A metabolomics approach exploring the function of the ESX-3 type VII secretion system of M. smegmatis

The genome of Mycobacterium, including Mycobacterium tuberculosis, contains five copies of a cluster of genes encoding a novel type VII secretion system, named the ESX gene cluster region. This ESX-3 gene cluster is essential for in vitro growth and is thought to play a role in iron and zinc homeostasis, however, its exact functionality remains an enigma. A metabolomics research approach was subsequently used to compare the metabolite profiles of a M. smegmatis ESX-3 knockout strain to that a wild type parental strain, in order to elucidate its functionality from a metabolic perspective. Statistical analysis of the GC–MS generated data showed a clear separation between the wild type and knockout sample groups, based on the analysed metabolite profiles of these organisms. Of all the metabolite markers identified, various amino acids and metabolite pathways related to these, appeared to be most affected by the ESX-3 knockout, especially those with enzymes regulated by iron and zinc, supporting previous genomics and proteomics generated hypotheses and findings. This study is the first to demonstrate the capacity of using metabolomics, in conjunction with previous genomics and proteomic findings, to identify underlying metabolic changes and confirm previous hypotheses related to the functionality of ESX-3 in Mycobacterium growth and survival.     

Dexamethasone Attenuates VEGF Expression and Inflammation but Not Barrier Dysfunction in a Murine Model of Ventilator–Induced Lung Injury

Background

Ventilator–induced lung injury (VILI) is characterized by vascular leakage and inflammatory responses eventually leading to pulmonary dysfunction. Vascular endothelial growth factor (VEGF) has been proposed to be involved in the pathogenesis of VILI. This study examines the inhibitory effect of dexamethasone on VEGF expression, inflammation and alveolar–capillary barrier dysfunction in an established murine model of VILI.

Methods

Healthy male C57Bl/6 mice were anesthetized, tracheotomized and mechanically ventilated for 5 hours with an inspiratory pressure of 10 cmH₂O (“lower” tidal volumes of ∼7.5 ml/kg; LV_T) or 18 cmH₂O (“higher” tidal volumes of ∼15 ml/kg; HV_T). Dexamethasone was intravenously administered at the initiation of HV_T–ventilation. Non–ventilated mice served as controls. Study endpoints included VEGF and inflammatory mediator expression in lung tissue, neutrophil and protein levels in bronchoalveolar lavage fluid, PaO₂ to FiO₂ ratios and lung wet to dry ratios.

Results

Particularly HV_T–ventilation led to alveolar–capillary barrier dysfunction as reflected by reduced PaO₂ to FiO₂ ratios, elevated alveolar protein levels and increased lung wet to dry ratios. Moreover, VILI was associated with enhanced VEGF production, inflammatory mediator expression and neutrophil infiltration. Dexamethasone treatment inhibited VEGF and pro–inflammatory response in lungs of HV_T–ventilated mice, without improving alveolar–capillary permeability, gas exchange and pulmonary edema formation.

Conclusions

Dexamethasone treatment completely abolishes ventilator–induced VEGF expression and inflammation. However, dexamethasone does not protect against alveolar–capillary barrier dysfunction in an established murine model of VILI.     

Vascular Multiplicity Should Not Be a Contra-Indication for Live Kidney Donation and Transplantation

BackgroundWhether vascular multiplicity should be considered as contraindication and therefore ‘extended donor criterion’ is still under debate.MethodsData from all live kidney donors from 2006–2013 (n = 951) was retrospectively reviewed. Vascular anatomy as imaged by MRA, CTA or other modalities was compared with intraoperative findings. Furthermore, the influence of vascular multiplicity on outcome of donors and recipients was studied.ResultsIn 237 out of 951 donors (25%), vascular multiplicity was present. CTA had the highest accuracy levels regarding vascular anatomy assessment. Regarding outcome of donors with vascular multiplicity, warm ischemia time (WIT) and skin-to-skin time were significantly longer if arterial multiplicity (AM) was present (5.1 vs. 4.0 mins and 202 vs. 178 mins). Skin-to-skin time was significantly longer, and complication rates were higher in donors with venous multiplicity (203 vs. 180 mins and 17.2% vs. 8.4%). Outcome of renal transplant recipients showed a significantly increased WIT (30 vs. 26.7 minutes), higher rate of DGF (13.9% vs. 6.9%) and lower rate of BPAR (6.9% vs. 13.9%) in patients receiving a kidney with AM compared to kidneys with singular anatomy.ConclusionsWe conclude that vascular multiplicity should not be a contra-indication, since it has little impact on clinical outcome in the donor as well as in renal transplant recipients.     

Definition of a short region of XPG necessary for TFIIH interaction and stable recruitment to sites of UV damage

XPG is the human endonuclease that cuts 3' to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individual's XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.     

Occasional sex in an 'asexual' polyploid hermaphrodite

Asexual populations are usually considered evolutionary dead-ends because they lack the mechanisms to generate and maintain sufficient genetic diversity. Yet, some asexual forms are remarkably widespread and genetically diverse. This raises the question whether asexual systems are always truly clonal or whether they have cryptic forms of sexuality that enhance their viability. In the planarian flatworm Schmidtea polychroa parthenogens are functional hermaphrodites (as are their sexual conspecifics), copulate and exchange sperm. Sperm is required for initiation of embryogenesis but usually does not contribute genetically to the offspring (sperm-dependent parthenogenesis). Using karyology and genotyping of parents and offspring, we show that in a purely parthenogenetic population an estimated 12% of all offspring are the result of partial genetic exchange. Several processes of chromosome addition and loss are involved. Some of these result in an alternation between a common triploid and a rare tetraploid state. We conclude that genetic recombination does not necessarily require segregation and fusion within the same generation, as is the case in most sexual species. These occasional sexual processes help to explain the geographical dominance of parthenogens in our study species.     

Improved protein-ligand docking using GOLD

The Chemscore function was implemented as a scoring function for the protein-ligand docking program GOLD, and its performance compared to the original Goldscore function and two consensus docking protocols, "Goldscore-CS" and "Chemscore-GS," in terms of docking accuracy, prediction of binding affinities, and speed. In the "Goldscore-CS" protocol, dockings produced with the Goldscore function are scored and ranked with the Chemscore function; in the "Chemscore-GS" protocol, dockings produced with the Chemscore function are scored and ranked with the Goldscore function. Comparisons were made for a "clean" set of 224 protein-ligand complexes, and for two subsets of this set, one for which the ligands are "drug-like," the other for which they are "fragment-like." For "drug-like" and "fragment-like" ligands, the docking accuracies obtained with Chemscore and Goldscore functions are similar. For larger ligands, Goldscore gives superior results. Docking with the Chemscore function is up to three times faster than docking with the Goldscore function. Both combined docking protocols give significant improvements in docking accuracy over the use of the Goldscore or Chemscore function alone. "Goldscore-CS" gives success rates of up to 81% (top-ranked GOLD solution within 2.0 A of the experimental binding mode) for the "clean list," but at the cost of long search times. For most virtual screening applications, "Chemscore-GS" seems optimal; search settings that give docking speeds of around 0.25-1.3 min/compound have success rates of about 78% for "drug-like" compounds and 85% for "fragment-like" compounds. In terms of producing binding energy estimates, the Goldscore function appears to perform better than the Chemscore function and the two consensus protocols, particularly for faster search settings. Even at docking speeds of around 1-2 min/compound, the Goldscore function predicts binding energies with a standard deviation of approximately 10.5 kJ/mol.     

B1 cells contribute to serum IgM,but not to intestinal IgA,production in gnotobiotic Ig allotype chimeric mice

B1 cells are a significant source of natural serum IgM, thereby serving as a first line of defense against systemic bacterial and viral infections. They can migrate to the intestinal lamina propria and differentiate into IgA-producing plasma cells and thus might play a similar role in mucosal immunity. To investigate the contribution of B1 cells to the intestinal IgA response induced by the commensal flora in immunocompetent animals, we generated gnotobiotic and conventionally reared Ig allotype chimeric mice. In this system B1- and B2-derived Abs can be distinguished based on different allotypes. FACS analysis of peritoneal cavity cells and analysis of B1- and B2-derived serum IgM indicated stable B1/B2 chimerism and the establishment of a functional B1 population. Monoassociation with either Morganella morganii, Bacteroides distasonis, or segmented filamentous bacteria induced germinal center reactions in Peyer's patches and led to the production of intestinal IgA, partially reactive with bacterial Ag. A considerable amount of serum IgM was B1 cell derived in both monoassociated and conventionally reared mice. However, most of the total as well as bacteria-specific intestinal IgA was produced by B2 cells. These data suggest that intestinal IgA production induced by commensal bacteria is mainly performed by B2, not B1, cells.     

Enhanced Lacto-Tri-Peptide Bio-Availability by Co-Ingestion of Macronutrients

Some food-derived peptides possess bioactive properties, and may affect health positively. For example, the C-terminal lacto-tri-peptides Ile-Pro-Pro (IPP), Leu-Pro-Pro (LPP) and Val-Pro-Pro (VPP) (together named here XPP) are described to lower blood pressure. The bioactivity depends on their availability at the site of action. Quantitative trans-organ availability/kinetic measurements will provide more insight in C-terminal tri-peptides behavior in the body. We hypothesize that the composition of the meal will modify their systemic availability. We studied trans-organ XPP fluxes in catheterized pigs (25 kg; n=10) to determine systemic and portal availability, as well as renal and hepatic uptake of a water-based single dose of synthetic XPP and a XPP containing protein matrix (casein hydrolyte, CasH). In a second experiment (n=10), we compared the CasH-containing protein matrix with a CasH-containing meal matrix and the modifying effects of macronutrients in a meal on the availability (high carbohydrates, low quality protein, high fat, and fiber). Portal availability of synthetic XPP was 0.08 ± 0.01% of intake and increased when a protein matrix was present (respectively 3.1, 1.8 and 83 times for IPP, LPP and VPP). Difference between individual XPP was probably due to release from longer peptides. CasH prolonged portal bioavailability with 18 min (absorption half-life, synthetic XPP: 15 ± 2 min, CasH: 33 ± 3 min, p<0.0001) and increased systemic elimination with 20 min (synthetic XPP: 12 ± 2 min; CasH: 32 ± 3 min, p<0.0001). Subsequent renal and hepatic uptake is about 75% of the portal release. A meal containing CasH, increased portal 1.8 and systemic bioavailability 1.2 times. Low protein quality and fiber increased XPP systemic bioavailability further (respectively 1.5 and 1.4 times). We conclude that the amount and quality of the protein, and the presence of fiber in a meal, are the main factors that increase the systemic bioavailability of food-derived XPP.