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

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

Mouse nuclear myosin I knock-out shows interchangeability and redundancy of myosin isoforms in the cell nucleus.

Nuclear myosin I (NM1) is a nuclear isoform of the well-known "cytoplasmic" Myosin 1c protein (Myo1c). Located on the 11(th) chromosome in mice, NM1 results from an alternative start of transcription of the Myo1c gene adding an extra 16 amino acids at the N-terminus. Previous studies revealed its roles in RNA Polymerase I and RNA Polymerase II transcription, chromatin remodeling, and chromosomal movements. Its nuclear localization signal is localized in the middle of the molecule and therefore directs both Myosin 1c isoforms to the nucleus. In order to trace specific functions of the NM1 isoform, we generated mice lacking the NM1 start codon without affecting the cytoplasmic Myo1c protein. Mutant mice were analyzed in a comprehensive phenotypic screen in cooperation with the German Mouse Clinic. Strikingly, no obvious phenotype related to previously described functions has been observed. However, we found minor changes in bone mineral density and the number and size of red blood cells in knock-out mice, which are most probably not related to previously described functions of NM1 in the nucleus. In Myo1c/NM1 depleted U2OS cells, the level of Pol I transcription was restored by overexpression of shRNA-resistant mouse Myo1c. Moreover, we found Myo1c interacting with Pol II. The ratio between Myo1c and NM1 proteins were similar in the nucleus and deletion of NM1 did not cause any compensatory overexpression of Myo1c protein. We observed that Myo1c can replace NM1 in its nuclear functions. Amount of both proteins is nearly equal and NM1 knock-out does not cause any compensatory overexpression of Myo1c. We therefore suggest that both isoforms can substitute each other in nuclear processes

MyD88 signalling in myeloid cells is sufficient to prevent chronic mycobacterial infection

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis that is responsible for almost 1.5 million deaths per year. Sensing of mycobacteria by the host's immune system relies on different families of receptors present on innate immune cells. Amongst them, several members of the TLR family are involved in the activation of immune cells by mycobacteria, yet the in vivo contribution of individual TLRs to the protective immune response remains controversial. On the contrary, MyD88, the adaptor molecule for most TLRs, plays a non-redundant role in the protection against tuberculosis and mice with a complete germline deletion of MyD88 succumb very early to infection. MyD88 is expressed in both immune and non-immune cells, but it is not clear whether control of mycobacteria requires ubiquitous or cell-type specific MyD88 expression. Therefore, using novel conditional switch-on mouse models, we aimed to investigate the importance of MyD88 signalling in DCs and macrophages for the induction of protective effector mechanisms against mycobacterial infection. We conclude that specific reactivation of MyD88 signalling in CD11c- or lysozyme M-expressing myeloid cells during Mycobacterium bovis Bacille Calmette-Guerin infection is sufficient to restore systemic and local inflammatory cytokine production and to control pathogen burden.Fil: Berod, Luciana. Helmholtz Centre for Infection Research; Alemania. Medical School Hanover; AlemaniaFil: Stüve, Philipp. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Swallow, Maxine. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Arnold Schrauf, Catharina. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Kruse, Friederike. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Gentilini, Maria Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Helmholtz Centre for Infection Research; Alemania. Medical School Hanover; AlemaniaFil: Freitag, Jenny. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Holzmann, Bernhard. Universitat Technical Zu Munich; Alemania. Helmholtz Centre for Infection Research; AlemaniaFil: Sparwasser, Tim. Medical School Hanover; Alemania. Helmholtz Centre for Infection Research; Alemani

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

Modeling of liquid flow in surface discontinuities

Polymer composite and metallic materials have found wide application in various industries such as aviation, rocket, car manufacturing, ship manufacturing, etc. Many design elements need permanent quality control. Ensuring high quality and reliability of products is impossible without effective nondestructive testing methods. One of these methods is penetrant testing using penetrating substances based on liquid penetration into defect cavities. In this paper, we propose a model of liquid flow to determine the rates of filling the defect cavities with various materials and, based on this, to choose optimal control modes

Identifying metabolite markers for preterm birth in cervicovaginal fluid by magnetic resonance spectroscopy

Introduction Preterm birth (PTB) may be preceded by changes in the vaginal microflora and metabolite profiles. Objectives We sought to characterise the metabolite profile of cervicovaginal fluid (CVF) of pregnant women by 1H NMR spectroscopy, and assess their predictive value for PTB. Methods A pair of high-vaginal swabs was obtained from pregnant women with no evidence of clinical infection and grouped as follows: asymptomatic low risk (ALR) women with no previous history of PTB, assessed at 20–22 gestational weeks, g.w., n = 83; asymptomatic high risk (AHR) women with a previous history of PTB, assessed at both 20–22 g.w., n = 71, and 26–28 g.w., n = 58; and women presenting with symptoms of preterm labor (PTL) (SYM), assessed at 24–36 g.w., n = 65. Vaginal secretions were dissolved in phosphate buffered saline and scanned with a 9.4 T NMR spectrometer. Results Six metabolites (lactate, alanine, acetate, glutamine/glutamate, succinate and glucose) were analysed. In all study cohorts vaginal pH correlated with lactate integral (r = -0.62, p\0.0001). Lactate integrals were higher in the term ALR compared to the AHR (20–22 g.w.) women (p = 0.003). Acetate integrals were higher in the preterm versus term women for the AHR (20–22 g.w.) (p = 0.048) and SYM (p = 0.003) groups; and was predictive of PTB\37 g.w. (AUC 0.78; 95 % CI 0.61–0.95), and delivery within 2 weeks of the index assessment (AUC 0.84; 95 % CI 0.64–1) in the SYM women, whilst other metabolites were not. Conclusion High CVF acetate integral of women with symptoms of PTL appears predictive of preterm delivery, as well as delivery within 2 weeks of presentation