In situ proteo-metabolomics reveals metabolite secretion by the acid mine drainage bio-indicator,Euglena mutabilis

Euglena mutabilis is a photosynthetic protist found in acidic aquatic environments such as peat bogs, volcanic lakes and acid mine drainages (AMDs). Through its photosynthetic metabolism, this protist is supposed to have an important role in primary production in such oligotrophic ecosystems. Nevertheless, the exact contribution of E. mutabilis in organic matter synthesis remains unclear and no evidence of metabolite secretion by this protist has been established so far. Here we combined in situ proteo-metabolomic approaches to determine the nature of the metabolites accumulated by this protist or potentially secreted into an AMD. Our results revealed that the secreted metabolites are represented by a large number of amino acids, polyamine compounds, urea and some sugars but no fatty acids, suggesting a selective organic matter contribution in this ecosystem. Such a production may have a crucial impact on the bacterial community present on the study site, as it has been suggested previously that prokaryotes transport and recycle in situ most of the metabolites secreted by E. mutabilis. Consequently, this protist may have an indirect but important role in AMD ecosystems but also in other ecological niches often described as nitrogen-limited.     

Single collateral reconstructions reveal distinct phases of corticospinal remodeling after spinal cord injury

Background

Injuries to the spinal cord often result in severe functional deficits that, in case of incomplete injuries, can be partially compensated by axonal remodeling. The corticospinal tract (CST), for example, responds to a thoracic transection with the formation of an intraspinal detour circuit. The key step for the formation of the detour circuit is the sprouting of new CST collaterals in the cervical spinal cord that contact local interneurons. How individual collaterals are formed and refined over time is incompletely understood.

Methodology/Principal Findings

We traced the hindlimb corticospinal tract at different timepoints after lesion to show that cervical collateral formation is initiated in the first 10 days. These collaterals can then persist for at least 24 weeks. Interestingly, both major and minor CST components contribute to the formation of persistent CST collaterals. We then developed an approach to label single CST collaterals based on viral gene transfer of the Cre recombinase to a small number of cortical projection neurons in Thy1-STP-YFP or Thy1-Brainbow mice. Reconstruction and analysis of single collaterals for up to 12 weeks after lesion revealed that CST remodeling evolves in 3 phases. Collateral growth is initiated in the first 10 days after lesion. Between 10 days and 3–4 weeks after lesion elongated and highly branched collaterals form in the gray matter, the complexity of which depends on the CST component they originate from. Finally, between 3–4 weeks and 12 weeks after lesion the size of CST collaterals remains largely unchanged, while the pattern of their contacts onto interneurons matures.

Conclusions/Significance

This study provides a comprehensive anatomical analysis of CST reorganization after injury and reveals that CST remodeling occurs in distinct phases. Our results and techniques should facilitate future efforts to unravel the mechanisms that govern CST remodeling and to promote functional recovery after spinal cord injury.     

Determination of the absolute configuration and evaluation of the in vitro antitumor activity of dilospirane B

The absolute configuration of dilospirane B (1), a diterpene featuring a novel carbon framework recently isolated from the brown alga Dilophus spiralis, was established on the basis of theoretical calculations of its electronic circular dichroism spectra applying the time-dependent density functional theory method. Metabolite 1, evaluated for its in vitro antitumor activity against six human cancer cell lines displaying various levels of sensitivity to pro-apoptotic stimuli, exhibited growth inhibitory activity with a mean IC₅₀ value of 66 μM. Quantitative videomicroscopy analyses indicated that 1 initially displays cytostatic effects which later become cytotoxic.     

Comparative effectiveness research: an empirical study of trials registered in ClinicalTrials.gov

Background

The $1.1 billion investment in comparative effectiveness research will reshape the evidence-base supporting decisions about treatment effectiveness, safety, and cost. Defining the current prevalence and characteristics of comparative effectiveness (CE) research will enable future assessments of the impact of this program.

Methods

We conducted an observational study of clinical trials addressing priority research topics defined by the Institute of Medicine and conducted in the US between 2007 and 2010. Trials were identified in ClinicalTrials.gov. Main outcome measures were the prevalence of comparative effectiveness research, nature of comparators selected, funding sources, and impact of these factors on results.

Results

231 (22.3%; 95% CI 19.8%–24.9%) studies were CE studies and 804 (77.7%; 95% CI, 75.1%–80.2%) were non-CE studies, with 379 (36.6%; 95% CI, 33.7%–39.6%) employing a placebo control and 425 (41.1%; 95% CI, 38.1%–44.1%) no control. The most common treatments examined in CE studies were drug interventions (37.2%), behavioral interventions (28.6%), and procedures (15.6%). Study findings were favorable for the experimental treatment in 34.8% of CE studies and greater than twice as many (78.6%) non-CE studies (P<0.001). CE studies were more likely to receive government funding (P = 0.003) and less likely to receive industry funding (P = 0.01), with 71.8% of CE studies primarily funded by a noncommercial source. The types of interventions studied differed based on funding source, with 95.4% of industry trials studying a drug or device. In addition, industry-funded CE studies were associated with the fewest pediatric subjects (P<0.001), the largest anticipated sample size (P<0.001), and the shortest study duration (P<0.001).

Conclusions

In this sample of studies examining high priority areas for CE research, less than a quarter are CE studies and the majority is supported by government and nonprofits. The low prevalence of CE research exists across CE studies with a broad array of interventions and characteristics.     

Synthesis and glycosidase inhibitory activities of 2-(aminoalkyl)pyrrolidine-3,4-diol derivatives

Several 2-(aminomethyl)-and 2-(2-aminoethyl)-pyrrolidine-3,4-diol derivatives have been assayed for their inhibitory activities towards glycosidases. Good inhibitors of alpha-mannosidases must have the (2R,3R,4S) configuration and possess 2-(benzylamino)methyl substituents. Stereomers with the (2S,3R,4S) configuration are also competitive inhibitors of alpha-mannosidases, but less potent as they share the configuration of C(1), C(2), C(3) of beta-D-mannosides rather than that of alpha-D-mannosides. Interestingly, (2S,3R,4S)-2-[2-[(4-phenyl)phenylamino]ethyl]pyrrolidine-3,4-diol (12g) inhibits several enzymes, for instance alpha-L-fucosidase from bovine epididymis (K(i)=6.5microM, competitive), alpha-galactosidase from bovine liver (K(i)=5microM, mixed) and alpha-mannosidase from jack bean (K(i)=102microM, mixed). Diamines such as (2R,3S,4R)-2-[2-(phenylamino) or 2-(benzylamino)ethyl]pyrrolidine-3,4-diol (ent-12a, ent-12b) inhibit beta-glucosidase from almonds (K(i)=13-40microM, competitive).     

Rapid Method for Enumeration of Viable Legionella pneumophila and Other Legionella spp. in Water

A sensitive and specific method has been developed to enumerate viable L. pneumophila and other Legionella spp. in water by epifluorescence microscopy in a short period of time (a few hours). This method allows the quantification of L. pneumophila or other Legionella spp. as well as the discrimination between viable and nonviable Legionella. It simultaneously combines the specific detection of Legionella cells using antibodies and a bacterial viability marker (ChemChrome V6), the enumeration being achieved by epifluorescence microscopy. The performance of this immunological double-staining (IDS) method was investigated in 38 natural filterable water samples from different aquatic sources, and the viable Legionella counts were compared with those obtained by the standard culture method. The recovery rate of the IDS method is similar to, or higher than, that of the conventional culture method. Under our experimental conditions, the limit of detection of the IDS method was <176 Legionella cells per liter. The examination of several samples in duplicates for the presence of L. pneumophila and other Legionella spp. indicated that the IDS method exhibits an excellent intralaboratory reproducibility, better than that of the standard culture method. This immunological approach allows rapid measurements in emergency situations, such as monitoring the efficacy of disinfection shock treatments. Although its field of application is as yet limited to filterable waters, the double-staining method may be an interesting alternative (not equivalent) to the conventional standard culture methods for enumerating viable Legionella when rapid detection is required.     

Hydrogen peroxide evolution during V-UV photolysis of water.

Hydrogen peroxide evolution during the vacuum-ultraviolet (V-UV, 172 nm) photolysis of water is considerably affected by the presence of oxalic acid (employed as a model water pollutant) and striking differences are observed in the absence and in the presence of dioxygen.     

Nox1-dependent superoxide production controls colon adenocarcinoma cell migration

Reactive oxygen species are well-known mediators of various biological responses. Recently, new homologues of the catalytic subunit of NADPH oxidase have been discovered in non-phagocytic cells. These new homologues (Nox1-Nox5) produce low levels of superoxides compared to the phagocytic homologue Nox2/gp91phox. Using Nox1 siRNA, we show that Nox1-dependent superoxide production affects the migration of HT29-D4 colonic adenocarcinoma cells on collagen-I. Nox1 inhibition or down-regulation led to a decrease of superoxide production and alpha 2 beta 1 integrin membrane availability. An addition of arachidonic acid stimulated Nox1-dependent superoxide production and HT29-D4 cell migration. Pharmacological evidences using phospholipase A2, lipoxygenases and protein kinase C inhibitors show that upstream regulation of Nox1 relies on arachidonic acid metabolism. Inhibition of 12-lipoxygenase decreased basal and arachidonic acid induced Nox1-dependent superoxide production and cell migration. Migration and ROS production inhibited by a 12-lipoxygenase inhibitor were restored by the addition of 12(S)-HETE, a downstream product of 12-lipoxygenase. Protein kinase C delta inhibition by rottlerin (and also GO6983) prevented Nox1-dependent superoxide production and inhibited cell migration, while other protein kinase C inhibitors were ineffective. We conclude that Nox1 activation by arachidonic acid metabolism occurs through 12-lipoxygenase and protein kinase C delta, and controls cell migration by affecting integrin alpha 2 subunit turn-over.     

Efficient regioselective chemical modifications of maltotriose: an easy access to oligosaccharidic scaffold

Regioselective chlorination of fully unprotected maltotriose has given in high yield 1^I,2^I-III,3^I-III,4^III-octa-O-acetyl-6^I-III-trichloro-6^I-III-trideoxymaltotriose. Moreover, regioselective ditritylation of methyl β-maltotrioside has provided the two regioselectively C₆-disubstituted trisaccharides. Selective deprotection of these new compounds gives the corresponding diol and halogenated analogues, respectively, in good yield. All compounds have been completely characterized and the substitution pattern in the oligosaccharidic sequence has been elucidated. A new family of amphiphilic carbohydrates, namely the 6-deoxy-6-alkylthiomaltotriose derivatives, bearing either two or three thioalkyl hydrophobic chains, respectively, has been synthesized. Critical micellar concentration (CMC) values as well as the antimicrobial properties have been evaluated for amphiphilic compounds.