Two-Way Regionalized Classification of Multivariate Datasets and its Application to the Assessment of Hydrodynamic Dispersion

Zones of mixing between shallow groundwaters of different composition were unravelled by “two-way regionalized classification,” a technique based on correspondence analysis (CA), cluster analysis (ClA) and discriminant analysis (DA), aided by gridding, map-overlay and contouring tools. The shallow groundwaters are from a granitoid plutonite in the Fundão region (central Portugal). Correspondence analysis detected three natural clusters in the working dataset: 1, weathering; 2, domestic effluents; 3, fertilizers. Cluster analysis set an alternative distribution of the samples by the three clusters. Group memberships obtained by correspondence analysis and by cluster analysis were optimized by discriminant analysis, gridded over the entire Fundão region, and converted into “two-way regionalized classification” memberships as follows: codes 1, 2 or 3 were used when classification by correspondence analysis and cluster analysis produced the same results; code 0 when the grid node was first assigned to cluster 1 and then to cluster 2 or vice versa (mixing between weathering and effluents); code 4 in the other cases (mixing between agriculture and the other influences). Code-3 areas were systematically surrounded by code-4 areas, an observation attributed to hydrodynamic dispersion. Accordingly, the extent of code-4 areas in two orthogonal directions was assumed proportional to the longitudinal and transverse dispersivities of local soils. The results (0.7–16.8 and 0.4–4.3 m, respectively) are acceptable at the macroscopic scale. The ratios between longitudinal and transverse dispersivities (1.2–11.1) are also in agreement with results obtained by other studies

The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage

Helicobacter pylori Depletes Cholesterol in Gastric Glands to Prevent Interferon Gamma Signaling and Escape the Inflammatory Response

Background and Aims Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-aglucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. Methods MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-b-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Dcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1–/– mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Dcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. Results In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human b-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNGresponse gene IRF1 was substantially higher in PMSS1Dcgtinfected mice than PMSS1-infected mice. Ifngr1–/– mice were colonized by PMSS1 to a greater extent than control mice. Conclusions H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell–based vaccines against H pylori.</p

Helicobacter pylori depletes cholesterol in gastric glands to prevent interferon gamma signaling and escape the inflammatory response

Background and Aims Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-aglucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. Methods MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-b-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Dcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1–/– mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Dcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. Results In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human b-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNGresponse gene IRF1 was substantially higher in PMSS1Dcgtinfected mice than PMSS1-infected mice. Ifngr1–/– mice were colonized by PMSS1 to a greater extent than control mice. Conclusions H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell–based vaccines against H pylori.</p