BIOCIDE RESISTANCE AS A RESULT OF EXPOSURE TO BIOCIDES USED IN HYDRAULIC FRACTURING

Microbial resistance to antimicrobials is an important topic to investigate not only for our health but for the environment. There is a growing concern about expanding microbial resistance to both antibiotics and other antimicrobials such as biocides. We wanted to know how bacteria previously exposed to hydraulic fracturing fluids, including biocides, would react to biocide exposure and what the mechanism of resistance looked like. In order to test this hypothesis bacterial isolates were obtained from water and sediment from a stream that had been previously exposed to a spill of hydraulic fracturing water. Thirty bacterial isolates were obtained from these samples that could withstand a moderate dose of two biocides (Glutaraldehyde and 2,2-dibromo-3-nitrilopropionamide (DBNPA)). These isolates then had their DNA extracted and taxonomy identified using 16S rRNA sequencing. We also tested minimum inhibitory concentrations (MIC) for each isolate against each biocide. This data however was not conclusive. Attempts were made to perform whole genome sequencing on these isolates to better understand resistance mechanisms. Sequencing was also attempted but due to complications was not completed. To increase our understanding of the mechanism of resistance a comparative genomic study was undertaken to compare between hydraulic fracturing associate Bacillus spp. and non-hydraulic fracturing associated relatives. Overall, isolates resistant to glutaraldehyde, DBNPA or both were obtained and identified. More isolates were found resistant to either glutaraldehyde or DBNPA not both. Meaning that the mechanisms of resistance may be different for the two biocides

Analysis of the Function of DNA Polymerase Kappa and M17 in the Immune System

In T cell-dependent immune responses, activated B cells undergo a phase of rapid expansion and form distinct histological structures, the germinal centers (GC). GCs are the sites of secondary antibody diversification. Somatic hypermutation (SHM) introduces mutations into the rearranged V genes, whereas class switch recombination (CSR) alters the IgH constant region to modulate effector function. The current model of SHM postulates cytidine deamination by AID, followed by error-prone repair that involves short-patch DNA synthesis by error-prone DNA polymerases. The Pol k (DinB1) gene encodes a specialized mammalian DNA polymerase called DNA polymerase k. The mouse Pol k gene is expressed in most tissues of the body including B cells. The ability of Pol k to generate mutations when extending primers on undamaged DNA templates identifies this enzyme as a candidate for the introduction of nucleotide exchanges during SHM. Here, I show that Polk-deficient mice are viable, fertile and able to mount a normal immune response to the antigen (4-hydroxy-3-nitrophenyl) acetyl-chicken globulin (NPGC). Polk-deficient mice mutate their Ig genes normally. Pol k-/- Pol l-/- Pol i-/- mice also show no defects in SHM, indicating that these error-prone DNA polymerases do not substitute for each other's function during SHM. However, Polk-deficient embryonic fibroblasts are sensitive to cell death following exposure to ultraviolet radiation, suggesting a role for Polk in translesion DNA synthesis. The human gene HGAL serves as marker in the prognosis of patients with GC-derived diffuse large B cell lymphomas (DLBCL). The mouse gene M17 is the homologue of HGAL. M17 is predominantly expressed in the GCs, indicating a role in GC function. In the present study, I analyzed M17-/- mice to investigate the role of M17 in the GC reaction. M17-/- mice form normal GCs, undergo efficient CSR and SHM and mount a T cell-dependent immune response. Thus, M17 is dispensable for the GC reaction and the current data support a rather indirect role for HGAL as a prognostic marker in the biology of DLBCL

miRNAs Regulate Cytokine Secretion Induced by Phosphorylated S100A8/A9 in Neutrophils.

The release of cytokines by neutrophils constitutes an essential process in the development of inflammation by recruiting and activating additional cells. Neutrophils are also able to secrete a complex of S100A8 and S100A9 proteins (S100A8/A9), which can amplify the general inflammatory state of the host and is involved in the pathogenesis of several chronic inflammatory diseases, such as rheumatoid arthritis (RA). S100A8/A9 have received renewed attention due to their susceptibility to several function-altering post-translational modifications. In that context, it has been recently demonstrated that only the phosphorylated form of S100A8/A9 (S100A8/A9-P) is able to induce the secretion of several cytokines in neutrophils. Here, we investigate the mechanism by which this post-translational modification of S100A8/A9 can regulate the extracellular activity of the protein complex and its impact on the inflammatory functions of neutrophils. We found that S100A8/A9-P are present in large amounts in the synovial fluids from RA patients, highlighting the importance of this form of S100A8/A9 complex in the inflammation process. Using miRNA-sequencing on S100A8/A9-P-stimulated differentiated HL-60 cells, we identified a dysregulation of miR-146a-5p and miR-155-5p expression through TRL4 signaling pathways. Our data reveal that overexpression of these miRNAs in neutrophil-like cells reduces S100A8/A9-P-mediated secretion of pro-inflammatory cytokines

Regulation of Neutrophil Degranulation and Cytokine Secretion: A Novel Model Approach Based on Linear Fitting

Neutrophils participate in the maintenance of host integrity by releasing various cytotoxic proteins during degranulation. Due to recent advances, a major role has been attributed to neutrophil-derived cytokine secretion in the initiation, exacerbation, and resolution of inflammatory responses. Because the release of neutrophil-derived products orchestrates the action of other immune cells at the infection site and, thus, can contribute to the development of chronic inflammatory diseases, we aimed to investigate in more detail the spatiotemporal regulation of neutrophil-mediated release mechanisms of proinflammatory mediators. Purified human neutrophils were stimulated for different time points with lipopolysaccharide. Cells and supernatants were analyzed by flow cytometry techniques and used to establish secretion profiles of granules and cytokines. To analyze the link between cytokine release and degranulation time series, we propose an original strategy based on linear fitting, which may be used as a guideline, to (i) define the relationship of granule proteins and cytokines secreted to the inflammatory site and (ii) investigate the spatial regulation of neutrophil cytokine release. The model approach presented here aims to predict the correlation between neutrophil-derived cytokine secretion and degranulation and may easily be extrapolated to investigate the relationship between other types of time series of functional processes

MyD88 signalling in colonic mononuclear phagocytes drives colitis in IL-10-deficient mice

Commensal bacterial sensing by Toll-like receptors is critical for maintaining intestinal homeostasis, but can lead to colitis in the absence of interleukin-10. Although Toll-like receptors are expressed in multiple cell types in the colon, the cell type(s) responsible for the development of colitis are currently unknown. Here we generated mice that are selectively deficient in MyD88 in various cellular compartments in an interleukin-10[superscript −/−] setting. Although epithelial expression of MyD88 was dispensable, MyD88 expression in the mononuclear phagocyte compartment was required for colitis development. Specifically, phenotypically distinct populations of colonic mononuclear phagocytes expressed high levels of interleukin-1β, interleukin-23 and interleukin-6, and promoted T-helper 17 responses in the absence of interleukin-10. Thus, gut bacterial sensing through MyD88 in mononuclear phagocytes drives inflammatory bowel disease when unopposed by interleukin-10.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (NIH grant DK071754)National Institutes of Health (U.S.) (NIH grant AI046688)National Institutes of Health (U.S.) (NIH grant AI055502)National Institutes of Health (U.S.) (NIH grant RO1OD011141)National Institutes of Health (U.S.) (Training grant)National Cancer Institute (U.S.) (Irvington Fellowship

A20-Deficient Mast Cells Exacerbate Inflammatory Responses In Vivo

Mast cells are implicated in the pathogenesis of inflammatory and autoimmune diseases. However, this notion based on studies in mast cell-deficient mice is controversial. We therefore established an in vivo model for hyperactive mast cells by specifically ablating the NF-kappa B negative feedback regulator A20. While A20 deficiency did not affect mast cell degranulation, it resulted in amplified pro-inflammatory responses downstream of IgE/Fc epsilon RI, TLRs, IL-1R, and IL-33R. As a consequence house dust mite- and IL-33-driven lung inflammation, late phase cutaneous anaphylaxis, and collagen-induced arthritis were aggravated, in contrast to experimental autoimmune encephalomyelitis and immediate anaphylaxis. Our results provide in vivo evidence that hyperactive mast cells can exacerbate inflammatory disorders and define diseases that might benefit from therapeutic intervention with mast cell function

Polζ ablation in B cells impairs the germinal center reaction, class switch recombination, DNA break repair, and genome stability

Polζ is an error-prone DNA polymerase that is critical for embryonic development and maintenance of genome stability. To analyze its suggested role in somatic hypermutation (SHM) and possible contribution to DNA double-strand break (DSB) repair in class switch recombination (CSR), we ablated Rev3, the catalytic subunit of Polζ, selectively in mature B cells in vivo. The frequency of somatic mutation was reduced in the mutant cells but the pattern of SHM was unaffected. Rev3-deficient B cells also exhibited pronounced chromosomal instability and impaired proliferation capacity. Although the data thus argue against a direct role of Polζ in SHM, Polζ deficiency directly interfered with CSR in that activated Rev3-deficient B cells exhibited a reduced efficiency of CSR and an increased frequency of DNA breaks in the immunoglobulin H locus. Based on our results, we suggest a nonredundant role of Polζ in DNA DSB repair through nonhomologous end joining

DNA polymerase zeta is required for proliferation of normal mammalian cells

Unique among translesion synthesis (TLS) DNA polymerases, pol ζ is essential during embryogenesis. To determine whether pol ζ is necessary for proliferation of normal cells, primary mouse fibroblasts were established in which Rev3L could be conditionally inactivated by Cre recombinase. Cells were grown in 2% O2 to prevent oxidative stress-induced senescence. Cells rapidly became senescent or apoptotic and ceased growth within 3–4 population doublings. Within one population doubling following Rev3L deletion, DNA double-strand breaks and chromatid aberrations were found in 30–50% of cells. These breaks were replication dependent, and found in G1 and G2 phase cells. Double-strand breaks were reduced when cells were treated with the reactive oxygen species scavenger N-acetyl-cysteine, but this did not rescue the cell proliferation defect, indicating that several classes of endogenously formed DNA lesions require Rev3L for tolerance or repair. T-antigen immortalization of cells allowed cell growth. In summary, even in the absence of external challenges to DNA, pol ζ is essential for preventing replication-dependent DNA breaks in every division of normal mammalian cells. Loss of pol ζ in slowly proliferating mouse cells in vivo may allow accumulation of chromosomal aberrations that could lead to tumorigenesis. Pol ζ is unique amongst TLS polymerases for its essential role in cell proliferation