Monooxime reactivators of acetylcholinesterase with (E)-but-2-ene linker: preparation and reactivation of tabun- and paraoxon-inhibited acetylcholinesterase

Acetylcholinesterase reactivators are crucial antidotes for the treatment of organophosphate intoxication. Fifteen new monooxime reactivators of acetylcholinesterase with a (E)-but-2-ene linker were developed in an effort to extend the properties of K-oxime (E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide (K203). The known reactivators (pralidoxime, HI-6, obidoxime, K075, K203) and the new compounds were tested in vitro on a model of tabun- and paraoxon-inhibited AChE. Monooxime reactivators were not able to exceed the best known compounds for tabun poisoning, but some of them did show reactivation comparable with known compounds for paraoxon poisoning. However, extensive differences were found by a SAR study for various substitutions on the non-oxime part of the reactivator molecule.     

Acinetobacter bohemicus sp. nov. widespread in natural soil and water ecosystems in the Czech Republic

We investigated the taxonomic status of a phenetically unique group of 25 Acinetobacter strains which were isolated from multiple soil and water samples collected in natural ecosystems in the Czech Republic. Based on the comparative sequence analyses of the rpoB, gyrB, and 16S rRNA genes, the strains formed a coherent and well separated branch within the genus Acinetobacter. The genomic uniqueness of the group at the species level was supported by the low average nucleotide identity values (≤77.37%) between the whole genome sequences of strain ANC 3994^T (NCBI accession no. APOH00000000) and the representatives of the known Acinetobacter species. Moreover, all 25 strains created a tight cluster clearly separated from all hitherto described species based on whole-cell protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and shared a unique combination of metabolic and physiological properties. The capacity to assimilate l-histidine and the inability to grow at 35 °C differentiated them from their phenotypically closest neighbor, Acinetobacter johnsonii. We conclude that the 25 strains represent a novel Acinetobacter species, for which the name Acinetobacter bohemicus sp. nov. is proposed. The type strain of A. bohemicus is ANC 3994^T (=CIP 110496^T = CCUG 63842^T = CCM 8462^T).     

Omega-3 phospholipids from fish suppress hepatic steatosis by integrated inhibition of biosynthetic pathways in dietary obese mice

Non-alcoholic fatty liver disease (NAFLD) accompanies obesity and insulin resistance. Recent meta-analysis suggested omega-3 polyunsaturated fatty acids DHA and EPA to decrease liver fat in NAFLD patients. Anti-inflammatory, hypolipidemic, and insulin-sensitizing effects of DHA/EPA depend on their lipid form, with marine phospholipids showing better efficacy than fish oils. We characterized the mechanisms underlying beneficial effects of DHA/EPA phospholipids, alone or combined with an antidiabetic drug, on hepatosteatosis. C57BL/6N mice were fed for 7 weeks an obesogenic high-fat diet (cHF) or cHF-based interventions: (i) cHF supplemented with phosphatidylcholine-rich concentrate from herring (replacing 10% of dietary lipids; PC), (ii) cHF containing rosiglitazone (10 mg/kg diet; R), or (iii) PC + R. Metabolic analyses, hepatic gene expression and lipidome profiling were performed. Results showed that PC and PC + R prevented cHF-induced weight gain and glucose intolerance, while all interventions reduced abdominal fat and plasma triacylglycerols. PC and PC + R also lowered hepatic and plasma cholesterol and reduced hepatosteatosis. Microarray analysis revealed integrated down-regulation of hepatic lipogenic and cholesterol biosynthesis pathways by PC, while R-induced lipogenesis was fully counteracted in PC + R. Gene expression changes in PC and PC + R were associated with preferential enrichment of hepatic phosphatidylcholine and phosphatidylethanolamine fractions by DHA/EPA. The complex down-regulation of hepatic lipogenic and cholesterol biosynthesis genes and the antisteatotic effects were unique to DHA/EPA-containing phospholipids, since they were absent in mice fed soy-derived phosphatidylcholine. Thus, inhibition of lipid and cholesterol biosynthesis associated with potent antisteatotic effects in the liver in response to DHA/EPA-containing phospholipids support their use in NAFLD prevention and treatment.     

Postprandial Oxidative Stress and Gastrointestinal Hormones: Is There a Link?

Background

Abnormal postprandial elevation of plasma glucose and lipids plays an important role in the pathogenesis of diabetes and strongly predicts cardiovascular mortality. In patients suffering from type 2 diabetes (T2D) postprandial state is associated with oxidative stress, cardiovascular risk and, probably, with impairment of both secretion and the effect of gastrointestinal peptides. Evaluating postprandial changes of gastrointestinal hormones together with changes in oxidative stress markers may help to understand the mechanisms behind the postprandial state in diabetes as well as suggest new preventive and therapeutical strategies.

Methods

A standard meal test has been used for monitoring the postprandial concentrations of gastrointestinal hormones and oxidative stress markers in patients with T2D (n = 50) compared to healthy controls (n = 50). Blood samples were drawn 0, 30, 60, 120 and 180 minutes after the standard meal.

Results

Both basal and postprandial plasma concentrations of glucose and insulin proved to be significantly higher in patients with T2D, whereas plasma concentrations of ghrelin showed significantly lower values during the whole meal test. In comparison with healthy controls, both basal and postprandial concentrations of almost all other gastrointestinal hormones and lipoperoxidation were significantly increased while ascorbic acid, reduced glutathione and superoxide dismutase activity were decreased in patients with T2D. A positive relationship was found between changes in GIP and those of glucose and immunoreactive insulin in diabetic patients (p<0.001 and p<0.001, respectively) and between changes in PYY and those of glucose (p<0.01). There was a positive correlation between changes in GIP and PYY and changes in ascorbic acid in patients with T2D (p<0.05 and p<0.001, respectively).

Conclusion/Interpretation

Apart from a positive relationship of postprandial changes in GIP and PYY with changes in ascorbic acid, there was no direct link observed between gastrointestinal hormones and oxidative stress markers in diabetic patients.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01572402","term_id":"NCT01572402"}}NCT01572402     

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.     

Identification of bacteria utilizing biphenyl, benzoate, and naphthalene in long-term contaminated soil

Bacteria were identified associated with biodegradation of aromatic pollutants biphenyl, benzoate, and naphthalene in a long-term polychlorinated biphenyl- and polyaromatic hydrocarbon-contaminated soil. In order to avoid biases of culture-based approaches, stable isotope probing was applied in combination with sequence analysis of 16 S rRNA gene pyrotags amplified from (13)C-enriched DNA fractions. Special attention was paid to pyrosequencing data analysis in order to eliminate the errors caused by either generation of amplicons (random errors caused by DNA polymerase, formation of chimeric sequences) or sequencing itself. Therefore, sample DNA was amplified, sequenced, and analyzed along with the DNA of a mock community constructed out of 8 bacterial strains. This warranted that appropriate tools and parameters were chosen for sequence data processing. (13)C-labeled metagenomes isolated after the incubation of soil samples with all three studied aromatics were largely dominated by Proteobacteria, namely sequences clustering with the genera Rhodanobacter Burkholderia, Pandoraea, Dyella as well as some Rudaea- and Skermanella-related ones. Pseudomonads were mostly labeled by (13)C from naphthalene and benzoate. The results of this study show that many biphenyl/benzoate-assimilating bacteria derive carbon also from naphthalene, pointing out broader biodegradation abilities of some soil microbiota. The results also demonstrate that, in addition to traditionally isolated genera of degradative bacteria, yet-to-be cultured bacteria are important players in bioremediation. Overall, the study contributes to our understanding of biodegradation processes in contaminated soil. At the same time our results show the importance of sequencing and analyzing a mock community in order to more correctly process and analyze sequence data.     

Genetic Basis of a Violation of Dollo’s Law: Re-Evolution of Rotating Sex Combs in Drosophila bipectinata

Phylogenetic analyses suggest that violations of “Dollo’s law”—that is, re-evolution of lost complex structures—do occur, albeit infrequently. However, the genetic basis of such reversals has not been examined. Here, we address this question using the Drosophila sex comb, a recently evolved, male-specific morphological structure composed of modified bristles. In some species, sex comb development involves only the modification of individual bristles, while other species have more complex “rotated” sex combs that are shaped by coordinated migration of epithelial tissues. Rotated sex combs were lost in the ananassae species subgroup and subsequently re-evolved, ∼12 million years later, in Drosophila bipectinata and its sibling species. We examine the genetic basis of the differences in sex comb morphology between D. bipectinata and D. malerkotliana, a closely related species with a much simpler sex comb representing the ancestral condition. QTL mapping reveals that >50% of this difference is controlled by one chromosomal inversion that covers ∼5% of the genome. Several other, larger inversions do not contribute appreciably to the phenotype. This genetic architecture suggests that rotating sex combs may have re-evolved through changes in relatively few genes. We discuss potential developmental mechanisms that may allow lost complex structures to be regained.     

The order of PDZ3 and TrpCage in fusion chimeras determines their properties—a biophysical characterization

Most of the structural proteins known today are composed of domains that carry their own functions while keeping their structural properties. It is supposed that such domains, when taken out of the context of the whole protein, can retain their original structure and function to a certain extent. Information on the specific functional and structural characteristics of individual domains in a new context of artificial fusion proteins may help to reveal the rules of internal and external domain communication. Moreover, this could also help explain the mechanism of such communication and address how the mutual allosteric effect plays a role in a such multi‐domain protein system. The simple model system of the two‐domain fusion protein investigated in this work consisted of a well‐folded PDZ3 domain and an artificially designed small protein domain called Tryptophan Cage (TrpCage). Two fusion proteins with swapped domain order were designed to study their structural and functional features as well as their biophysical properties. The proteins composed of PDZ3 and TrpCage, both identical in amino acid sequence but different in composition (PDZ3‐TrpCage, TrpCage‐PDZ3), were studied using circualr dichroism (CD) spectrometry, analytical ultracentrifugation, and molecular dynamic simulations. The biophysical analysis uncovered different structural and denaturation properties of both studied proteins, revealing their different unfolding pathways and dynamics.     

A photobioreactor system for precision cultivation of photoautotrophic microorganisms and for high-content analysis of suspension dynamics

Small-scale photobioreactors for cultivation of photoautotrophic microbes are required for precise characterization of the growth parameters of wild-type and engineered strains of these organisms, for their screening, and for optimization of culture conditions. Here, we describe the design and use of a flat-cuvette photobioreactor that allows accurate control of culture irradiance, temperature, pH, and gas composition combined with real-time monitoring by a built-in fluorometer and densitometer. The high-power LED light source generates precise irradiance levels that are programmed by user-designed protocols. The irradiance, temperature, and gas composition may be static or dynamically modulated, while optical density and pH may be stabilized in turbidostat and pH-stat modes, respectively. We demonstrate that the instrument is able to detect minute variations of growth caused, for example, by sudden dilution or by circadian rhythms. The sensitivity of the instrument is sufficient to monitor suspension optical density as low as 10(-2). This newly designed photobioreactor can significantly contribute to the study and use of photoautotrophic microbes in systems biology and biotechnology.