Metabonomic investigation of liver profiles of nonpolar metabolites obtained from alcohol-dosed rats and mice using high mass accuracy MSn analysis

Alcoholism is a complex disorder that, in man, appears to be genetically influenced, although the underlying genes and molecular pathways are not completely known. Here, the intragastric alcohol feeding model in rodents was used together with high mass accuracy LC-MS(n) analysis to assess the metabonomic changes in nonpolar metabolite profiles for livers from control and alcohol-treated rats and mice. Ion signals with a peak area variance of less than 30% (based on repeat analysis of a pooled quality control sample analyzed throughout the batch) were submitted to multivariate statistical analysis (using principal components analysis, PCA). PCA revealed robust differences between profiles from control and alcohol-treated animals from both species. The major metabolites seen to differ between control and alcohol-treated animals were identified using high accuracy MS(n) data and verified using external search engines ( http://www.lipidmaps.org ; http://www.hmdb.ca; http://www.genome.jp/kegg/ ) and authentic standards. The main metabolite classes to show major changes in the alcoholic liver-derived samples were fatty acyls, fatty acid ethyl esters, glycerolipids, and phosphatidylethanol homologues. Significant metabolites that were up-regulated by alcohol treatment in both rat and mouse livers included fatty acyls, metabolites such as octadecatrienoic acid and eicosapentaenoic acid, a number of fatty acid ethyl esters such as ethyl arachidonate, ethyl docosahexaenoic acid, ethyl linoleate, and ethyl oleate and phosphatidylethanol (PEth) homologues (predominantly PEth 18:0/18:2 and PEth 16:0/18:2; PEth homologues are currently considered as potential biomarkers for harmful and prolonged alcohol consumption in man). A number of glycerophospholipids resulted in both up-regulation (m/z 903.7436 [M + H](+) corresponding to a triglyceride) and down-regulation (m/z 667.5296 [M + H](+) corresponding to a diglyceride). Metabolite profiles were broadly similar in both mouse and rat models. However, there were a number of significant differences in the alcohol-treated group particularly in the marked down-regulation of retinol and free cholesterol in the mouse compared to the rat. Unique markers for alcohol treatment included ethyl docosahexaenoic acid. Metabolites were identified with high confidence using predominantly negative ion MS(n) data for the fatty acyl components to match to www.lipidmaps.org MS and MS/MS databases; interpreting positive ion data needed to take into account possible adduct ions which may confound the identification of other lipid classes. The observed changes in lipid profiles were consistent with alcohol-induced liver injury in humans.     

Restituting intestinal epithelial cells exhibit increased transducibility by adenoviral vectors

BACKGROUND AND AIMS: While mature enterocytes are resistant to transduction by adenovirus type 5 (Ad5) vectors, undifferentiated cells are transduced much more efficiently. Our purpose was to study enterocyte transduction in models of intestinal wound healing. METHODS: Transduction was studied ex vivo using cultures of endoscopic biopsies and in vitro utilizing Caco-2 cells in models of mucosal wound healing. Vectors carried either the LacZ or the luciferase gene. CAR (coxsackievirus and adenovirus receptor) and integrins were studied with transduction inhibition and immunofluorescent staining. RESULTS: Increased transduction efficiency was observed for a subset of enterocytes with a flattened de-differentiated phenotype present at the edge of cultured biopsies. In the in vitro systems, expanding Caco-2 cell monolayers exhibited increased transducibility that was time- and dose-dependent, reaching virtually 100% in cells along the leading edge at high viral load. Bioluminescence activity of transduced expanding monolayers was up to 3-fold greater than that of non-expanding monolayers ('fence' system, 48 h, MOI 1000, p < 0.05). Mitomycin C pre-treatment did not affect levels of transduction in expanding monolayers. At the highest viral load tested, CAR or integrin blocking prior to virus application resulted in 39.4% and 45.4% reduction in transduction levels (p < 0.05). Immunofluorescence revealed altered expression of CAR on the migrating edge of the Caco-2 cultures and the expression of CAR on the apical membrane of biopsy enterocytes. CONCLUSIONS: Increased CAR and integrin accessibility in migrating enterocytes mediates increased transduction by Ad5 vectors. This subset of enterocytes provides a target for the delivery of genes of interest for both research and gene therapy applications.     

Use of Preclinical Dog Studies and Absorption Modeling to Facilitate Late Stage Formulation Bridging for a BCS II Drug Candidate

Formulation changes are common during drug development either due to clinical or manufacturing considerations. These changes especially at later stages of drug development oftentimes raise questions on the potential impact of a new formulation on bioavailability. In this work, the preclinical assessment of formulation bridging risk for a Biopharmaceutics Classification System II development compound is presented. Early clinical studies were conducted using a liquid-filled capsule (LFC). To assess the feasibility of a conventional solid dosage form, an initial analysis was conducted using absorption modeling which indicated conventional formulation of micronized active pharmaceutical ingredient (API) could be a viable option. Subsequently, test formulations were prepared and tested in vivo in dogs. The solid formulations were able to match exposures of the LFC capsule in the dog model; in addition, a sensitivity to API PSD was observed in line with the modeling predictions. When tested in the clinic, the conventional solid formulation resulted in exposures of approximately 25% lower compared to the LFC on an equivalent dose basis; however, bridging with a small dose adjustment would be feasible. The outcome of the clinical study was better predicted by the modeling approach while the dog model appeared to somewhat overestimate absorption. Through the use of preclinical tools and modeling and simulation, a risk assessment around formulation bridging can be conducted and inform formulation decisions or subsequent clinical study designs.     

Co-Infection with Mycobacterium tuberculosis Impairs HIV-Specific CD8+ and CD4+ T Cell Functionality

The ability of antigen-specific T cells to simultaneously produce multiple cytokines is thought to correlate with the functional capacity and efficacy of T cells. These ‘polyfunctional’ T cells have been associated with control of HIV. We aimed to assess the impact of co-infection with Mycobacterium tuberculosis (MTB) on HIV-specific CD8+ and CD4+ T cell function. We assessed T cell functionality in 34 South African adults by investigating the IFN-y, IL-2, TNF-α, IL-21 and IL-17 cytokine secretion capacity, using polychromatic flow cytometry, following HIV Gag-specific stimulation of peripheral blood mononuclear cells. We show that MTB is associated with lower HIV-specific T cell function in co-infected as compared to HIV mono-infected individuals. This decline in function was greatest in co-infection with active Tuberculosis (TB) compared to co-infection with latent MTB (LTBI), suggesting that mycobacterial load may contribute to this loss of function. The described impact of MTB on HIV-specific T cell function may be a mechanism for increased HIV disease progression in co-infected subjects as functionally impaired T cells may be less able to control HIV.     

Molecular dynamics simulations of ion separation in nano-channel water flows using an electric field

ABSTRACT

Removal of undesired substances from water is a field of investigation recently focused at the nanoscale. Towards this direction, molecular dynamics simulations are conducted in this paper to investigate unwanted ion removal in nanochannel flows. The simulation method incorporates a Poiseuille-like water/ion flow system at the nanoscale where an electric field, of various magnitudes in the range of E?=?0.25–1.5?V/Å, is applied perpendicular to the flow, leading anions and cations close to the wall regions, similar to the Capacitive De-Ionization method. The time needed for ions to reach equilibrium, i.e. to flow in the region near the walls while pure water flows in the channel interior, is t?=?1.3?ns when E?=?1.5?V/Å and t?=?4.0?ns when E?=?0.25?V/Å, showing a dependency on the value of the electric field. Calculations on density, velocity, and temperature values report on fluid properties to be used in the proposed desalination configuration and could act as a basis to guide novel technological applications and extend to higher scales.     

Is the genetic diversity of small scattered forest tree populations at the southern limits of their range more prone to stochastic events? A wild cherry case study by microsatellite-based markers

Wild cherry (Prunus avium L.) is a widespread, partially asexual, noble hardwood European species characterized by a scattered distribution, small population sizes, and human exploitation for its valuable wood. These characteristics, especially at the southern limits of the species natural distribution where additional varying stresses may occur, render P. avium populations prone to potential stochastic, genetic, and demographic events. In this study, we used dominant inter simple sequence repeat (ISSR) and codominant simple sequence repeat (SSR) markers to infer the genetic structure of P. avium. Five populations from northern Greece were evaluated based on 46 ISSR and 11 SSR loci. Populations presented a relatively high level of genetic variation, with a mean genetic diversity of H _e?=?0.166 and H _e?=?0.740 regarding ISSR and SSR analysis, respectively. We observed moderate population differentiation for ISSR (G _ST?=?0.113) and SSR (F _ST?=?0.097) markers. AMOVA also detected significant differentiation among populations for ISSRs (?? _ST?=?0.338) and SRRs (?? _ST?=?0.162). According to linkage disequilibrium analysis, estimates of effective population size were generally sufficient for maintaining extant genetic variability and evolutionary potential. A possible bottleneck was detected for only one population. In general, it appears that despite the particular characteristics of the P. avium populations studied, genetic stochasticity events were not apparent. The studied populations, located at the rear edge of the species European distribution, reveal a wealth of genetic variation that is very valuable for the genetic conservation of local adaptive gene complexes, especially under contemporary climatic change scenarios.     

Comparative Genomics of Multiple Strains of Pseudomonas cannabina pv. alisalensis,a Potential Model Pathogen of Both Monocots and Dicots

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.     

Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection--novel approaches against Helicobacter pylori inflammation

Helicobacter pylori neutrophil-activating protein (HP-NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP-NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe(2+) is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP-NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C-terminal region of both mutant HP-NAP and wild-type dodecamer HP-NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69-Leu75) and H4 (Lys89-Leu114) or the linking coils (His63-Thr68 and Thr76-Ser88) are probably critical in stimulating neutrophil activation.