Structural Insights Into NMDA Ionotropic Glutamate Receptors via Molecular Modelling

Structural models have been produced for the agonist binding and transmembrane domains of two NMDA ionotropic glutamate receptors: homomeric NMDA-R2C and heteromeric NMDA-R1/R2C. These models--produced using homology modelling techniques in conjunction with distance restraints derived from the accessibility of substituted cysteines--have aided our understanding of (1) ligand selectivity and (2) channel activity. The model of the agonist binding domain of NMDA-R2C indicates that T691 forms an essential hydrogen bond with glutamate ligand. This interaction is absent in the NMDA-R1 model--where a valine replaces the threonine--explaining why NMDA-R1 binds glycine rather than glutamate. For the transmembrane region, the models suggest that a number of positive residues, located in the cytoplasmic loop between the M1 and M2 segments, create a large electrostatic energy barrier that could explain why homomeric NMDA-R2C channels are non-functional. Introducing NMDA-R1 to form heteromeric NMDA-R1/R2C channels is predicted to rescue channel activity because the corresponding region in NMDA-R1 contains negative residues that more than compensate for the electrostatic energy barrier in NMDA-R2C. These studies suggest that replacing the positively charged region in the M1-M2 loop of NMDA-R2C with the corresponding negatively charged region of NMDA-R1 could transform NMDA-R2C into a functional homomeric channel.     

Bird community response to filter strips in Maryland

Filter strips are strips of herbaceous vegetation planted along agricultural field margins adjacent to streams or wetlands and are designed to intercept sediment, nutrients, and agrichemicals. Roughly 16,000 ha of filter strips have been established in Maryland through the United States Department of Agriculture's Conservation Reserve Enhancement Program. Filter strips often represent the only uncultivated herbaceous areas on farmland in Maryland and therefore may be important habitat for early-successional bird species. Most filter strips in Maryland are planted to either native warm-season grasses or cool-season grasses and range in width from 10.7 m to 91.4 m. From 2004 to 2007 we studied the breeding and wintering bird communities in filter strips adjacent to wooded edges and non-buffered field edges and the effect that grass type and width of filter strips had on bird community composition. We used 5 bird community metrics (total bird density, species richness, scrub-shrub bird density, grassland bird density, and total avian conservation value), species-specific densities, nest densities, and nest survival estimates to assess the habitat value of filter strips for birds. Breeding and wintering bird community metrics were greater in filter strips than in non-buffered field edges but did not differ between cool-season and warm-season grass filter strips. Most breeding bird community metrics were negatively related to the percent cover of orchardgrass (Dactylis glomerata) in ≥1 yr. Breeding bird density was greater in narrow (<30 m) compared to wide (>60 m) filter strips. Our results suggest that narrow filter strips adjacent to wooded edges can provide habitat for many bird species but that wide filter strips provide better habitat for grassland birds, particularly obligate grassland species. If bird conservation is an objective, avoid planting orchardgrass in filter strips and reduce or eliminate orchardgrass from filter strips through management practices. © 2011 The Wildlife Society.     

RGB marking facilitates multicolor clonal cell tracking

We simultaneously transduced cells with three lentiviral gene ontology (LeGO) vectors encoding red, green or blue fluorescent proteins. Individual cells were thereby marked by different combinations of inserted vectors, resulting in the generation of numerous mixed colors, a principle we named red-green-blue (RGB) marking. We show that lentiviral vector-mediated RGB marking remained stable after cell division, thus facilitating the analysis of clonal cell fates in vitro and in vivo. Particularly, we provide evidence that RGB marking allows assessment of clonality after regeneration of injured livers by transplanted primary hepatocytes. We also used RGB vectors to mark hematopoietic stem/progenitor cells that generated colored spleen colonies. Finally, based on limiting-dilution and serial transplantation assays with tumor cells, we found that clonal tumor cells retained their specific color-code over extensive periods of time. We conclude that RGB marking represents a useful tool for cell clonality studies in tissue regeneration and pathology.     

Quiescin sulfhydryl oxidase 1 promotes invasion of pancreatic tumor cells mediated by matrix metalloproteinases

Quiescin sulfhydryl oxidase 1 (QSOX1) oxidizes sulfhydryl groups to form disulfide bonds in proteins. We previously mapped a peptide in plasma from pancreatic ductal adenocarcinoma (PDA) patients back to an overexpressed QSOX1 parent protein. In addition to overexpression in pancreatic cancer cell lines, 29 of 37 patients diagnosed with PDA expressed QSOX1 protein in tumor cells, but QSOX1 was not detected in normal adjacent tissues or in a transformed, but nontumorigenic cell line. To begin to evaluate the advantage QSOX1 might provide to tumors, we suppressed QSOX1 protein expression using short hairpin (sh) RNA in two pancreatic cancer cell lines. Growth, cell cycle, apoptosis, invasion, and matrix metalloproteinase (MMP) activity were evaluated. QSOX1 shRNA suppressed both short and long isoforms of the protein, showing a significant effect on cell growth, cell cycle, and apoptosis. However, QSOX1 shRNA dramatically inhibited the abilities of BxPC-3 and Panc-1 pancreatic tumor cells to invade through Matrigel in a modified Boyden chamber assay. Mechanistically, gelatin zymography indicated that QSOX1 plays an important role in activation of MMP-2 and MMP-9. Taken together, our results suggest that the mechanism of QSOX1-mediated tumor cell invasion is by activation of MMP-2 and MMP-9.     

A micro RNA processing defect in rapidly progressing idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis exhibits differential progression from the time of diagnosis but the molecular basis for varying progression rates is poorly understood. The aim of the present study was to ascertain whether differential miRNA expression might provide one explanation for rapidly versus slowly progressing forms of IPF.

Methodology and Principal Findings

miRNA and mRNA were isolated from surgical lung biopsies from IPF patients with a clinically documented rapid or slow course of disease over the first year after diagnosis. A quantitative PCR miRNA array containing 88 of the most abundant miRNA in the human genome was used to profile lung biopsies from 9 patients with rapidly progressing IPF, 6 patients with slowly progressing IPF, and 10 normal lung biopsies. Using this approach, 11 miRNA were significantly increased and 36 were significantly decreased in rapid biopsies compared with normal biopsies. Slowly progressive biopsies exhibited 4 significantly increased miRNA and 36 significantly decreased miRNA compared with normal lung. Among the miRNA present in IPF with validated mRNA targets were those with regulatory effects on epithelial-mesenchymal transition (EMT). Five miRNA (miR-302c, miR-423-5p, miR-210, miR-376c, and miR-185) were significantly increased in rapid compared with slow IPF lung biopsies. Additional analyses of rapid biopsies and fibroblasts grown from the same biopsies revealed that the expression of AGO1 and AGO2 (essential components of the miRNA processing RISC complex) were lower compared with either slow or normal lung biopsies and fibroblasts.

Conclusion

These findings suggest that the development and/or clinical progression of IPF might be the consequence of aberrant miRNA processing.     

Ultra-fast and sensitive detection of non-typhoidal Salmonella using microwave-accelerated metal-enhanced fluorescence ("MAMEF")

Certain serovars of Salmonella enterica subsp. enterica cause invasive disease (e.g., enteric fever, bacteremia, septicemia, meningitis, etc.) in humans and constitute a global public health problem. A rapid, sensitive diagnostic test is needed to allow prompt initiation of therapy in individual patients and for measuring disease burden at the population level. An innovative and promising new rapid diagnostic technique is microwave-accelerated metal-enhanced fluorescence (MAMEF). We have adapted this assay platform to detect the chromosomal oriC locus common to all Salmonella enterica subsp. enterica serovars. We have shown efficient lysis of biologically relevant concentrations of Salmonella spp. suspended in bacteriological media using microwave-induced lysis. Following lysis and DNA release, as little as 1 CFU of Salmonella in 1 ml of medium can be detected in <30 seconds. Furthermore the assay is sensitive and specific: it can detect oriC from Salmonella serovars Typhi, Paratyphi A, Paratyphi B, Paratyphi C, Typhimurium, Enteritidis and Choleraesuis but does not detect Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae or Acinetobacter baumanii. We have also performed preliminary experiments using a synthetic Salmonella oriC oligonucleotide suspended in whole human blood and observed rapid detection when the sample was diluted 1:1 with PBS. These pre-clinical data encourage progress to the next step to detect Salmonella in blood (and other ordinarily sterile, clinically relevant body fluids).