Sema3E/PlexinD1 regulates the migration of hem-derived Cajal-Retzius cells in developing cerebral cortex

International audienceDuring the development of the cerebral cortex, Cajal-Retzius (CR) cells settle in the preplate and coordinate the precise growth of the neocortex. Indeed, CR cells migrate tangentially from specific proliferative regions of the telencephalon (for example, the cortical hem (CH)) to populate the entire cortical surface. This is a very finely tuned process regulated by an emerging number of factors that has been sequentially revealed in recent years. However, the putative participation of one of the major families of axon guidance molecules in this process, the Semaphorins, was not explored. Here we show that Semaphorin-3E (Sema3E) is a natural negative regulator of the migration of PlexinD1-positive CR cells originating in the CH. Our results also indicate that Sema3E/PlexinD1 signalling controls the motogenic potential of CR cells in vitro and in vivo. Indeed, absence of Sema3E/PlexinD1 signalling increased the migratory properties of CR cells. This modulation implies negative effects on CXCL12/CXCR4 signalling and increased ADF/Cofilin activity

DFT study of the radical scavenging activity of isoxanthohumol, humulones (α-acids), and iso-α-acids from beer

Humulones and iso-humulones are potent natural antioxidants found in beer. In this study, density functional theory (DFT) method was applied for elucidating the structure-antioxidant activity relationship and molecular mechanism of antioxidant activity of eight bioactive humulones previously identified in different beer samples: isoxanthohumol, (R)- and (S)-adhumulone, cis- and trans-iso-adhumulone, cis- and trans-iso-n-humulone, and desdimethyl-octahydro-iso-cohumulone. The calculated bond dissociation enthalpies (BDEs) suggest that desdimethyl-octahydro-iso-cohumulone was the most potent compound with BDEs 5.1 and 23.9 kJ/mol lower compared to the values for resveratrol in gas phase and water, respectively. The enolic –OH is the most reactive site for hydrogen atom transfer (HAT). The presence of β-keto group with respect to enolic –OH diminishes the HAT potency via the formation of a strong intramolecular hydrogen bond. Another common antioxidant mechanism, single electron transfer followed by proton transfer (SET-PT), is only feasible for isoxanthohumol. The results of this study indicate a strong correlation between the increased antioxidant activity of beer products and the higher content of reduced iso-α-acids

RP-HPLC-DAD metoda za određivanje olmesartan medoksomila kao čiste supstancije i u tabletama izloženih razgradnji

A simple, sensitive and precise RP-HPLC-DAD method was developed and validated for the determination of olmesartan medoxomil (AT-II receptor blocker) in the presence of its degradation products. Olmesartan medoxomil and all the degradation products were resolved on a C18 column with the mobile phase composed of methanol, acetonitrile and water (60:15:25, V/V/V, pH 3.5 by orthophosphoric acid) at 260 nm using a photodiode array detector. The method was linear over the concentration range of 1–18 µg mL 1 and precise with RSD 2.0 for each peak and sensitive with LOD 0.03 µg mL−1 and LOQ 0.1 µg mL−1. The method was used to study the drug degradation behavior under forced conditions. Four degradation products (DP-I, II, III, IV) were formed during the degradation study in 0.1 mol L−1 HCl whereas only DP-I, II and III were formed in water, 0.01 mol L−1 NaOH and 3 % H2O2. No significant thermal or photolytic degradation was observed in solid drug. The method was applied successfully for the assay of olmesartan medoxomil in the tablet dosage form.U ovom radu razvijena je i validirana jednostavna, osjetljiva i precizna RP-HPLC-DAD metoda za određivanje olmesartan medoksomila (inhibitor AT-II receptora) u prisutnosti njegovih razgradnih produkata. Olmesartan medoksomil i razgradni produkti kromatografirani su na C18 koloni uz mobilnu fazu metanol/ acetonitril/vo da (60:15:25 V/V/V; pH 3,5 podešen ortofosfornom kiselinom) pri 260 nm uz detektor s fotodiodnim nizom. Metoda je linearna u koncentracijskom rasponu 1–18 µg mL 1 i precizna s RSD < 1 % tijekom ispitivanja repetabilnosti i intermedijarne ponovljivosti. Povrat od 99,3 ± 0,9 do 100,8 ± 1,2 % dokazuje točnost metode. Razvijena metoda je specifična na što ukazuje kromatografsku rezoluciju veću od 2,0 i osjetljiva (LOD = 0,03 µg mL−1 i LOQ = 0,1 µg mL−1). Metoda je upotrebljena za praćenje razgradnje olmesartan medoksomila u uvjetima potencirane razgradnje. U 0,1 mol L−1 HCl detektirana su četiri razgradna produkta (DP-I, II, III, IV), a u vodi, 0,01 mol L−1 NaOH i 3 % H2O2 samo DP-I, II i III. U čvrstom agregatnom stanju nije primjećena značajna termička ni fotolitička razgradnja ljekovite tvari. Metoda je uspješno primijenjena za određivanje olmesartan medoksomila u tabletama

Practical computational toolkits for dendrimers and dendrons structure design

Dendrimers and dendrons offer an excellent platform for developing novel drug delivery systems and medicines. The rational design and further development of these repetitively branched systems are restricted by difficulties in scalable synthesis and structural determination, which can be overcome by judicious use of molecular modelling and molecular simulations. A major difficulty to utilise in silico studies to design dendrimers lies in the laborious generation of their structures. Current modelling tools utilise automated assembly of simpler dendrimers or the inefficient manual assembly of monomer precursors to generate more complicated dendrimer structures. Herein we describe two novel graphical user interface (GUI) toolkits written in Python that provide an improved degree of automation for rapid assembly of dendrimers and generation of their 2D and 3D structures. Our first toolkit uses the RDkit library, SMILES nomenclature of monomers and SMARTS reaction nomenclature to generate SMILES and mol files of dendrimers without 3D coordinates. These files are used for simple graphical representations and storing their structures in databases. The second toolkit assembles complex topology dendrimers from monomers to construct 3D dendrimer structures to be used as starting points for simulation using existing and widely available software and force fields. Both tools were validated for ease-of-use to prototype dendrimer structure and the second toolkit was especially relevant for dendrimers of high complexity and size.Peer reviewe

Bacteriophage application restores ethanol fermentation characteristics disrupted by Lactobacillusfermentum

BACKGROUND: Contamination of corn mash by lactic acid bacteria (LAB) reduces the efficiency of the ethanol fermentation process. The industry relies heavily on antibiotics for contamination control and there is a need to develop alternative methods. The goals of this study were to determine the diversity and abundance of bacteria contaminating commercial ethanol fermentations, and to evaluate the potential of anti-LAB bacteriophages in controlling production losses. RESULTS: Bacterial populations in 27 corn mash samples collected from nine different commercial plants were determined by pyrosequencing of 16S rRNA amplicons. The results showed that the most abundant bacteria (>50 % of total population) in 24 of the 27 samples included LAB genera such as Lactobacillus, Streptococcus, Lactococcus, Weissella, Enterococcus, and Pediococcus. Lactobacillus was identified as the most prevalent genus at all fermentation stages in all plants, accounting for between 2.3 and 93.7 % of each population and constituting the major genus (>50 %) in nine samples from five plants and the most abundant genus in five other samples. Lactobacillus species, including L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri were the most well-represented species. Two bacteriophages that target L. fermentum strains from ethanol plants, vB_LfeS_EcoSau and vB_LfeM_EcoInf (EcoSau and EcoInf), were isolated and characterized as a siphophage and a myophage, respectively. Analysis of the 31,703 bp genome of EcoSau revealed its similarity to the P335-like phage group, and the 106,701 bp genome of phage EcoInf was determined to be a novel phage type despite its distant relationship to the SPO1-like phages. Addition of phages EcoSau and EcoInf to L. fermentum-contaminated corn mash fermentation models restored the yields of ethanol and reduced levels of residual glucose, lactic acid, and acetic acid to that comparable to the infection-free control. CONCLUSIONS: This study provides detailed insight into the microbiota contaminating commercial ethanol fermentations, and highlights the abundance of LAB, especially L. delbrueckii, L. fermentum, L. mucosae, and L. reuteri, in the process. This study suggests that phages with broad coverage of major LAB species can be applied directly to corn mash for antibiotic-free control of contamination in the ethanol fermentation industry. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-015-0325-9) contains supplementary material, which is available to authorized users

Conformation-dependent GAD65 autoantibodies in diabetes

Aims/hypothesis. Conformation-dependent autoantibodies directed against GAD65 are markers of Type 1 diabetes. In this study we aimed to determine whether the substitution of GAD65 with GAD67 amino acids would affect the binding of conformation-dependent GAD65 autoantibodies. Methods. We used PCR-based site-directed mutagenesis to generate a series of mutated GAD65 cDNA constructs in which specific GAD65 coding sequences for regions of the protein critical for autoantibody binding were replaced with GAD67 coding sequences. Results. The introduction of a point mutation at position 517, substituting glutamic acid with proline, markedly reduced the binding of disease-associated GAD65 antibodies. The binding of GAD65 antibodies to the E517P mutant was reduced in the sera of all newly diagnosed Type 1 diabetes patients (n=85) by a mean of 72% (p<0.0001) compared with binding to wild-type GAD65. Patients with latent autoimmune diabetes in adults (n=24) showed a similar reduction in binding (79% reduction, p<0.0001). First-degree relatives who subsequently progressed to Type 1 diabetes (n=12) showed a reduction in binding of 80% compared with a reduction of only 65% among relatives who had not progressed to disease (n=38; p=0.025). In healthy GAD65Ab-positive individuals who did not progress to diabetes during a 9-year follow-up period (n=51), binding to GAD65-E517P was reduced by only 28% compared with binding to wild-type GAD65. Conclusions/interpretation. Differences in autoantibody binding to wild-type GAD65 versus GAD65-E517P may provide predictive information about Type 1 diabetes risk beyond that provided by the presence or absence of GAD65 autoantibodies. Lack of binding to mutant GAD65-E517P defines GAD65-positive individuals who are at higher risk of developing diabetes

RP-LC and HPTLC Methods for the Determination of Olmesartan Medoxomil and Hydrochlorothiazide in Combined Tablet Dosage Forms

Two new, rapid, precise, accurate and specific chromatographic methods were described for the simultaneous determination of olmesartan medoxomil and hydrochlorothiazide in combined tablet dosage forms. The first method was based on reversed phase liquid chromatography using an Eurosphere 100 RP C18 column (250 × 4.6 mm ID, 5 μm). The mobile phase was methanol–0.05% o-phosphoric acid (60:40 v/v) at a flow rate of 1.0 mL min−1. Commercially available tablets and laboratory mixtures containing both drugs were assayed and detected using a UV detector at 270 nm. The second method involved silica gel 60 F254 high performance thin layer chromatography and densitometric detection at 254 nm using acetonitrile–ethyl acetate–glacial acid (7:3:0.4 v/v/v) as the mobile phase. Calibration curves ranged between 200–600 and 125–375 ng spot−1 for olmesartan and hydrochlorothiazide, respectively

A Spatio-Temporal Analysis of Matrix Protein and Nucleocapsid Trafficking during Vesicular Stomatitis Virus Uncoating

To study VSV entry and the fate of incoming matrix (M) protein during virus uncoating we used recombinant viruses encoding M proteins with a C-terminal tetracysteine tag that could be fluorescently labeled using biarsenical (Lumio) compounds. We found that uncoating occurs early in the endocytic pathway and is inhibited by expression of dominant-negative (DN) Rab5, but is not inhibited by DN-Rab7 or DN-Rab11. Uncoating, as defined by the separation of nucleocapsids from M protein, occurred between 15 and 20 minutes post-entry and did not require microtubules or an intact actin cytoskeleton. Unexpectedly, the bulk of M protein remained associated with endosomal membranes after uncoating and was eventually trafficked to recycling endosomes. Another small, but significant fraction of M distributed to nuclear pore complexes, which was also not dependent on microtubules or polymerized actin. Quantification of fluorescence from high-resolution confocal micrographs indicated that after membrane fusion, M protein diffuses across the endosomal membrane with a concomitant increase in fluorescence from the Lumio label which occurred soon after the release of RNPs into the cytoplasm. These data support a new model for VSV uncoating in which RNPs are released from M which remains bound to the endosomal membrane rather than the dissociation of M protein from RNPs after release of the complex into the cytoplasm following membrane fusion

Pichia pastoris versus Saccharomyces cerevisiae:a case study on the recombinant production of human granulocyte-macrophage colony-stimulating factor

BACKGROUND: Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is a glycoprotein that has been approved by the FDA for the treatment of neutropenia and leukemia in combination with chemotherapies. Recombinant hGM-CSF is produced industrially using the baker's yeast, Saccharomyces cerevisiae, by large-scale fermentation. The methylotrophic yeast, Pichia pastoris, has emerged as an alternative host cell system due to its shorter and less immunogenic glycosylation pattern together with higher cell density growth and higher secreted protein yield than S. cerevisiae. In this study, we compared the pipeline from gene to recombinant protein in these two yeasts. RESULTS: Codon optimization in silico for both yeast species showed no difference in frequent codon usage. However, rhGM-CSF expressed from S. cerevisiae BY4742 showed a significant discrepancy in molecular weight from those of P. pastoris X33. Analysis showed purified rhGM-CSF species with molecular weights ranging from 30 to more than 60 kDa. Fed-batch fermentation over 72 h showed that rhGM-CSF was more highly secreted from P. pastoris than S. cerevisiae (285 and 64 mg total secreted protein/L, respectively). Ion exchange chromatography gave higher purity and recovery than hydrophobic interaction chromatography. Purified rhGM-CSF from P. pastoris was 327 times more potent than rhGM-CSF from S. cerevisiae in terms of proliferative stimulating capacity on the hGM-CSF-dependent cell line, TF-1. CONCLUSION: Our data support a view that the methylotrophic yeast P. pastoris is an effective recombinant host for heterologous rhGM-CSF production