Role of Spectral Studies in Detection of Antibacterial Phytoelements and Phytochemicals of Moringa oleifera

This paper reports, the Laser Induced Breakdown Spectroscopy (LIBS) studies and structure elucidation of compounds isolated from the fruit extract of Moringa oleifera and also deals with their possible effects on some bacterial strains viz. Staphylococcus aureus, Klebsiella pneumonia, Escherichia coli and Pseudomonas aeruginosa. The extract was found to be active against all four microorganisms used. Extent of inhibitory effect of extract was assessed at different concentrations of 25, 50, 75 mg/ml by measuring diameter of inhibition zone (DIZ). Our results clearly showed that the 75 mg/ml concentration of the extract had 14, 12 and 18 mm of the DIZ against Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa and 14 mm with 50 mg/ml concentration against Escherichia coli. The results were compared with the standard antibiotic ‘ampicillin’ of 1 mg/ml concentration. LIBS was recorded with high power pulsed laser beam from Nd: YAG Laser (Continuum Surelite III-10), focused on the surface of the material, which was in liquid form, to generate plasma on the surface of the sample. LIBS data clearly demonstrate the presence of trace elements, magnesium and iron, in high concentration in the extract. Whereas, from the phytochemical profile reveals the presence of two new compounds, S-ethyl-N-{4-[(α-L-rhamnosyloxy) benzyl]} thiocarbamate and 2-acetoxy {4-[(2′,3′,4′-tri-O-acetyl-α-L-rhamnosyloxy) benzyl]} acetonitrile as the major constituents. This study is the first report on synergetic effect of the phytoconstituents and certain set of elements present in their defined role in bacterial management against different bacterial strains.     

Elemental composition in vesicles of an arbuscular mycorrhizal fungus, as revealed by PIXE analysis

We investigated element accumulation in vesicles of the arbuscular mycorrhizal (AM) fungus Glomus intraradices, extracted from the roots of inoculated leek plants. The elemental composition (elements heavier than Mg) was quantified using particle-induced X-ray emission (PIXE), in combination with scanning transmission ion microscopy (STIM). In vesicles, P was the most abundant of the elements analysed, followed by Ca, S, Si and K. We analysed 12 vesicles from two root systems and found that the variation between vesicles was particularly high for P and Si. The P content related positively to Si, Zn and K, while its relation to Cl fitted to a negative power function. Vesicle transects showed that P and K were present in central parts, while Ca was present mainly near the vesicle surfaces. The results showed that P is an important part (0.5% of the dry weight) of the vesicle content and that the distribution of some elements, within mycelia, may be strongly correlated.     

Abdominal fat mass is associated with adaptive immune activation: the CODAM Study

Abdominal fat-related activation of the innate immune system and insulin resistance (IR) are implicated in the pathogenesis of cardiovascular diseases. Recent data support an important role of the adaptive immune system as well. In this study, we investigate the association between waist circumference and markers of systemic adaptive immune activation, and the potential mediating role of innate immune activation and/or IR herein. The study population consisted of 477 (304 men) individuals (mean age: 59.4 ± 7.0 years) in whom waist circumference, HOMA2-IR (IR derived from homeostasis model assessment), and markers of innate (C-reactive protein (CRP), interleukin (IL)-6, serum amyloid A (SAA)) and adaptive (neopterin, soluble CD25 (sCD25)) immune activation were measured. These markers were compiled into an adaptive and innate immune activation score by averaging the respective z-scores. After adjustments for age, sex, glucose metabolism, smoking status, prior cardiovascular disease, and other risk factors, waist circumference was associated with the adaptive (standardized regression coefficient β = 0.12 (95% confidence intervals: 0.04-0.20)) and the innate immune activation scores (β = 0.24 (0.17-0.31)), and with HOMA2-IR (β = 0.49 (0.42-0.56)). The innate immune activation score and HOMA2-IR were also positively associated with the adaptive immune activation score (β = 0.31 (0.21-0.40) and β = 0.11 (0.02-0.21), respectively). The association between waist circumference and the adaptive immune activation score was completely abolished when further adjusted for innate immune activation and HOMA2-IR (to β = -0.01 (-0.10-0.08)), and the specific mediation "effects" attributable to each of these variables were 58% and 42%, respectively. We conclude that abdominal obesity is associated with systemic adaptive immune activation and that innate immune activation and IR constitute independent and equally important pathways explaining this association.     

Responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes: a randomized trial

Background

The ability of subjects to respond to nutritional challenges can reflect the flexibility of their biological system. Nutritional challenge tests could be used as an indicator of health status but more knowledge on metabolic and immune responses of different subjects to nutritional challenges is needed. The aim of this study was to compare the responses to high-fat challenges varying in fat type in subjects with different metabolic risk phenotypes.

Methodology/Principal Findings

In a cross-over design 42 men (age 50–70 y) consumed three high-fat shakes containing saturated fat (SFA), monounsaturated fat (MUFA) or n-3 polyunsaturated (PUFA). Men were selected on BMI and health status (lean, obese or obese diabetic) and phenotyped with MRI for adipose tissue distribution. Before and 2 and 4 h after shake consumption blood was drawn for measurement of expression of metabolic and inflammation-related genes in peripheral blood mononuclear cells (PBMCs), plasma triglycerides (TAG), glucose, insulin, cytokines and ex vivo PBMC immune response capacity. The MUFA and n-3 PUFA challenge, compared to the SFA challenge, induced higher changes in expression of inflammation genes MCP1 and IL1β in PBMCs. Obese and obese diabetic subjects had different PBMC gene expression and metabolic responses to high-fat challenges compared to lean subjects. The MUFA challenge induced the most pronounced TAG response, mainly in obese and obese diabetic subjects.

Conclusion/Significance

The PBMC gene expression response and metabolic response to high-fat challenges were affected by fat type and metabolic risk phenotype. Based on our results we suggest using a MUFA challenge to reveal differences in response capacity of subjects.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00977262","term_id":"NCT00977262"}}NCT00977262     

Effect of adjuvants on the humoral immune response to congopain in mice and cattle

ABSTRACT: BACKGROUND: We investigated several adjuvants for their effects on the humoral immune response in both mice and cattle using the central domain of congopain (C2), the major cysteine protease of Trypanosoma congolense, as a model for developing a vaccine against animal trypanosomosis. The magnitude and sustainability of the immune response against C2 and the occurrence of a booster effect of infection, an indirect measure of the presence of memory cells, were determined by ELISA, while spectrofluorometry was used to determine and measure the presence of enzyme- inhibiting antibodies. RESULTS: Mice immunized with recombinant C2 in AdjuphosTM, TiterMaxTM, purified saponin Quil ATM or GERBUTM showed the best response according to the evaluation criteria and these three were chosen for the cattle vaccination study. The animals were challenged with T. congolense four and a half months after the last booster. Cattle immunized with recombinant C2 in purified saponin Quil ATM showed the best antibody response according to the measured parameters. CONCLUSIONS: We identified purified saponin Quil ATM as a good adjuvant for immunizations with C2. The results from this study will be useful in future attempts to develop an effective anti-disease vaccine against African trypanosomosis.     

Universal fingerprinting chip server

The Virtual Hybridization approach predicts the most probable hybridization sites across a target nucleic acid of known sequence, including both perfect and mismatched pairings. Potential hybridization sites, having a user-defined minimum number of bases that are paired with the oligonucleotide probe, are first identified. Then free energy values are evaluated for each potential hybridization site, and if it has a calculated free energy of equal or higher negative value than a user-defined free energy cut-off value, it is considered as a site of high probability of hybridization. The Universal Fingerprinting Chip Applications Server contains the software for visualizing predicted hybridization patterns, which yields a simulated hybridization fingerprint that can be compared with experimentally derived fingerprints or with a virtual fingerprint arising from a different sample. AVAILABILITY: The database is available for free at http://bioinformatica.homelinux.org/UFCVH/     

Reduced Prostasin (CAP1/PRSS8) Activity Eliminates HAI-1 and HAI-2 Deficiency-Associated Developmental Defects by Preventing Matriptase Activation

Loss of either hepatocyte growth factor activator inhibitor (HAI)-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8), restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2), or the epithelial sodium channel (ENaC) alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.     

Expression of human CYP27B1 in Escherichia coli and characterization in phospholipid vesicles

CYP27B1 is a mitochondrial cytochrome P450 that catalyses the hydroxylation of 25-hydroxyvitamin D3 at the C1α-position to give the hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3. We successfully expressed human CYP27B1 in Escherichia?coli and partially purified this labile enzyme and carried out a detailed characterization of its kinetic properties in a reconstituted membrane environment. The phospholipid concentration did not affect the enzyme activity in the vesicle-reconstituted system, although it was influenced by the phospholipid composition, with the addition of cardiolipin lowering the K(m) for 25-hydroxyvitamin D3. These data are consistent with the enzyme accessing substrate from the hydrophobic domain of the vesicle membrane. Cardiolipin also caused the appearance of inhibition of activity at high substrate concentrations. This substrate inhibition fitted a model for one catalytic and two inhibitory sites on the enzyme for the binding of substrate. The K(m) for human adrenodoxin was observed to decrease with decreasing substrate concentration, with the catalytic efficiency (k(cat) /K(m) ) being largely independent of adrenodoxin concentration. Human CYP27B1 was also active on 25-hydroxyvitamin D(2) and on intermediates of the CYP24A1-mediated inactivation pathway, 24R,25-dihydroxyvitamin D3, 24-oxo-25-hydroxyvitamin D3 and 24-oxo-23,25-dihydroxyvitamin D3, with all these substrates showing comparable k(cat) values of 50-71?min(-1) , similar to 25-hydroxyvitamin D3. The latter two substrates gave higher K(m) values than that for 25-hydroxy-vitamin D3. The present study shows that human CYP27B1 can be partially purified in an active form with the enzyme displaying high activity towards a range of substrates in a phospholipid vesicle-reconstituted system that mimics the inner-mitochondrial membrane.     

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera

No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.