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We integrated a magnetohydrodynamic fluid extractor with an amperometric glucose biosensor to develop a wearable device for non-invasive glucose monitoring. Reproducible fluid extraction through the skin and efficient transport of the extracted fluid to the biosensor surface are prerequisites for non-invasive glucose monitoring. We optimized the enzyme immobilization and the interface layer between the sensing device and the skin. The monitoring device was evaluated by extracting fluid through porcine skin followed by glucose detection at the biosensor. The biosensor featured a screen-printed layer of Prussian Blue that was coated with a layer containing glucose oxidase. Both physical entrapment of glucose oxidase in chitosan and tethering of glucose oxidase to electrospun nanofibers were evaluated. Binding of glucose oxidase to nanofibers under mild conditions provided a stable biosensor with analytical performance suitable for accurate detection of micromolar concentrations of glucose. Hydrogels of varying thickness (95-2000 mu m) as well as a thin (30 mu m) nanofibrous polycaprolactone mat were studied as an interface layer between the biosensor and the skin. The effect of mass transfer phenomena at the biosensor-skin interface on the analytical performance of the biosensor was evaluated. The sensing device detected glucose extracted through porcine skin with an apparent (overall) sensitivity of-0.8 mA/(M.cm(2)), compared to a sensitivity of-17 mA/(M.cm(2)) for measurement in solution. The amperometric response of the biosensor correlated with the glucose concentration in the fluid that had been extracted through porcine skin with the magnetohydrodynamic technique.

Indexación: Web of Science; Scopus. The Gram-positive Renibacterium salmoninarum causes bacterial kidney disease, a serious threat to Chilean salmon farming. To aid in vaccine development, this study used biochemical, antigenic, and genetic techniques to characterise 39 R. salmoninarum isolates from diseased Salmo salar and Oncorhynchus kisutch. Regardless of host, R. salmoninarum isolates were highly homogeneous pheno-typically. However, the isolates presented varied hydrophobicity (10-90%), as found by hydrocarbon adhesions, and distinct siderophores production, as evaluated on chrome azurol S agar. Serological assays established antigenic homogeneity among isolates, which can facilitate vaccine development. While western-blot profiles (using antiserum against iron-limited R. salmoninarum) differed, all isolates belonged to the same serogroup. Finally, RAPD, ERIC-PCR, and REP-PCR supported genetic R. salmoninarum homogeneity, suggesting clonal relationships. In conclusion, high homogeneity might facilitate vaccine development, but in vivo studies are needed to clarify the relationship of R. salmoninarum virulence with hydrophobicity/siderophores synthesis. https://eafp.org/bulletin-archive/

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

Background In wheat, the vernalization requirement is mainly controlled by the VRN genes. Different species of hexaploid and tetraploid wheat are widely used as genetic source for new mutant variants and alleles for fundamental investigations and practical breeding programs. In this study, VRN-A1 and VRN-B1 were analysed for 178 accessions representing six tetraploid wheat species (Triticum dicoccoides, T. dicoccum, T. turgidum, T. polonicum, T. carthlicum, T. durum) and five hexaploid species (T. compactum, T. sphaerococcum, T. spelta, T. macha, T. vavilovii). Results Novel allelic variants in the promoter region of VRN-A1 and VRN-B1 were identified based on the change in curvature and flexibility of the DNA molecules. The new variants of VRN-A1 (designated as Vrn-A1a.2, Vrn-A1b.2 – Vrn-A1b.6 and Vrn-A1i) were found to be widely distributed in hexaploid and tetraploid wheat, and in fact were predominant over the known VRN-A1 alleles. The greatest diversity of the new variants of VRN-B1 (designated as VRN-B1.f, VRN-B1.s and VRN-B1.m) was found in the tetraploid and some hexaploid wheat species. For the first time, minor differences within the sequence motif known as the VRN-box of VRN1 were correlated with wheat growth habit. Thus, vrn-A1b.3 and vrn-A1b.4 were revealed in winter wheat in contrast to Vrn-A1b.2, Vrn-A1b.5, Vrn-A1b.6 and Vrn-A1i. It was found that single nucleotide mutation in the VRN-box can influence the vernalization requirement and growth habit of wheat. Our data suggest that both the A-tract and C-rich segment within the VRN-box contribute to its functionality, and provide a new view of the hypothesised role of the VRN-box in regulating transcription of the VRN1 genes. Specifically, it is proposed that combination of mutations in this region can modulate vernalization sensitivity and flowering time of wheat. Conclusions New allelic variants of the VRN-A1 and VRN-B1 genes were identified in hexaploid and tetraploid wheat. Mutations in A-tract and C-rich segments within the VRN-box of VRN-A1 are associated with modulation of the vernalization requirement and flowering time. New allelic variants will be useful in fundamental investigations into the regulation of VRN1 expression, and provide a valuable genetic resource for practical breeding of wheat. Electronic supplementary material The online version of this article (doi:10.1186/s12870-015-0691-2) contains supplementary material, which is available to authorized users.

Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host–pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate–polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10–50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.

This study aims to identify cancer-associated proteins in the secretome of oral cancer cell lines. We have successfully established four primary cell cultures of normal cells with a limited lifespan without human telomerase reverse transcriptase (hTERT) immortalization. The secretome of these primary cell cultures were compared with that of oral cancer cell lines using 2DE. Thirty five protein spots were found to have changed in abundance. Unambiguous identification of these proteins was achieved by MALDI TOF/TOF. In silico analysis predicted that 24 of these proteins were secreted via classical or nonclassical mechanisms. The mRNA expression of six genes was found to correlate with the corresponding protein abundance. Ingenuity Pathway Analysis (IPA) core analysis revealed that the identified proteins were relevant in, and related to, cancer development with likely involvements in tumor growth, metastasis, hyperproliferation, tumorigenesis, neoplasia, hyperplasia, and cell transformation. In conclusion, we have demonstrated that a comparative study of the secretome of cancer versus normal cell lines can be used to identify cancer-associated proteins.

Wilson disease (WD) is a rare hereditary condition that is caused by a genetic defect in the copper-transporting ATPase ATP7B that results in hepatic copper accumulation and lethal liver failure. The present study focuses on the structural mitochondrial alterations that precede clinical symptoms in the livers of rats lacking Atp7b, an animal model for WD. Liver mitochondria from these Atp7b–/– rats contained enlarged cristae and widened intermembrane spaces, which coincided with a massive mitochondrial accumulation of copper. These changes, however, preceded detectable deficits in oxidative phosphorylation and biochemical signs of oxidative damage, suggesting that the ultrastructural modifications were not the result of oxidative stress imposed by copper- dependent Fenton chemistry. In a cell-free system containing a reducing dithiol agent, isolated mitochondria exposed to copper underwent modifications that were closely related to those observed in vivo. In this cell-free system, copper induced thiol modifications of three abundant mitochondrial membrane proteins, and this correlated with reversible intramitochondrial membrane crosslinking, which was also observed in liver mitochondria from Atp7b–/– rats. In vivo, copper-chelating agents reversed mitochondrial accumulation of copper, as well as signs of intra-mitochondrial membrane crosslinking, thereby preserving the functional and structural integrity of mitochondria. Together, these findings suggest that the mitochondrion constitutes a pivotal target of copper in WD.

Background All studies aimed at understanding complex molecular changes occurring at synapses face the problem of how a complete view of the synaptic proteome and of its changes can be efficiently met. This is highly desirable when synaptic plasticity processes are analyzed since the structure and the biochemistry of neurons and synapses get completely reshaped. Because most molecular studies of synapses are nowadays mainly or at least in part based on protein extracts from neuronal cultures, this is not a feasible option: these simplified versions of the brain tissue on one hand provide an homogeneous pure population of neurons but on the other yield only tiny amounts of proteins, many orders of magnitude smaller than conventional brain tissue. As a way to overcome this limitation and to find a simple way to screen for protein changes at cultured synapses, we have produced and characterized two dimensional electrophoresis (2DE) maps of the synaptic proteome of CA3-CA1 hippocampal neurons in culture. Results To obtain 2D maps, hippocampal cultures were mass produced and after synaptic maturation, proteins were extracted following subfractionation procedures and separated by 2D gel electrophoresis. Similar maps were obtained for the crude cytosol of cultured neurons and for synaptosomes purified from CA3-CA1 hippocampal tissue. To efficiently compare these different maps some clearly identifiable reference points were molecularly identified by mass spectrometry and immunolabeling methods. This information was used to run a differential analysis and establish homologies and dissimilarities in these 2D protein profiles. Conclusion Because reproducible fingerprints of cultured synapses were clearly obtained, we believe that our mapping effort could represent a simple tool to screen for protein expression and/or protein localization changes in CA3-CA1 hippocampal neurons following plasticity.

Small molecule sorption and slow release in and out of polyacrylamide (PAAm) gels were studied by using steady state fluorescence (SSF) technique. Pyranine (P-y) dissolved in water used as a probe. Fluorescence emission intensity, I-p from P-y was used to monitor for studying sorption and slow release processes at various temperatures. Sorption and slow release processes were analyzed by using Fickian diffusion model and coefficients were obtained. Related activation energies were also calculated for the corresponding physical processes. Publisher's Version Q4 WOS:000252408800003

The antiapoptotic role of Bcl-2 can be regulated by its phosphorylation in serine and threonine residues located in a nonstructured loop that links BH3 and BH4 domains. p38 MAPK has been identified as one of the kinases able to mediate such phosphorylation, through direct interaction with Bcl-2 protein in the mitochondrial compartment. In this study, we identify, by using mass spectrometry techniques and specific anti-phosphopeptide antibodies, Ser(87) and Thr(56) as the Bcl-2 residues phosphorylated by p38 MAPK and show that phosphorylation of these residues is always associated with a decrease in the antiapoptotic potential of Bcl-2 protein. Furthermore, we obtained evidence that p38 MAPK-induced Bcl-2 phosphorylation plays a key role in the early events following serum deprivation in embryonic fibroblasts. Both cytochrome c release and caspase activation triggered by p38 MAPK activation and Bcl-2 phosphorylation are absent in embryonic fibroblasts from p38alpha knock-out mice (p38alpha(-/-) MEF), whereas they occur within 12 h of serum withdrawal in p38alpha(+/+) MEF; moreover, they can be prevented by p38 MAPK inhibitors and are not associated with the synthesis of the proapoptotic proteins Bax and Fas. Thus, Bcl-2 phosphorylation by activated p38 MAPK is a key event in the early induction of apoptosis under conditions of cellular stress.

Surfaces carrying hydrophilic polymer brushes were prepared from poly(styrene)-poly(acrylic acid) and poly(styrene)-poly(ethylene oxide) diblock copolymers, respectively, using a Langmuir-Blodgett technique and employing poly(styrene)-coated planar glass as substrates. The electrical properties of these surfaces in aqueous electrolyte were analyzed as a function of pH and KCl concentration using streaming potential/streaming current measurements. From these data, both the zeta potential and the surface conductivity could be obtained. The poly(acrylic acid) brushes are charged due to the dissociation of carboxylic acid groups and give theoretical surface potentials of -160 mV at full dissociation in 10(-3) M solutions. The surface conductivity of these brushes is enormous under these conditions, accounting for more than 93% of the total measured surface conductivity. However, the mobility of the ions within the brush was estimated from the density of the carboxylic acid groups and the surface conductivity data to be only about 14% of that of free ions. The poly(ethylene oxide) (PEO) brushes effectively screen the charge of the underlying substrate, giving a very low zeta potential except when the ionic strength is very low. From the data, a hydrodynamic layer thickness of the PEO brushes could be estimated which is in good agreement with independent experiments (neutron reflectivity) and theoretical estimates. The surface conductivity in this system was slightly lower than that of the polystyren substrate. This also indicates that no significant amount of preferentially, i.e., nonelectrostatically attracted, ions taken up in the brush.

Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of A infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on, 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.