Molecular and dimensional profiling of highly purified extracellular vesicles by fluorescence fluctuation spectroscopy.

Cells secrete extracellular vesicles (EVs) into their microenvironment that act as mediators of intercellular communication under physiological conditions and in this context also actively participate in spreading various diseases. Large efforts are currently made to produce reliable EV samples and to develop, improve, and standardize techniques allowing their biophysical characterization. Here, we used ultrafiltration and size-exclusion chromatography for the isolation and a model-free fluorescence fluctuation analysis for the investigation of the physical and biological properties of EVs secreted by mammalian cells.

Structure-based engineering of a minimal porin reveals loop-independent channel closure.

Porins, like outer membrane protein G (OmpG) of Escherichia coli, are ideal templates among ion channels for protein and chemical engineering because of their robustness and simple architecture. OmpG shows fast transitions between open and closed states, which were attributed to loop 6 (L6). As flickering limits single-channel-based applications, we pruned L6 by either 8 or 12 amino acids.

New insights into the analysis of the electrode kinetics of flavin adenine dinucleotide redox center of glucose oxidase immobilized on carbon electrodes.

New insights into electrochemical kinetics of the flavin adenine dinucleotide (FAD) redox center of glucose-oxidase (GlcOx) immobilized on reduced graphene oxide (rGO), single- and multiwalled carbon nanotubes (SW and MWCNT), and combinations of rGO and CNTs have been gained by application of Fourier transformed AC voltammetry (FTACV) and simulations based on a range of models. A satisfactory level of agreement between experiment and theory, and hence establishment of the best model to describe the redox chemistry of FAD, was achieved with the aid of automated e-science tools. Although still not perfect, use of Marcus theory with a very low reorganization energy (≤0.

Flexibility of the N-terminal mVDAC1 segment controls the channel's gating behavior.

Since the solution of the molecular structures of members of the voltage dependent anion channels (VDACs), the N-terminal α-helix has been the main focus of attention, since its strategic location, in combination with its putative conformational flexibility, could define or control the channel's gating characteristics. Through engineering of two double-cysteine mVDAC1 variants we achieved fixing of the N-terminal segment at the bottom and midpoint of the pore. Whilst cross-linking at the midpoint resulted in the channel remaining constitutively open, cross-linking at the base resulted in an "asymmetric" gating behavior, with closure only at one electric field's orientation depending on the channel's orientation in the lipid bilayer.

Chemical engineering of Mycobacterium tuberculosis dodecin hybrids.

The suitability for chemical engineering of the highly symmetrical Mycobacterium tuberculosis dodecin was investigated, its inner cavity providing a large compartment shields introduced compounds from bulk solvent. Hybrids were obtained by S-alkylation of cysteine mutants and characterized by spectroscopic methods, including the crystal structures of wild type and biohybrid dodecins.

Strategies and perspectives in ion-channel engineering.

Membranes form natural barriers that need to be permeable to diverse matter like ions and substrates. This permeability is controlled by ion-channel proteins, which have attracted great interest for pharmaceutical applications. Ion-channel engineering (ICE) modifies biological ion channels by chemical/biological synthetis means.

Reversible shape memory of nanoscale deformations in inherently conducting polymers without reprogramming.

By using inherently conducting polymers, we introduce new shape memory functionality for stimuli-responsive polymers. The shape memory process is unique in that it utilizes electrochemical control of the polymer redox state to conceal, and temporarily store, preformed nanoscale surface patterns, which can later be recalled. Unlike classical thermoset and thermoplastic shape memory polymers, the electrochemical control does not completely perturb the low entropy state of the deformed polymer chains, thus enabling the concept of reversible transition between the permanent and temporary shapes.

Structural characterization of a beta-turn mimic within a protein-protein interface.

β-Turns are secondary structure elements not only exposed on protein surfaces, but also frequently found to be buried in protein-protein interfaces. Protein engineering so far considered mainly the backbone-constraining properties of synthetic β-turn mimics as parts of surface-exposed loops. A β-turn mimic, Hot═Tap, that is available in gram amounts, provides two hydroxyl groups that enhance its turn-inducing properties besides being able to form side-chain-like interactions.

Structural and functional characterization of a synthetically modified OmpG.

Chemical modification of ion channels has recently attracted attention due to their potential use in stochastic sensing and neurobiology. Among the available channel templates stable β-barrel proteins have shown their potential for large scale chemical modifications due to their wide pore lumen. Ion-channel hybrids using the outer membrane protein OmpG were generated by S-alkylation with a synthetic modulator and functionally as well as structurally characterized.

[Intraoperative coronary artery spasm with cardiac arrest: cardiologic acute intervention].

Ventricular fibrillation occurred intraoperatively during a laparoscopy-assisted resection of the rectum in a 63-year-old patient. A coronary artery spasm was identified as the cause. It was possible to rectify the coronary artery spasm and restore a stable circulatory situation with an intracoronary injection of nitroglycerine.

Global transcriptional profiling demonstrates the combination of type I and type II interferon enhances antiviral and immune responses at clinically relevant doses.

A role for type II interferon (IFN-gamma) in resolving viral infection is suggested by the correlation of hepatitis C virus (HCV) clearance with enhancement of IFN-gamma-producing activated T cells in the resolution of acute HCV infection. Using vesicular stomatitis virus (VSV), a synergistic direct antiviral effect was documented using IFN-gamma1b and a potent, consensus type I IFN (IFN alfacon-1). Global expression profiling following EC50 exposure to IFN alfacon-1, IFN-gamma1b, or a cocktail of the two allowed the antiviral state to be correlated with induction of a subset of IFN-stimulated genes (ISGs).

Gene expression profiling of bovine macrophages in response to Escherichia coli O157:H7 lipopolysaccharide.

The aim of this study was to identify changes in bovine macrophage gene expression in response to treatment with Escherichia coli 0157:H7 lipopolysaccharide (LPS), utilizing a human gene microarray. Bovine cDNA from control and LPS-treated primary macrophages hybridized to greater than 5644 (79.8%) of the non-control gene targets on a commercially available microarray containing greater than 7075 targets (Incyte Genomics, St.

Global effect of PEG-IFN-alpha and ribavirin on gene expression in PBMC in vitro.

Using oligonucleotide microarrays, we have examined the expression of 22,000 genes in peripheral blood cells treated with pegylated interferon-alpha2b (PEG-IFN-alpha) and ribavirin. Treatment with ribavirin had very little effect on gene expression, whereas treatment with PEG-IFN-alpha had a dramatic effect, modulating the expression of approximately 1000 genes (at p < 0.001).

Association of bovine neonatal Fc receptor alpha-chain gene (FCGRT) haplotypes with serum IgG concentration in newborn calves.

This report describes allelic variation in FCGRT (which encodes the alpha-chain of FcRn) and its association with variation of IgG concentration in neonatal calves. Five SNPs were identified by sequencing 1305 bp of FCGRT genomic DNA from a multi-breed panel of 96 cattle and 27 founders of a reference population. These SNPs defined five FCGRT haplotypes that were verified by segregation and used to test association of FCGRT with neonatal IgG concentration in a case-control study.

Xenobiotics removal from polluted water by a multifunctional constructed wetland.

Removal efficiencies on xenobiotics from polluted water in a twin-shaped constructed wetland consisting of a vertical flow chamber with the crop plant Colocasia esculenta L. Schott and a reverse vertical flow one with Ischaemum aristatum var. glaucum Honda, were assessed by chemical analysis and bioassays.

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle.

DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations.

Use of bovine EST data and human genomic sequences to map 100 gene-specific bovine markers.

A system to use bovine EST data in conjunction with human genomic sequence to improve the bovine linkage map over the entire genome or on specific chromosomes was evaluated. Bovine EST sequence was used to provide primer sequences corresponding to bovine genes, while human genomic sequence directed primer design to flank introns and produce amplicons of appropriate size for efficient direct sequencing. The sequence tagged sites (STS) produced in this way from the four sires of the MARC reference families were examined for single nucleotide polymorphisms (SNPs) that could be used to map the corresponding genes.

Sulphonated aromatic pollutants. Limits of microbial degradability and potential of phytoremediation.

Many synthetic sulphonated aromatic compounds are used as starting material to produce dyes and pigments, or are released as by-products in the effluents of the textile and dye industry. A large number of these chemicals are poorly biodegradable and cannot be eliminated by classical wastewater treatment plants. To limit the impact of these pollutants on the environment, new processes, based on the use of higher plants (constructed wetlands or hydroponic systems), are under development.

A bisexually reproducing all-triploid vertebrate.

Green toads are common in the Palaearctic region, where they have differentiated into several taxa. The toads exist with variable amounts of ploidy, similar to other anuran species or reptiles. In vertebrate biology, the very rare occurrence of triploidy is coupled with infertility or unisexuality, or requires the coexistence of individuals of different ploidy in a reproductive community.

Sequence evaluation of four pooled-tissue normalized bovine cDNA libraries and construction of a gene index for cattle.

An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained.

Interleukin-8 haplotype structure from nucleotide sequence variation in commercial populations of U.S. beef cattle.

The aim of the present study was twofold: first, to design a panel of 96 sires that reflects the breadth of genetic diversity in U.S. beef cattle, and second, to use this panel to discover nucleotide sequence diversity and haplotype structures of interleukin (IL)-8 in commercial populations.

Estimation of DNA sequence diversity in bovine cytokine genes.

DNA sequence variation provides the fundamental material for improving livestock through selection. In cattle, single nucleotide polymorphisms and small insertions/deletions (collectively referred to here as SNPs) have been identified in cytokine genes and scored in a reference population to determine linkage map positions. The aim of the present study was twofold: first, to estimate the SNP frequency in a reference population of beef cattle, and second, to determine cytokine haplotypes in a group of sires from commercial populations.

Linkage mapping and comparative analysis of bovine expressed sequence tags (ESTs).

Bovine expressed sequence tags (ESTs) containing microsatellites are suitable markers for both linkage and comparative maps. We isolated clones from a bovine fetal thigh skeletal muscle cDNA library that were positive for a (CA)10 probe. Thirty individual clones were isolated and characterised by sequencing.

Canine thyrotropin beta-subunit gene: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and transient expression in the Chinese hamster ovary cells.

The gene encoding the mature beta subunit of canine thyroid stimulating hormone (cTSH beta) was cloned, sequenced and expressed in Escherichia coli and in Chinese hamster ovary (CHO) cells, and monoclonal antibodies against the recombinant cTSH beta purified from E. coli were generated. The gene fragment that encodes mature TSH beta was cloned from the canine genomic DNA by direct polymerase chain reaction (PCR) using primers that were designed based on the consensus sequences from other species.

Latest on enzymology of serotonin biosynthesis in walnut seeds.

Serotonin (5-HT) accumulation in walnut cotyledons is seen as a detoxification mechanism protecting the sensitive plant tissues of seeds from highly toxic ammonia concentrations following seed desiccation. Different metabolic pathways and cell compartments are involved in biosynthesis and storage of 5-HT. Ammonia fixation and incorporation into the indole moiety of tryptophan is followed by 5-HT biosynthesis via tryptamine in a two-step pathway with the adaptive tryptophan decarboxylase and the constitutive tryptamine 5-hydroxylase.