Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.     

Synthesis and antimalarial activity of new 4-amino-7-chloroquinolyl amides,sulfonamides, ureas and thioureas

We report the synthesis and in vitro antimalarial activities of more than 50 7-chloro-4-aminoquinolyl-derived sulfonamides 3–8 and 11–26, ureas 19–22, thioureas 23–26, and amides 27–54. Many of the CQ analogues prepared for this study showed submicromolar antimalarial activity versus HB3 (chloroquine sensitive) and Dd2 (chloroquine resistant strains of Plasmodium falciparum) and low resistance indices were obtained in most cases. Systematic variation of the side chain length and introduction of fluorinated aliphatic and aromatic termini revealed promising leads that overcome CQ resistance. In particular, sulfonamide 3 exhibiting a short side chain with a terminal dansyl moiety combined high antiplasmodial potency with a low resistance index and showed IC₅₀s of 17.5 and 22.7 nM against HB3 and Dd2 parasites.     

Density functional theory investigation of cocaine water complexes

Twenty cocaine–water complexes were studied using density functional theory (DFT) B3LYP/6-311++G** level to understand their geometries, energies, vibrational frequencies, charge transfer and topological parameters. Among the 20 complexes, 12 are neutral and eight are protonated in the cocaine-water complexes. Based on the interaction energy, the protonated complexes are more stable than the neutral complexes. In both complexes, the most stable structure involves the hydrogen bond with water at nitrogen atom in the tropane ring and C?=?O groups in methyl ester. Carbonyl groups in benzoyl and methyl ester is the most reactive site in both forms and it is responsible for the stability order. The calculated topological results show that the interactions involved in the hydrogen bond are electrostatic dominant. Natural bond orbital (NBO) analysis confirms the presence of hydrogen bond and it supports the stability order. Atoms in molecules (AIM) and NBO analysis confirms the C-H?·?·?·?O hydrogen bonds formed between the cocaine-water complexes are blue shifted in nature.     

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl-CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells.     

The N-acylation-phosphodiesterase pathway and cell signalling

Long-chain N-acylethanolamines (NAEs) elicit a variety of biological and pharmacological effects, Anandamide (20:4n-6 NAE) and other polyunsaturated NAEs bind to the cannabinoid receptor and may thus serve as highly specific lipid mediators of cell signalling. NAEs can be formed by phospholipase D-catalyzed hydrolysis of N-acylethanolamine phospholipids or by direct condensation of ethanolamine and fatty acid, So far, most of the latter biosynthetic activity has been shown to be the reverse reaction of the NAE amidohydrolase that catalyzes NAE degradation. Thus, increasing evidence supports the hypothesis that the N-acylation-phosphodiesterase pathway yields not only saturated-monounsaturated NAEs, but polyunsaturated ones, including anandamide, as well.     

Computer modeling of deployment and mechanical expansion of neurovascular flow diverter in patient-specific intracranial aneurysms

Flow diverter (FD) is an emerging neurovascular device based on self-expandable braided stent for treating intracranial aneurysms. Variability in FD outcome has underscored a need for investigating the hemodynamic effect of fully deployed FD in patient-specific aneurysms. Image-based computational fluid dynamics, which can provide important hemodynamic insight, requires accurate representation of FD in deployed states. We developed a finite element analysis (FEA) based workflow for simulating mechanical deployment of FD in patient-specific aneurysms. We constructed FD models of interlaced wires emulating the Pipeline Embolization Device, using 3D finite beam elements to account for interactions between stent strands, and between the stent and other components. The FEA analysis encompasses all steps that affect the final deployed configuration including stent crimping, delivery and expansion. Besides the stent, modeling also includes key components of the FD delivery system such as microcatheter, pusher, and distal coil. Coordinated maneuver of these components allowed the workflow to mimic clinical operation of FD deployment and to explore clinical strategies. The workflow was applied to two patient-specific aneurysms. Parametric study indicated consistency of the deployment result against different friction conditions, but excessive intra-stent friction should be avoided. This study demonstrates for the first time mechanical modeling of braided FD stent deployment in cerebral vasculature to produce realistic deployed configuration, thus paving the way for accurate CFD analysis of flow diverters for reliable prediction and optimization of treatment outcome.     

Downregulation of connexin40 and increased prevalence of atrial arrhythmias in transgenic mice with cardiac-restricted overexpression of tumor necrosis factor

Atrial arrhythmias, primarily atrial fibrillation, have been independently associated with structural remodeling and with inflammation. We hypothesized that sustained inflammatory signaling by tumor necrosis factor (TNF) would lead to alterations both in underlying atrial myocardial structure and in atrial electrical conduction. We performed ECG recording, intracardiac electrophysiology studies, epicardial mapping, and connexin immunohistochemical analyses on transgenic mice with targeted overexpression of TNF in the cardiac compartment (MHCsTNF) and on wild-type (WT) control mice (age 8-16 wk). Atrial and ventricular conduction abnormalities were always evident on ECG in MHCsTNF mice, including a shortened atrioventricular interval with a wide QRS duration secondary to junctional rhythm. Supraventricular arrhythmias were observed in five of eight MHCsTNF mice, whereas none of the mice demonstrated ventricular arrhythmias. No arrhythmias were observed in WT mice. Left ventricular conduction velocity during apical pacing was similar between the two mouse groups. Connexin40 was significantly downregulated in MHCsTNF mice. In contrast, connexin43 density was not significantly altered in MHCsTNF mice, but rather dispersed away from the intercalated disks. In conclusion, sustained inflammatory signaling contributed to atrial structural remodeling and downregulation of connexin40 that was associated with an increased prevalence of atrial arrhythmias.     

Cytostatic drug activity in plasma, a bioassay for detecting mutagenicity of directly and indirectly acting chemicals, an evaluation of 20 chemicals

We have utilized in vivo drug metabolism for detecting the mutagenicity of known indirectly-acting chemical mutagens. Exponentially growing Chinese hamster ovary (CHO) cells were incubated with plasma derived from treated rats containing active metabolites of the test chemicals. The genotoxicity was assessed by the 18 of tested chemical mutagens. Plasma from rats treated with known non-mutagens did not increase the frequencies of SCEs. The results indicate that this method could be useful for the demonstration of genotoxicity of chemicals which need metabolic activation to be effective, and especially those which are not effective, when in vitro activation conditions (S9 mixture) are used.     

Isolation and molecular characterization of genetically diverse antagonistic, diazotrophic red-pigmented vibrios from different mangrove rhizospheres

Genetic diversity of red-pigmented vibrios from different mangrove rhizospheres ( Porteresia coarctata, Avicennia marina and Rhizophora mucronata ) collected from Pichavaram mangrove, India was investigated. Twenty red–pink pigmented strains were isolated, 16S rRNA gene analyses indicted that these isolates belong to the genus Vibrio and were phylogenetically closely related to the type strains of Vibrio rhizosphaerae and Vibrio ruber . The rep-PCR analysis using GTG₅ and BOX primers had similar groupings, and segregated these pigmented Vibrio isolates including two type strains into seven unique genotypic groups (REP groups A1–A7). The rhizosphere of P. coarctata harbors highly genetically diverse groups of red-pigmented vibrios compared to other plants. Multilocus sequence analysis using four genetic loci ( pyrH, recA, rpoA , 16S rRNA) clearly supported the hypothesis that strains MSSRF38 (REP group A5) and MSSRF39 (REP group A6) could represent new Vibrio species. Biological functions of these vibrios were also determined and it was found that all these isolates have antagonistic activity against phytopathogens, and isolates belonging to REP groups A5 and A6 were positive for nifH gene by PCR. In conclusion, this study for the first time demonstrates the occurrence of genetically diverse groups of antagonistic, diazotrophic red-pigmented vibrios from different mangrove plants and suggests a new ecological role for vibrios as heterotrophic plant associated rhizobacteria.     

Ketomethylureas. A new class of angiotensin converting enzyme inhibitors

The design rationale for a new series of tripeptide derived angiotensin converting enzyme (ACE) inhibitors, which we term "ketomethylureas", is described. Analogs of tripeptide substrates (i.e. N-benzoyl-Phe-Ala-Pro) in which the nitrogen atom of the scissile amide bond and the adjacent asymmetric carbon atom of the penultimate amino acid residue are formally transposed give rise to this novel class of inhibitors. The most potent ketomethylureas inhibit ACE with I50 values in the nM range.