Mutagenesis of Claudins to Probe in vivo Interactions and Assemblies

Paracellular transport of solutes and the control of the flow of molecules through the intracellular space in vertebrate epithelia is directed by tight junctions (TJs). Claudins form paracellular barriers and pores that determine tight junction permeability. This investigation attempts to explain the molecular bases for destruction and reconstruction of tight junctions within epithelial cells, occurring via both natural and disease-causing mechanisms. This interdisciplinary research is important in the advancement of our understanding of human biology and health, as different disruptions to epithelial tight junctions are hallmarks of many human diseases. The overall objective was to take previously made Claudin containing vectors (pEGFP-N3) containing a Green Fluorescent Protein (GFP) sequence and utilize site specific mutations to change the sequences to express Cyan Fluorescent Protein (CFP) and Yellow Fluorescent Protein (YFP). Proteins tested in this experiment were Human Claudins 1, 9, 19, Tricellulin, and Occludin. Once the specified claudins and TAMPs are tagged with CFP and YFP, FRET microscopy techniques can be utilized to determine generic interactions within the formation of TJs, forming a baseline for future research

Rapid Bacterial and Fungal Successional Dynamics in First Year After Chaparral Wildfire

The rise in wildfire frequency and severity across the globe has increased interest in secondary succession. However, despite the role of soil microbial communities in controlling biogeochemical cycling and their role in the regeneration of post-fire vegetation, the lack of measurements immediately post-fire and at high temporal resolution has limited understanding of microbial secondary succession. To fill this knowledge gap, we sampled soils at 17, 25, 34, 67, 95, 131, 187, 286, and 376 days after a southern California wildfire in fire-adapted chaparral shrublands. We assessed bacterial and fungal biomass with qPCR of 16S and 18S and richness and composition with Illumina MiSeq sequencing of 16S and ITS2 amplicons. Fire severely reduced bacterial biomass by 47%, bacterial richness by 46%, fungal biomass by 86%, and fungal richness by 68%. The burned bacterial and fungal communities experienced rapid succession, with 5-6 compositional turnover periods. Analogous to plants, turnover was driven by fire-loving pyrophilous microbes, many of which have been previously found in forests worldwide and changed markedly in abundance over time. Fungal secondary succession was initiated by the Basidiomycete yeast Geminibasidium, which traded off against the filamentous Ascomycetes Pyronema, Aspergillus, and Penicillium. For bacteria, the Proteobacteria Massilia dominated all year, but the Firmicute Bacillus and Proteobacteria Noviherbaspirillum increased in abundance over time. Our high-resolution temporal sampling allowed us to capture post-fire microbial secondary successional dynamics and suggest that putative tradeoffs in thermotolerance, colonization, and competition among dominant pyrophilous microbes control microbial succession with possible implications for ecosystem function

Pathways of Resistance to Thymineless Death in Escherichia coli and the Function of UvrD

Thymineless death (TLD) is the rapid loss of viability in bacterial, yeast, and human cells starved of thymine. TLD is the mode of action of common anticancer drugs and some antibiotics. TLD in Escherichia coli is accompanied by blocked replication and chromosomal DNA loss and recent work identified activities of recombination protein RecA and the SOS DNA-damage response as causes of TLD. Here, we examine the basis of hypersensitivity to thymine deprivation (hyper-TLD) in mutants that lack the UvrD helicase, which opposes RecA action and participates in some DNA repair mechanisms, RecBCD exonuclease, which degrades double-stranded linear DNA and works with RecA in double-strand-break repair and SOS induction, and RuvABC Holliday-junction resolvase. We report that hyper-TLD in ∆uvrD cells is partly RecA dependent and cannot be attributed to accumulation of intermediates in mismatch repair or nucleotide-excision repair. These data imply that both its known role in opposing RecA and an additional as-yet-unknown function of UvrD promote TLD resistance. The hyper-TLD of ∆ruvABC cells requires RecA but not RecQ or RecJ. The hyper-TLD of recB cells requires neither RecA nor RecQ, implying that neither recombination nor SOS induction causes hyper-TLD in recB cells, and RecQ is not the sole source of double-strand ends (DSEs) during TLD, as previously proposed; models are suggested. These results define pathways by which cells resist TLD and suggest strategies for combating TLD resistance during chemotherapies

A family-based study of gene variants and maternal folate and choline in neuroblastoma: a report from the Children’s Oncology Group

Neuroblastoma is a childhood cancer of the sympathetic nervous system with embryonic origins. Previous epidemiologic studies suggest maternal vitamin supplementation during pregnancy reduces the risk of neuroblastoma. We hypothesized offspring and maternal genetic variants in folate-related and choline-related genes are associated with neuroblastoma and modify the effects of maternal intake of folate, choline and folic acid

Hydrogenation of Organic Matter as a Terminal Electron Sink Sustains High CO2:CH4 Production Ratios During Anaerobic Decomposition

Once inorganic electron acceptors are depleted, organic matter in anoxic environments decomposes by hydrolysis, fermentation, and methanogenesis, requiring syntrophic interactions between microorganisms to achieve energetic favorability. In this classic anaerobic food chain, methanogenesis represents the terminal electron accepting (TEA) process, ultimately producing equimolar CO2 and CH4 for each molecule of organic matter degraded. However, CO2:CH4 production in Sphagnum-derived, mineral-poor, cellulosic peat often substantially exceeds this 1:1 ratio, even in the absence of measureable inorganic TEAs. Since the oxidation state of C in both cellulose-derived organic matter and acetate is 0, and CO2 has an oxidation state of +4, if CH4 (oxidation state -4) is not produced in equal ratio, then some other compound(s) must balance CO2 production by receiving 4 electrons. Here we present evidence for ubiquitous hydrogenation of diverse unsaturated compounds that appear to serve as organic TEAs in peat, thereby providing the necessary electron balance to sustain CO2:CH4 \u3e1. While organic electron acceptors have previously been proposed to drive microbial respiration of organic matter through the reversible reduction of quinone moieties, the hydrogenation mechanism that we propose, by contrast, reduces C-C double bonds in organic matter thereby serving as 1) a terminal electron sink, 2) a mechanism for degrading complex unsaturated organic molecules, 3) a potential mechanism to regenerate electron-accepting quinones, and, in some cases, 4) a means to alleviate the toxicity of unsaturated aromatic acids. This mechanism for CO2 generation without concomitant CH4 production has the potential to regulate the global warming potential of peatlands by elevating CO2:CH4 production ratios

Genomic Profiling of Childhood Tumor Patient-Derived Xenograft Models to Enable Rational Clinical Trial Design.

Accelerating cures for children with cancer remains an immediate challenge as a result of extensive oncogenic heterogeneity between and within histologies, distinct molecular mechanisms evolving between diagnosis and relapsed disease, and limited therapeutic options. To systematically prioritize and rationally test novel agents in preclinical murine models, researchers within the Pediatric Preclinical Testing Consortium are continuously developing patient-derived xenografts (PDXs)-many of which are refractory to current standard-of-care treatments-from high-risk childhood cancers. Here, we genomically characterize 261 PDX models from 37 unique pediatric cancers; demonstrate faithful recapitulation of histologies and subtypes; and refine our understanding of relapsed disease. In addition, we use expression signatures to classify tumors for TP53 and NF1 pathway inactivation. We anticipate that these data will serve as a resource for pediatric oncology drug development and will guide rational clinical trial design for children with cancer

Characteristic functional equations of polynomials and the morera-carleman theorem

Several characteristic functional equations satisfied by classes of polynomials of bounded degree are examined in connection with certain generalizations of the Morera-Carleman Theorem. Certain functional equations which have nonanalytic polynomial solutions are also considered.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43923/1/10_2005_Article_BF02188016.pd