Differential Expression of Alpha 4 Integrins on Effector Memory T Helper Cells during Bordetella Infections. Delayed Responses in Bordetella pertussis

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX) to test the hypothesis that effector memory T-helper cells (emTh) display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i.) with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs) from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7⁺ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4⁺ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease.     

Proteolytic breakdown of gliadin by <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> isolated from Tunisian fermented dough

The aim of this work was to select strains with proteolytic activity on wheat gliadin, among lactic acid bacteria, previously isolated from Tunisian fermented wheat dough. Hydrolysis of gliadin, as sole nitrogen source, in an agar medium was visualized by a clear zone surrounding colonies. The increase in absorbance due to gliadin breakdown was measured spectrophotometrically using O-phthaldialdehyde (OPA) on Gliadin Glucose Broth medium. Fermented liquid dough inoculated with individual selected Enterococcus faecalis, showed a decrease of the gliadin concentration from 45 g/kg to 18 g/kg determined by sandwich ELISA test (R-7001). Only the enterococci strains show an hydrolysis of gliadin proteins. Strains showing proteolytic activity are gaining more and more importance in cereal based fermented foods and may help to reduce gliadin involved in coeliac disease.     

Molecular Formula and METLIN Personal Metabolite Database Matching Applied to the Identification of Compounds Generated by LC/TOF-MS

In an effort to simplify and streamline compound identification from metabolomics data generated by liquid chromatography time-of-flight mass spectrometry, we have created software for constructing Personalized Metabolite Databases with content from over 15,000 compounds pulled from the public METLIN database (http://metlin.scripps.edu/). Moreover, we have added extra functionalities to the database that (a) permit the addition of user-defined retention times as an orthogonal searchable parameter to complement accurate mass data; and (b) allow interfacing to separate software, a Molecular Formula Generator (MFG), that facilitates reliable interpretation of any database matches from the accurate mass spectral data. To test the utility of this identification strategy, we added retention times to a subset of masses in this database, representing a mixture of 78 synthetic urine standards. The synthetic mixture was analyzed and screened against this METLIN urine database, resulting in 46 accurate mass and retention time matches. Human urine samples were subsequently analyzed under the same analytical conditions and screened against this database. A total of 1387 ions were detected in human urine; 16 of these ions matched both accurate mass and retention time parameters for the 78 urine standards in the database. Another 374 had only an accurate mass match to the database, with 163 of those masses also having the highest MFG score. Furthermore, MFG calculated a formula for a further 849 ions that had no match to the database. Taken together, these results suggest that the METLIN Personal Metabolite database and MFG software offer a robust strategy for confirming the formula of database matches. In the event of no database match, it also suggests possible formulas that may be helpful in interpreting the experimental results.     

Establishment of an efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation system in <Emphasis Type="Italic">Pelargonium graveolens</Emphasis>: an important aromatic plant

Rose-scented geranium is an important aromatic herb, have eminent for oil. The oil of geranium commercially utilized in the perfumery, cosmetic and aromatherapy industries all over the world. It is also helpful to cure many of the diseases, since it possess antibacterial, antifungal, antioxidant, anti-inflammatory and anticancer activities. However rose scented geranium suffer from several biotic and abiotic stresses, which reduced the yield of oil. So there is need to genetically improve the geranium using biotechnological approaches. The present study demonstrates the establishment of direct regeneration and Agrobacterium tumefaciens (LBA4404) mediated transformation protocol in Pelargonium graveolens (cv. CIM-BIO 171). Different media combinations such as benzyl amino purine (BAP), kinetin, naphthalene acetic acid (NAA), and adenine di-sulphate (ADS) were standardised to induce direct regeneration in P. graveolens. The maximum regeneration frequency i.e. 90.56?±?1.2% per explant was achieved from petiolar segments in medium containing 2.5 mg/l BAP, 0.1 mg/l NAA, 1 mg/l ADS. However, with the leaf explants only 45.94?±?2.91% frequency was achieved. In the present study, A. tumefaciens strain LBA4404 was used carrying binary vector pBI121 with the gusA as a reporter gene and neomycin phosphotransferase II (nptII) gene as a plant selectable marker. Parameters like bacterial optical density, infection time, acetosyringone concentration and kanamycin concentration were optimised to achieve maximum transformation frequency (69.5?±?2.3%).The putative transgenic shoots were subsequently rooted on half strength MS medium and successfully transferred to the greenhouse. The transgenic plants were characterised by gus histochemical assay, PCR analysis (nptII-786 bp and gus A- 1707 bp) and Southern hybridization tests using gusA gene probe. The regeneration as well as transformation protocol will no doubt provide the basis to decipher the insights of metabolic pathways in geranium. Also could be useful for genetic improvement, to make it more tolerant/resistant against biotic and abiotic stresses and ultimately fruitful for Indian farmers in agronomic traits like high biomass, oil content, yield and better quality.     

Salt stress reveals differential physiological,biochemical and molecular responses in <Emphasis Type="Italic">T. monococcum</Emphasis> and <Emphasis Type="Italic">T. durum</Emphasis> wheat genotypes

Salt stress responses implicate a complex mechanism and differ from plant species to another. In this study, we analyzed the physiological, biochemical and molecular responses to salt stress of the diploid wheat (T. monococcum) and compared to the tetraploid wheat (T. durum). Our results showed that the diploid wheat cultivar (cv. Turkey) is relatively tolerant to different salt stress conditions than the tetraploid wheat cultivar (cv. Om Rabia3). This tolerance was manifested by significant germination, plant growth and uptake of water generating cell turgor and development. Moreover, total chlorophyll content was higher in the diploid wheat than that in the tetraploid wheat. The Na⁺ content in leaf blade of the cv. Om Rabia3 was significantly higher than that of the cv. Turkey, suggesting that the diploid cultivar accumulates less toxic sodium in the photosynthetic tissues. This mechanism could be explained by the recirculation of the toxic ions Na⁺ into the xylem sap by SOS1 protein, which coordinates with HKT-like proteins to reduce the accumulation of Na⁺ ions in leaf blade. Interestingly, the expression of the three genes SOS1, HKT and NHX was enhanced under salinity especially in leaf blade of the cv. Turkey. Moreover, this wheat cultivar induced the antioxidative enzymes CAT and SOD activity more efficiently than the other cultivar.     

Phytoremediative potential of salt-tolerant grass species for cadmium and lead under contaminated nutrient solution

Abstract

Phytoremediation of heavy metal contaminated soils represents a promising technique and salt-tolerant hyperaccumulators for multiple metals are the need of time. Therefore, phytoremediation potential of four salt-tolerant grass species [Dhab (Desmostachya bipinnata), Kallar (Leptochloa fusca), Para (Brachiaria mutica) and Sporobolus (Sporobolus arabicus Boiss)] was evaluated for cadmium (Cd) and lead (Pb) in a hydroponic study. The plants were harvested after a growth period of 3 months in a nutrient solution containing different levels of Cd (0, 5, and 25?mg?L^?1) and Pb (0, 25, and 125?mg L^?1). Results indicated that Dhab grass showed the highest root and shoot dry matter yield followed by Para, Kallar and Sporobolus grass irrespective of metal or its level under which they were grown. All the grass species showed considerable Cd-accumulating potential with an accumulation of >150?mg kg^?1of shoot dry matter at a higher level of Cd-contamination (25?mg?L^?1). While in case of shoot Pb-accumulation only Para grass performed well and accumulated Pb >1000?mg kg^?1 of shoot dry matter at the higher level of Pb-contamination (125?mg?L^?1). Moreover, Para and Dhab grasses performed better for shoot Cd-uptake, while only Para grass showed promising shoot Pb uptake potential. In conclusion, these grass species could be penitentially used for phytoremediation of salt-affected Cd and Pb contaminated soils.     

Ligand-guided selection of aptamers against T-cell Receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells

We recently introduced a screening technology termed ligand-guided selection, (LIGS), to selectively identify target-specific aptamers from an evolved cell-SELEX library. Cell-SELEX utilizes a large combinatorial single-stranded oligonucleotide library and progressively selects DNA ligands against whole cells with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. LIGS exploits the partition step and introduces a secondary, pre-existing high-affinity monoclonal antibody (mAb) ligand to outcompete and elute specific aptamers towards the binding target of the antibody, not the cell. Here, using anti-CD3ε mAb against the cluster of differentiation 3 (CD3ε), as the guiding ligand against one of the domains of the T-cell Receptor (TCR) complex expressed on Jurkat.E6 cells, we discovered three specific aptamers against TCR complex expressed on an immortalized line of human T lymphocyte cells. In sum, we demonstrate that specific aptamers can be identified utilizing an antibody against a single domain of a multidomain protein complex in their endogenous state with neither post- nor pre-SELEX protein manipulation.     

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 10³ M⁻¹, which indicates significant binding. The ΔG value was found to be − 7.56 kcal mol⁻¹ at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.     

The Impact of Myeloperoxidase and Activated Macrophages on Metaphase II Mouse Oocyte Quality

Myeloperoxidase (MPO), an abundant heme-containing enzyme present in neutrophils, monocytes, and macrophages, is produced in high levels during inflammation, and associated with poor reproductive outcomes. MPO is known to generate hypochlorous acid (HOCl), a damaging reactive oxygen species (ROS) utilizing hydrogen peroxide (H₂O₂) and chloride (Cl^-). Here we investigate the effect of activated immune cells and MPO on oocyte quality. Mouse metaphase II oocytes were divided into the following groups: 1) Incubation with a catalytic amount of MPO (40 nM) for different incubation periods in the presence of 100 mM Cl^- with and without H₂O₂ and with and without melatonin (100 μM), at 37°C (n = 648/648 total number of oocytes in each group for oocytes with and without cumulus cells); 2) Co-cultured with activated mouse peritoneal macrophage and neutrophils cells (1.0 x 10⁶ cells/ml) in the absence and presence of melatonin (200 μM), an MPO inhibitor/ROS scavenger, for different incubation periods in HTF media, at 37°C (n = 200/200); 3) Untreated oocytes incubated for 4 hrs as controls (n = 73/64). Oocytes were then fixed, stained and scored based on the microtubule morphology and chromosomal alignment. All treatments were found to negatively affect oocyte quality in a time dependent fashion as compared to controls. In all cases the presence of cumulus cells offered no protection; however significant protection was offered by melatonin. Similar results were obtained with oocytes treated with neutrophils. This work provides a direct link between MPO and decreased oocyte quality. Therefore, strategies to decrease MPO mediated inflammation may influence reproductive outcomes.