Investigation of the biosynthesis of the pipecolate moiety of neuroprotective polyketide meridamycin

Biogenesis of the pipecolate moiety of neuroprotective agent meridamycin in Streptomyces sp. NRRL30748 was investigated in feeding studies using lysine specifically labeled with (15)N at the α-amino or the ε-amino nitrogen position. Fourier transform mass spectrometry analysis with ultra-high mass resolving power and accurate mass measurement capability was employed to resolve the (15)N peak of labeled meridamycin from the (13)C peak of unlabeled meridamycin, allowing the precise calculation of labeling contents under each condition. The relative enrichment of (15)N-labeled meridamycin was ~43% with L-[α-(15)N]-lysine feeding and ~14% with L-[α-(15)N]-lysine feeding, suggesting two distinguishable pathways, with concomitant loss of either the ε-amino group or the α-amino group of lysine, were involved in the generation of the pipecolate moiety of meridamycin in this bacterium. PCR cloning using degenerate primers identified a proC gene encoding a putative pyrroline-5-carboxylate reductase, which was expected to catalyze the conversion of piperideine-6-carboxylate to pipecolate. However, inactivation of this locus did not significantly affect the incorporation of α-(15)N- or ε-(15)N-labeled lysine into meridamycin, indicating the existence of an alternative route for the last step of the lysine ε-transamination pathway. This work revealed the diversity and complexity of the biosynthetic pathways for pipecolate synthesis in the meridamycin producing bacterium Streptomyces sp. NRRL30748.     

Flocculate removal after alkaline lysis in plasmid DNA production

Alkaline lysis is the most commonly used method following harvest of bacterial cells for production of plasmid DNA. The method was originally developed for laboratory scale experiments and has shown to be challenging at larger scales. A major problem prior to further downstream processing is the risk of filter clogging without efficient removal of the flocculate that occurs after neutralization. For this purpose we here present a scalable method where the clarification of alkaline lysate is greatly simplified. Through a rapid procedure, involving the addition of ammonium hydrogen carbonate to the neutralized alkaline lysate, the flocculate is lifted to the surface of the solution by the released carbon dioxide and ammonium. After this step a clarified solution can be drained from the bottom of the vessel. The procedure does not impact pH, plasmid DNA concentration or the ratio of open circular to supercoiled plasmid DNA in the solution.     

An international validation study of the EORTC QLQ-INFO25 questionnaire: An instrument to assess the information given to cancer patients

AimThe EORTC Quality of Life (QOL) Group has developed an instrument to evaluate the information received by cancer patients. This study assessed the psychometric characteristics of the EORTC INFO module in a large international/multi-cultural sample of cancer patients.MethodsThe provisional 26-item information module (EORTC INFO26) was administered with the EORTC QLQ-C30 and the information scales of the inpatient satisfaction module EORTC IN-PATSAT32 on two occasions during the patients’ treatment and follow-up period. Questionnaire-hypothesised scale structure, reliability, validity and responsiveness to changes were evaluated through standard psychometric analyses. Patient acceptability was assessed with a debriefing questionnaire.ResultsThe study comprised 509 patients from 8 countries (7 European countries and Taiwan) with different cancers and disease stages. Multi-trait scaling analysis led to the deletion of one item but confirmed the hypothesised 4 multi-item scales (information about disease, medical tests, treatment and other services) and eight single items. Internal consistency for all scales was good (α > 0.70), as was test–retest reliability (intraclass correlations > 0.70). All items can be combined to generate a single score (α > 0.90). Convergent validity was supported by significant correlations with related areas of IN-PATSAT32 (r > 0.40). Low correlations with EORTC QLQ-C30 scales confirmed divergent validity (r < 0.30) The EORTC INFO-25 module discriminated among groups based on gender, age, education, levels of anxiety and depression, information wishes and satisfaction. Only one scale captured changes over time.ConclusionsThe EORTC QLQ-INFO 25 is a reliable and valid self-reported instrument. The module can be used in cross-cultural observational and intervention studies.     

The aetiology of upper aerodigestive tract cancers among young adults in Europe: the ARCAGE study

Background

The incidence of cancers of the upper aerodigestive tract (UADT) is increasing throughout the world. To date the increases have been proportionally greatest among young people. Several reports have suggested that they often do not have a history of tobacco smoking or heavy alcohol consumption.

Objective

To determine the contribution of lifestyle factors to the etiology of UADT cancers occurring in those aged less than 50 years.

Methods

A case–control study was conducted in 10 European countries. Cases were cancers of the oral cavity and pharynx, larynx and esophagus, and hospital or population controls were age and sex matched.

Results

There were 356 cases younger than 50 years and 419 controls. Risk was strongly related to current smoking [odds ratio (OR) 5.5 95%; confidence interval (CI) (3.3, 9.2)], and risk increased with number of pack-years smoked. Risk was also related to alcohol consumption for both current (OR 1.8; 0.97, 3.3) and past (OR 3.4; 1.6, 7.4) drinkers, and risk increased with number of drink-years. Persons frequently consuming fruits and vegetables were at significantly reduced risk.

Conclusions

Risk factors already identified as being important for UADT cancers in adults are also important influences on risk in younger adults. The implication of these results is that the public health message in preventing UADT cancers remains the same to young and old alike.     

Dietary fiber intake and head and neck cancer risk: A pooled analysis in the International Head and Neck Cancer Epidemiology consortium

The possible role of dietary fiber in the etiology of head neck cancers (HNCs) is unclear. We used individual‐level pooled data from ten case‐control studies (5959 cases and 12,248 controls) participating in the International Head and Neck Cancer Epidemiology (INHANCE) consortium, to examine the association between fiber intake and cancer of the oral cavity/pharynx and larynx. Odds Ratios (ORs) and their 95% Confidence Intervals (CIs) were estimated using unconditional multiple logistic regression applied to quintile categories of non‐alcohol energy‐adjusted fiber intake and adjusted for tobacco and alcohol use and other known or putative confounders. Fiber intake was inversely associated with oral and pharyngeal cancer combined (OR for 5th vs. 1st quintile category = 0.49, 95% CI: 0.40–0.59; p for trend <0.001) and with laryngeal cancer (OR = 0.66, 95% CI: 0.54–0.82, p for trend <0.001). There was, however, appreciable heterogeneity of the estimated effect across studies for oral and pharyngeal cancer combined. Nonetheless, inverse associations were consistently observed for the subsites of oral and pharyngeal cancers and within most strata of the considered covariates, for both cancer sites. Our findings from a multicenter large‐scale pooled analysis suggest that, although in the presence of between‐study heterogeneity, a greater intake of fiber may lower HNC risk.     

Sputum eosinophilia: an early marker of bronchial response to occupational agents

Background: False‐negative responses to specific inhalation challenge (SIC) with occupational agents may occur. We explored whether assessing changes in sputum cell counts would help improve the identification of bronchial reactivity to occupational agents during SICs. Methods: The predictive value of the changes in sputum cell counts after a negative FEV₁ response to a first challenge exposure to an occupational agent was determined using the changes in airway calibre observed during repeated challenges as the ‘gold standard’. The study included 68 subjects investigated for work‐related asthma in a tertiary centre. After a control day, the subjects were challenged with the suspected occupational agent(s) for up to 2 h. All subjects who did not show an asthmatic reaction were re‐challenged on the following day. Additional challenges were proposed to those who demonstrated a ≥ 2% increase in sputum eosinophils or an increase in nonspecific bronchial hyperresponsiveness to histamine after the second challenge day. Results: Six of the 35 subjects without changes in FEV₁ on the first challenge developed an asthmatic reaction on subsequent challenges. ROC analysis revealed that a >3% increase in sputum eosinophils at the end of the first challenge day was the most accurate parameter for predicting the development of an asthmatic response on subsequent challenges with a sensitivity of 67% and a specificity of 97%. Conclusions: An increase in sputum eosinophils is an early marker of specific bronchial reactivity to occupational agents, which may help to identify subjects who will develop an asthmatic reaction only after repeated exposure.