Genome sequence analysis of Helicobacter pylori strains associated with gastric ulceration and gastric cancer

<p>Abstract</p> <p>Background</p> <p>Persistent colonization of the human stomach by <it>Helicobacter pylori </it>is associated with asymptomatic gastric inflammation (gastritis) and an increased risk of duodenal ulceration, gastric ulceration, and non-cardia gastric cancer. In previous studies, the genome sequences of <it>H. pylori </it>strains from patients with gastritis or duodenal ulcer disease have been analyzed. In this study, we analyzed the genome sequences of an <it>H. pylori </it>strain (98-10) isolated from a patient with gastric cancer and an <it>H. pylori </it>strain (B128) isolated from a patient with gastric ulcer disease.</p> <p>Results</p> <p>Based on multilocus sequence typing, strain 98-10 was most closely related to <it>H. pylori </it>strains of East Asian origin and strain B128 was most closely related to strains of European origin. Strain 98-10 contained multiple features characteristic of East Asian strains, including a type s1c <it>vacA </it>allele and a <it>cagA </it>allele encoding an EPIYA-D tyrosine phosphorylation motif. A core genome of 1237 genes was present in all five strains for which genome sequences were available. Among the 1237 core genes, a subset of alleles was highly divergent in the East Asian strain 98-10, encoding proteins that exhibited <90% amino acid sequence identity compared to corresponding proteins in the other four strains. Unique strain-specific genes were identified in each of the newly sequenced strains, and a set of strain-specific genes was shared among <it>H. pylori </it>strains associated with gastric cancer or premalignant gastric lesions.</p> <p>Conclusion</p> <p>These data provide insight into the diversity that exists among <it>H. pylori </it>strains from diverse clinical and geographic origins. Highly divergent alleles and strain-specific genes identified in this study may represent useful biomarkers for analyzing geographic partitioning of <it>H. pylori </it>and for identifying strains capable of inducing malignant or premalignant gastric lesions.</p

Isolation and characterization of <i>Aag</i>^-/- cerebellar granule neurons.

<p>(A) Neurons were isolated from mouse cerebella at post-natal day 5–8. Representative immunocytochemical image depicts neurons (Map2, green) and astrocytes (GFAP, red). Nuclei are shown in blue. (B) Map2+ neurons were quantified from images from 10 pups isolated on different days. Error bars represent standard deviation from the mean. (C) <i>Aag</i> transcripts were counted per cell and are plotted by genotype, where each data point represents one cell. Errors bars denote standard deviation from the mean, n ≥ 50 cells, *** p<0.001 using Student’s standard two-tailed T-test. (D) Cartoon representation of the host-cell reactivation method to measure Aag glycosylase activity in cells. (E) Glycosylase activity over time by cellular fluorescent output from host cell reactivation assay. Solid lines denote mean while dashed lines indicate standard deviation from the mean. (F) WT, <i>Aag</i>^-/- and <i>mAagTg</i> neurons have significantly different Aag glycosylase activity at 24 hours post-transfection. Errors bars denote standard deviation from the mean. WT n = 3, <i>mAagTg</i> n = 2, <i>Aag</i>^-/- n = 2. ** p<0.01 using Student’s standard two-tailed T-test). (G) Fold changes between WT, <i>Aag</i>^-/- and <i>mAagTg</i> Aag expression and glycosylase activity.</p

ALKBH7 drives a tissue and sex-specific necrotic cell death response following alkylation-induced damage

Regulated necrosis has emerged as a major cell death mechanism in response to different forms of physiological and pharmacological stress. The AlkB homolog 7 (ALKBH7) protein is required for regulated cellular necrosis in response to chemotherapeutic alkylating agents but its role within a whole organism is unknown. Here, we show that ALKBH7 modulates alkylation-induced cellular death through a tissue and sex-specific mechanism. At the whole-animal level, we find that ALKBH7 deficiency confers increased resistance to MMS-induced toxicity in male but not female mice. Moreover, ALKBH7-deficient mice exhibit protection against alkylation-mediated cytotoxicity in retinal photoreceptor and cerebellar granule cells, two cell types that undergo necrotic death through the initiation of the base excision repair pathway and hyperactivation of the PARP1/ARTD1 enzyme. Notably, the protection against alkylation-induced cerebellar degeneration is specific to ALKBH7-deficient male but not female mice. Our results uncover an in vivo role for ALKBH7 in mediating a sexually dimorphic tissue response to alkylation damage that could influence individual responses to chemotherapies based upon alkylating agents

mAagTg CGN sensitivity to MMS is not dependent on NAD+, pyruvate, caspases, Rip1K, calcium fluxes, or MPT.

<p>(A) NAD⁺ pre-treatment did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 3. (B) Pyruvate did not rescue <i>mAagTg</i> neuron sensitivity to MMS. Errors bars denote standard deviation from the mean. <i>mAagTg</i> n = 1. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS and MMS + Veliparib. (C) Primary <i>mAagTg</i> CGN sensitivity to MMS is not dependent on caspase activation, Rip1 kinase activity, calcium fluxes, or mitochondrial permeability transition (MPT). Inhibitors used include zVad-fmk (zVad), n = 6, Necrostatin-1 (Nec-1), n = 3, BAPTA-AM, n = 3, and cyclosporin A (CsA), n = 3. Errors bars denote standard error from the mean. *** p<0.001 using Student’s standard two-tailed T-test comparing MMS to MMS+Veliparib.</p

No loss in mitochondrial permeability or translocation of AIF in neurons after MMS treatment.

<p>(A) There is no loss of mitochondrial permeability in <i>Aag</i>^-/-, WT, or <i>mAagTg</i> neurons 1–7 hours after MMS treatment. Errors bars denote standard deviation from the mean. <i>Aag</i>^-/- n = 2, WT n = 3, <i>mAagTg</i> n = 2. ** p<0.01 using Student’s standard two-tailed T-test comparing FCCP to control. * p<0.05 using Student’s standard one-tailed T-test comparing FCCP to control. (B) WT CGNs treated with MMS do not show evidence of AIF (red) translocation from the mitochondria (green; CoxIV) to the nucleus (blue; Hoechst).</p

Parp inhibition rescues CGN sensitivity to MMS.

<p><i>Aag</i>^-/-, WT, and <i>mAagTg</i> neurons are rescued from MMS toxicity (1 mM) after pretreatment with Parp inhibitor Veliparib (A) or Olaparib (B). Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test compared to neurons of the same genotype treated with 1 mM MMS (control). (C) Parp inhibitor Veliparib rescues cell sensitivity at all doses of MMS in WT and <i>mAagTg</i> neurons. Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test comparing sensitivity with or without Veliparib for a genotype at a particular dose of MMS. (D) Expression of BER genes in CGNs. Errors bars denote standard deviation from the mean, <i>Aag</i>^-/- n = 4, WT n = 5, <i>mAagTg</i> n = 1. (E) Aag expression was assessed either before, immediately after, or an hour after MMS treatment using <i>Aag</i> mRNA FISH in WT CGNs. Errors bars denote standard deviation from the mean.</p

CGN sensitivity to MMS is dependent on Aag and involves activation of Parp.

<p>(A) Overview of CGN toxicity assay based on high content imaging. (B) Sensitivity to MMS treatment <i>ex vivo</i> is dependent on Aag. Errors bars denote standard error from the mean, <i>Aag</i>^-/-: n = 6, WT: n = 5, <i>mAagTg</i>: n = 3. * p<0.05, ** p<0.01, *** p<0.0001 using Student’s standard two-tailed T-test comparing a single dose to WT. (C) PAR formation (green) was visualized in <i>Aag</i>^-/-, WT, and <i>mAagTg</i> neurons 0, 15, 30, or 60 minutes after the addition of MMS (1 mM) by immunocytochemical staining. (D) Fold changes in nuclear PAR staining was quantified and reflects qualitative trends in (C). Errors bars denote standard error from the mean. <i>Aag</i>^-/-: n = 4, WT: n = 3, <i>mAagTg</i>: n = 2. **p<0.01 using Student’s standard two-tailed T-test comparing nuclear fluorescence at a particular timepoint compared to untreated cells. (E) Representative images of PAR formation in <i>mAagTg</i> neurons either left untreated, or 15 minutes after MMS treatment (1 mM) with or without the addition of Veliparib (5 μM). (F) Quantification of changes in nuclear PAR formation in neurons with or without Veliparib pretreatment. Errors bars denote standard error from the mean. <i>Aag</i>^-/-: n = 4, WT: n = 3, <i>mAagTg</i>: n = 2. * p<0.05 using Student’s standard two-tailed T-test comparing nuclear fluorescence with or without Veliparib at a particular time post MMS treatment.</p