Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.

To examine the effect of dynamic changes in neutrophil-to-lymphocyte ratio (NLR) on tumor response and overall survival (OS) in patients with hepatocellular carcinoma (HCC) undergoing transarterial chemoembolization (TACE). Patients and Methods: Data from 181 patients with HCC were retrospectively collected. White blood cell, neutrophil and lymphocyte counts, and the NLR were obtained 1-3 days before as well as 3-6 weeks and 3 months after TACE. Patients were divided into two groups at each time point according to the mean value of NLR, and also divided into continuous decrease, fluctuating increase-decrease (I-D), fluctuating decrease-increase (D-I), and continuous increase groups according to the dynamic changes in the NLR. The dynamic changes in blood counts and NLR were analyzed using repeated-measures ANOVA. The odds ratios (ORs) for tumor response in different NLR groups were examined using a multivariate logistic regression model. Finally, the prognostic value of the dynamic changes in the NLR was examined using Cox regression models. Results: Continuous decline of white blood cell counts, neutrophil counts and lymphocyte counts were observed at 3-6 weeks and 3 months after TACE treatment. The NLR increased slightly and then decreased substantially in responders, while it increased slightly and then significantly in nonresponders, with a significant interaction effect of Time × Tumor response (P = 0.005). NLR grouping before TACE, 3-6 weeks and 3 months after TACE was not associated with tumor response, and only 3 months after TACE did, it shows a significant difference in univariate survival analyses (NLR > 2.5 vs NLR ≤ 2.5, hazard ratio [HR] = 2.442, 95% confidence interval (CI): 1.545, 3.860). The changes in the NLR were significantly correlated with tumor response and OS. Nonresponders for TACE were more common in the continuous NLR increase group (OR = 6.230, 95% CI: 1.848-21.001) and in the fluctuating D-I group (OR = 5.702, 95% CI: 1.480-21.957). Multivariate analyses revealed that these two patient groups also showed poorer OS (HR = 2.351, 95% CI: 1.120-4.605 and HR = 2.320, 95% CI: 1.187-4.533, respectively). Conclusion: Dynamic changes in the NLR may be better predictors of tumor response and OS than static NLR values, but more data are needed.

Background An association between a nonmedicinal herbal diet and nasopharyngeal carcinoma (NPC) has often been hypothesized but never thoroughly investigated. Methods This study enrolled a total of 2469 patients with incident NPC and 2559 population controls from parts of Guangdong and Guangxi Provinces in southern China between 2010 and 2014. Questionnaire information was collected on the intake of traditional herbal tea and herbal soup as well as the specific herbal plants used in soups and other potentially confounding lifestyle factors. Multivariate logistic regression models were used to estimate odds ratios (ORs) with 95% confidence intervals (CIs) for the NPC risk in association with herbal tea and soup intake. Results Ever consumption of herbal tea was not associated with NPC risk (OR, 1.03; 95% CI, 0.91‐1.17). An inverse association was observed for NPC among ever drinkers of herbal soup (OR, 0.78; 95% CI, 0.67‐0.90) but without any monotonic trend with an increasing frequency or duration of herbal soup consumption. Inverse associations with NPC risk were detected with 9 herbal plants used in herbal soup, including Ziziphus jujuba, Fructus lycii, Codonopsis pilosula, Astragalus membranaceus, Semen coicis, Smilax glabra, Phaseolus calcaratus, Morinda officinalis, and Atractylodes macrocephala (OR range, 0.31‐0.79). Conclusions Consuming herbal soups including specific plants, but not herbal tea, was inversely associated with NPC. If replicated, these results might provide potential for NPC prevention in endemic areas.

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