media reduced the protease productivity. In addition, all the three grades of fish meal yielded almost the same amounts of protease when employed as the sole source of carbon and nitrogen. Nevertheless, the best results were observed in fish meal SL1 medium. Furthermore, protease production was enhanced to 6,966 U/ml and 7,047 U/ml on scaling up from flask (4,914 U/ml) to 3.7 and 20 L fermenters, respectively, using fish meal (10 g/l). Similarly, the corresponding improvement in productivities over flask (102.38 U/ml/h) was 193.5 and 195.75 U/ml/h in 3.7 and 20 L fermenters, respectively. The crude protease was found to have dehairing ability in leather processing, which is bound to have great environmental benefits.     

Scanning conductance microscopy investigations on fixed human chromosomes

Scanning conductance microscopy investigations were carried out in air on human chromosomes fixed on pre-fabricated SiO2 surfaces with a backgate. The point of the investigation was to estimate the dielectric constant of fixed human chromosomes in order to use it for microfluidic device optimization. The phase shift caused by the electrostatic forces, together with geometrical measurements of the atomic force microscopy (AFM) cantilever and the chromosomes were used to estimate a value for the dielectric constant of different human chromosomes.     

Association of rasGAPSH3 binding protein 1, G3BP1, and rasGap120 with integrin containing complexes induced by an adhesion blocking antibody

The adhesion blocking antibody 3S3 was used to probe the regulation of alpha5beta1 integrin mediated adhesion in K562 cells. This antibody prevented cellular adherence but it did not interfere with ligand binding by cells or purified integrin. Interaction with 3S3 induced change in the cytoskeletal organization resulting in extensive filopodia formation. The antibody also prevented ligand and anti-integrin antibody induced phosphorylation of FAK in a trans acting fashion. MS based analysis of 3S3 induced integrin containing complexes identified rasGAP SH3 binding protein 1, G3BP1, as a component of these structures. The G3BP1 binding molecule, rasGap120, was also identified in the complexes. Microscopic examination confirmed the recruitment of a component of cellular G3BP1 and rasGap120 pools to sites of integrin cross-linking. G3BP1 was also observed in the 3S3 induced filopodia. In untreated cells, G3BP1 was shown to associate with submembranous regions involved in cellular polarization. Collectively, these results suggest that G3BP1 and rasGap120 can be recruited to sites of integrin ligation where they may play a role in cytoskeletal reorganization. Such changes may result in reduced adhesive potential and account for the 3S3 effects on cellular adhesion. It should be emphasized that these results do not necessarily indicate a direct interaction of integrin with G3BP1 and rasGap120.     

Proteomics analysis of human coronary atherosclerotic plaque: a feasibility study of direct tissue proteomics by liquid chromatography and tandem mass spectrometry

Cardiovascular disease presents significant variations in human populations with respect to the atherosclerotic plaque progression, inflammation, thrombosis, and rupture. To gain a more comprehensive picture of the pathogenic mechanism of atherosclerosis and the variations seen in patients, efficient methods to identify proteins from the normal and diseased arteries need to be developed. To accomplish this goal, we tested the feasibility and efficiency of protein identification by a recently developed method, termed direct tissue proteomics (DTP). We analyzed frozen and paraformaldehyde-fixed archival coronary arteries with the DTP method. We also validated the distinct expression of four proteins by immunohistochemistry. In addition, we demonstrated the compatibility of the DTP method with laser capture microdissection and the possibility of monitoring specific cytokines and growth factors by the absolute quantification of abundance method. Major findings from this feasibility study are that 1) DTP can be used to efficiently identify proteins from paraformaldehyde-fixed, paraffin-embedded, and frozen coronary arteries; 2) approximately twice the number of proteins were identified from the frozen sections when compared with the paraformaldehyde-fixed sections; 3) laser capture microdissection is compatible with DTP; and 4) detection of low abundance cytokines and growth factors in the coronary arteries required selective reaction monitoring experiments coupled to absolute quantification of abundance. The analysis of 35 human coronary atherosclerotic samples allowed identification of a total of 806 proteins. The present study provides the first large scale proteomics map of human coronary atherosclerotic plaques.