Molecular epidemiologic studies on helicobacter pylori infection and stomach cancer risk

Helicobacter pylori (H. pylori) infection increases stomach cancer risk. The aim of this thesis was to study genetic susceptibility from the host and to develop molecular methods for future characterization of bacterial virulence factors in longitudinal cohorts. In Study I, we investigated the association between genetic variation in an O-glycan transferase encoding gene (a4GnT) and H. pylori infection and gastric cancer risk in a Polish population-based case–control study (273 gastric cancer patients and 377 controls). A haplotype at rs2622694–rs397266 was associated with H. pylori infection, with the A-A haplotype associated with a higher risk compared with the most frequent G-G haplotype (odds ratio 2.30; 95% confidence intervals 1.35– 3.92). Neither this haplotype nor the tagSNPs were associated with overall gastric cancer risk. In Study II, we characterized genomic evolution of H. pylori over 20 years in the stomach. Whole genome of 21 sequential isolates 20 years apart, from 7 patients, were sequenced using 454 sequencing platform. There were on average 260 point mutations (range 70 to 488) per isolate over 20 years, and 45 recombinations (range 18 to 92). Genes in the cell motility category were overrepresented in point mutations and recombinations. Specifically, mutations often affected genes involved in chemotaxis, vacuolating cytotoxin-like protein, restriction and type IV secretory pathway; and recombinations affected glycosyltransferase involved in lipopolysaccharide biosynthesis. The major form of single nucleotide substitutions was transition (85%) and the minor form was transversion (15%). Mutation was sequence contextdependent. Clinical samples are often precious and of trace amounts. In Study III, we developed novel methods for DNA shotgun library construction and quantification. As compared with the standard procedure, our double-stranded and Y library construction methods are simpler and more efficient. A highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) was developed to quantify the amount of effective library. We also demonstrated that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labor intensive and costly titration can be eliminated and trace amounts of starting material is applicable. Archived formalin-fixed and paraffin-embedded (FFPE) biopsies, coupled with long term follow-up, are valuable resources for molecular epidemiologic studies. Study IV presented a method based on laser capture micro-dissection and modified whole genome sequencing methods to obtain metagenomic profiles of H. pylori from 15-year old FFPE biopsy sections

Rapid Screening of Complex DNA Samples by Single-Molecule Amplification and Sequencing

Microbial cloning makes Sanger sequencing of complex DNA samples possible but is labor intensive. We present a simple, rapid and robust method that enables laboratories without special equipment to perform single-molecule amplicon sequencing, although in a low-throughput manner, from sub-picogram quantities of DNA. The method can also be used for quick quality control of next-generation sequencing libraries, as was demonstrated for a metagenomic sample

LIN28 Is Involved in Glioma Carcinogenesis and Predicts Outcomes of Glioblastoma Multiforme Patients

LIN28, an evolutionarily conversed RNA binding protein which can bind to the terminal loops of let-7 family microRNA precursors and block their processing to maturation, is highly expressed in several subsets of tumors that carry poor prognoses, such as ovarian carcinoma, hepatocellular carcinoma, colon carcinoma and germ cell carcinoma. However, there has been no study on the expression of LIN28 in glioma tissues or their importance as a prognostic predictor of glioma patients. This study aimed to examine the expression of LIN28 in glioma and correlate the results to patient outcome. We found that LIN28 expression was significantly higher in the group of patients with a poor prognosis compared to patients with a good prognosis by gene microarray. Log-rank analysis showed patients with higher LIN28 expression level in tumor had a shorter progression-free survival and overall survival times compared to those with lower LIN28 expression level. Similar results were also obtained from the tissue microarray analysis. Univariate and multivariate analyses showed high LIN28 expression was an independent prognostic factor for a shorter progression-free survival and overall survival in GBM patients. Furthermore in vitro experiments showed that down-regulation of LIN28 in U251 and U373 cells caused cell cycle arrest in the G1 phase, delayed cell proliferation, increased apoptosis, and resulted in fewer colonies compared to controls. Summarily, our data provides a potential target for cancer therapy as an approach to overcome the poor options currently available for GBM patients

Titration-free massively parallel pyrosequencing using trace amounts of starting material

Continuous efforts have been made to improve next-generation sequencing methods for increased robustness and for applications on low amounts of starting material. We applied double-stranded library protocols for the Roche 454 platform to avoid the yield-reducing steps associated with single-stranded library preparation, and applied a highly sensitive Taqman MGB-probe-based quantitative polymerase chain reaction (qPCR) method. The MGB-probe qPCR, which can detect as low as 100 copies, was used to quantify the amount of effective library, i.e. molecules that form functional clones in emulsion PCR. We also demonstrate that the distribution of library molecules on capture beads follows a Poisson distribution. Combining the qPCR and Poisson statistics, the labour-intensive and costly titration can be eliminated and trace amounts of starting material such as precious clinical samples, transcriptomes of small tissue samples and metagenomics on low biomass environments is applicable

Erector spinae plane block versus caudal block for postoperative analgesia in pediatric patients undergoing inguinal hernia repair: a randomized controlled trial

AbstractBackground Erector spinae plane block is a promising strategy for pain management in some settings. However, the effectiveness of erector spinae plane block versus caudal block in pediatric inguinal hernia repair has yet to be formally investigated.Methods One hundred and two patients aged 2–5 years undergoing unilateral open inguinal hernia repair randomly received unilateral erector spinae plane block (0.2% ropivacaine 0.5 mL kg−1), caudal block (0.2% ropivacaine 1 mL kg−1), or no block. The primary outcome was time to the first rescue analgesia, defined as the interval from the end of surgery to the Face, Legs, Activity, Cry, and Consolability scale greater than three. Secondary outcomes included the number of patients requiring rescue analgesia, the area under the curve of pain scores over time, satisfaction of guardians, and adverse events.Results The median time to the first rescue analgesia was longer in the erector spinae plane block group than in the caudal block group [10.0 h (interquartile range, 6.6–24.0 h) vs. 5.0 h (interquartile range, 2.9–7.3 h); p < .001]. The Cox regression model demonstrated that the risk of postoperative rescue analgesia requirement was 0.38 in children receiving erector spinae plane block compared with caudal block (95% confidence interval 0.23–0.64; p < .001). Additionally, the area under the curve of the pain scores over time was lower in the erector spinae plane block group than in the caudal block group (44.3 [36.6–50.7] vs. 59.0 [47.1–64.5]; p < .001).Conclusions Erector spinae plane block provided superior postoperative analgesia compared to caudal block in children undergoing inguinal hernia repair.Trial registration: Chinese Clinical Trial Registry; ChiCTR210004830

Is there a link between the lipopolysaccharide of Helicobacter pylori gastric MALT lymphoma associated strains and lymphoma pathogenesis?

The aim of this study was to investigate the Lewis antigen expression in Helicobacter pylori gastric MALT lymphoma associated strains in comparison to chronic gastritis only strains. Forty MALT strains (19 cagPAI (-) and 21 cagPAI (+)) and 39 cagPAI frequency-matched gastritis strains (17 cagPAI (-) and 22 cagPAI (+)) were included in this study. The lipopolyssacharide for each strain was extracted using a hot phenol method and the expression of Le(x) and Le(y) were investigated using Western Blot. The data were analyzed according to the strains' cagPAI status and vacA genotype. Le(x) was identified in 21 (52.5%) MALT strains and 29 (74.3%) gastritis strains. Le(y) was identified in 30 (75%) MALT strains and 31 (79.5%) gastritis strains. There was an association between cagPAI positivity and Le(x) expression among MALT strains (p<0.0001), but not in gastritis strains (p = 0.64). Among cagPAI (-) strains, isolates expressing solely Le(y) were associated with MALT with an odds ratio of 64.2 (95% CI 4.9-841.0) when compared to strains expressing both Le(x) and Le(y). vacA genotypes did not modify the association between Lewis antigen expression and disease status. In conclusion, cagPAI (-) MALT strains have a particular Lewis antigen profile which could represent an adaptive mechanism to the host response or participate in MALT lymphomagenesis

Formation mechanism of an undesirable by-product in the mild hydro-chemical process for the extraction of alumina from fly ash and its mitigation

The mild hydro-chemical process has been commercially demonstrated as an effective method to extract alumina from high alumina fly ash. This study investigated the formation mechanism of an undesirable aluminum containing by-product, hydrated sodium calcium aluminosilicate (HSCA, 1.2Na(2)O center dot 0.8CaO center dot Al2O3 center dot 2SiO(2)center dot H2O), produced during the mild hydro-chemical process and its mitigation strategy. The effects of reaction temperature, residence time and mole ratio of calcium to silicon on the alumina extraction and phase transformation of the leaching residues have been investigated, confirming the formation of HSCA phase being responsible for the decrease of alumina extraction efficiency. At the low Ca(OH)(2) addition, the HSCA phase could be easily transformed from Na8Al6Si6O24(OH)(2)(H2O)(2) combining with Ca(OH)(2). On the other hand, when the addition of Ca(OH)(2) was abundant, the existence of the NaCaHSiO4 product layer would insulate most of Ca(OH)(2) from the Na8Al6Si6O24(OH)(2)(H2O)(2) causing formation of HSCA. Based on the reaction mechanism and coating effect analysis, the mitigation strategy was also proposed. By increasing stirring speed to break the NaCaHSiO4 coating layer, HSCA phase could be completely avoided and a qualified alumina extraction efficiency of 90.84% could be achieved