The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD

Modern microwave methods in solid state inorganic materials chemistry: from fundamentals to manufacturing

No abstract available

Antibiotic Transport in Resistant Bacteria: Synchrotron UV Fluorescence Microscopy to Determine Antibiotic Accumulation with Single Cell Resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibitiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack

Force spectroscopy in studying infection

Biophysical force spectroscopy tools - for example optical tweezers, magnetic tweezers, atomic force microscopy, - have been used to study elastic, mechanical, conformational and dynamic properties of single biological specimens from single proteins to whole cells to reveal information not accessible by ensemble average methods such as X-ray crystallography, mass spectroscopy, gel electrophoresis and so on. Here we review the application of these tools on a range of infection-related questions from antibody-inhibited protein processivity to virus-cell adhesion. In each case we focus on how the instrumental design tailored to the biological system in question translates into the functionality suitable for that particular study. The unique insights that force spectroscopy has gained to complement knowledge learned through population averaging techniques in interrogating biomolecular details prove to be instrumental in therapeutic innovations such as those in structure-based drug design

The methanol dehydrogenase gene, mxaF, as a functional and phylogenetic marker for proteobacterial methanotrophs in natural environments

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 8 (2013): e56993, doi:10.1371/journal.pone.0056993.The mxaF gene, coding for the large (α) subunit of methanol dehydrogenase, is highly conserved among distantly related methylotrophic species in the Alpha-, Beta- and Gammaproteobacteria. It is ubiquitous in methanotrophs, in contrast to other methanotroph-specific genes such as the pmoA and mmoX genes, which are absent in some methanotrophic proteobacterial genera. This study examined the potential for using the mxaF gene as a functional and phylogenetic marker for methanotrophs. mxaF and 16S rRNA gene phylogenies were constructed based on over 100 database sequences of known proteobacterial methanotrophs and other methylotrophs to assess their evolutionary histories. Topology tests revealed that mxaF and 16S rDNA genes of methanotrophs do not show congruent evolutionary histories, with incongruencies in methanotrophic taxa in the Methylococcaceae, Methylocystaceae, and Beijerinckiacea. However, known methanotrophs generally formed coherent clades based on mxaF gene sequences, allowing for phylogenetic discrimination of major taxa. This feature highlights the mxaF gene’s usefulness as a biomarker in studying the molecular diversity of proteobacterial methanotrophs in nature. To verify this, PCR-directed assays targeting this gene were used to detect novel methanotrophs from diverse environments including soil, peatland, hydrothermal vent mussel tissues, and methanotroph isolates. The placement of the majority of environmental mxaF gene sequences in distinct methanotroph-specific clades (Methylocystaceae and Methylococcaceae) detected in this study supports the use of mxaF as a biomarker for methanotrophic proteobacteria.This work was supported in part by grants from the U.S. National Science Foundation Ecosystems Studies program (awards # DEB9708092 and DEB0089738)

Generation of a Novel Regulatory NK Cell Subset from Peripheral Blood CD34+ Progenitors Promoted by Membrane-Bound IL-15

BACKGROUND: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells. CONCLUSIONS/SIGNIFICANCE: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells

Conservation and Diversity of Seed Associated Endophytes in Zea across Boundaries of Evolution, Ethnography and Ecology

Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration