Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways

Microbial systems are being increasingly developed as production hosts for a wide variety of chemical compounds. Broader adoption of microbial synthesis is hampered by a limited number of high-yielding natural pathways for molecules with the desired physical properties, as well as the difficulty in functionally assembling complex biosynthetic pathways in heterologous hosts. Here, we address both of these challenges by reporting the adaptation of the butanol biosynthetic pathway for the synthesis of odd-chain molecules and the development of a complementary modular toolkit to facilitate pathway construction, characterization, and optimization in engineered Escherichia coli. The modular feature of our pathway enables multientry and multiexit biosynthesis of various odd-chain compounds at high efficiency. By varying combinations of the pathway and toolkit enzymes, we demonstrate controlled production of propionate, trans-2-pentenoate, valerate, and pentanol, compounds with applications that include biofuels, antibiotics, biopolymers, and aroma chemicals. Importantly, and in contrast to a previously used method to identify limitations in heterologous amorphadiene production, our bypass strategy was effective even without the presence of freely membrane-diffusible substrates. This approach should prove useful for optimization of other pathways that use CoA-derivatized intermediates, including fatty acid β-oxidation and the mevalonate pathway for isoprenoid synthesis.     

Alcohol and vagal tone as triggers for paroxysmal atrial fibrillation

Alcohol and vagal activity may be important triggers for paroxysmal atrial fibrillation (PAF), but it remains unknown if these associations occur more often than would be expected by chance alone because of the lack of a comparator group in previous studies. We compared self-reported frequency of these triggers in patients with PAF to those with other supraventricular tachycardias (SVTs). Consecutive consenting patients presenting for electrophysiology procedures at a single university medical center underwent a structured interview regarding arrhythmia triggers. Two hundred twenty-three patients with a documented arrhythmia (133 with PAF and 90 with SVT) completed the survey. After multivariable adjustment, patients with PAF had a 4.42 greater odds (95% confidence interval [CI] 1.35 to 14.44) of reporting alcohol consumption (p = 0.014) and a 2.02 greater odds (95% CI 1.02 to 4.00) of reporting vagal activity (p = 0.044) as an arrhythmia trigger compared to patients with SVT. In patients with PAF, drinking primarily beer was associated with alcohol as a trigger (odds ratio [OR] 4.49, 95% CI 1.41 to 14.28, p = 0.011), whereas younger age (OR 0.68, 95% CI 0.49 to 0.95, p = 0.022) and a family history of AF (OR 5.73, 95% CI 1.21 to 27.23, p = 0.028) each were independently associated with having vagal activity provoke an episode. Patients with PAF and alcohol triggers were more likely to have vagal triggers (OR 10.32, 95% CI 1.05 to 101.42, p = 0.045). In conclusion, alcohol consumption and vagal activity elicit PAF significantly more often than SVT. Alcohol and vagal triggers often were found in the same patients with PAF, raising the possibility that alcohol may precipitate AF by vagal mechanisms.     

Arecoline induced cell cycle arrest, apoptosis, and cytotoxicity to human endothelial cells

Betel quid (BQ) chewing is a common oral habit in South Asia and Taiwan. BQ consumption may increase the risk of oral squamous cell carcinoma (OSCC), oral submucous fibrosis (OSF), and periodontitis as well as systemic diseases (atherosclerosis, hypertension, etc.). However, little is known about the toxic effect of BQ components on endothelial cells that play important roles for angiogenesis, carcinogenesis, tissue fibrosis, and cardiovascular diseases. EAhy 926 (EAHY) endothelial cells were exposed to arecoline, a major BQ alkaloid, for various time periods. Cytotoxicity was estimated by 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The cell cycle distribution of EAHY cells residing in sub-G0/G1, G0/G1, S-, and G2/M phases was analyzed by propidium iodide staining of cellular DNA content and flow cytometry. Some EAHY cells retracted, became round-shaped in appearance, and even detached from the culture plate after exposure to higher concentrations of arecoline (> 0.4 mM). At concentrations of 0.4 and 0.8 mM, arecoline induced significant cytotoxicity to EAHY cells. At similar concentrations, arecoline induced G2/M cell cycle arrest and increased sub-G0/G1 population, a hallmark of apoptosis. Interestingly, prolonged exposure to arecoline (0.1 mM) for 12 and 21 days significantly suppressed the proliferation of EAHY cells, whereas EAHY cells showed adaptation and survived when exposed to 0.05 mM arecoline. These results suggest that BQ components may contribute to the pathogenesis of OSF and BQ chewing-related cardiovascular diseases via toxicity to oral or systemic endothelial cells, leading to impairment of vascular function. During BQ chewing, endothelial damage may be induced by areca nut components and associate with the pathogenesis of OSF, periodontitis, and cardiovascular diseases.     

Diabetes but not insulin is associated with higher colon cancer mortality

AIM: To evaluate whether diabetic patients had a higher risk of colon cancer mortality and its associated risk factors.METHODS: The sex-specific crude and age-standardized (to the 2000 World Health Organization population) mortality rates of colon cancer in the Taiwanese general population were first calculated from 1995 to 2006. The trends were evaluated by linear regression. A total of 113 347 diabetic men and 131 573 diabetic women aged ≥ 25 years at recruitment from 1995 to 1998 were followed up until the end of 2006. Age/sex-specific colon cancer mortality rate ratios were calculated comparing the mortality rates of the diabetic patients with the average mortality rates of the general population within 12 years (1995-2006). A sub-cohort of diabetic patients (42 260 men and 49 405 women) was interviewed using a baseline questionnaire and Cox’s regression was used to evaluate the risk factors for colon cancer mortality in these diabetic patients.RESULTS: The crude and age-standardized trends of colon cancer mortality from 1995 to 2006 increased significantly for both sexes in the general population. A total of 641 diabetic men and 573 diabetic women died of colon cancer, with a mortality rate of 74.4 and 54.3 per 100 000 person-years, respectively. Mortality rate ratios [95% confidence intervals (CIs)] showed a significantly higher risk of mortality from colon cancer for the diabetic patients compared to the general population, with the magnitude increasing with decreasing age: 1.65 (1.40-1.95), 2.01 (1.78-2.27), 2.75 (2.36-3.21) and 5.69 (4.65-6.96) for ≥ 75, 65-74, 55-64 and 25-54 years old, respectively, for men; and 1.46 (1.24-1.72), 2.09 (1.84-2.38), 2.67 (2.27-3.14) and 3.05 (2.29-4.06), respectively, for women. Among the sub-cohort of diabetic patients who had been interviewed with the baseline questionnaire, including information on age, sex, diabetes duration, diabetes type, body mass index, smoking, insulin use and area of residence, age and smoking were significantly predictive for colon cancer mortality, with respective adjusted hazard ratios (HRs) (95% CIs) of 1.077 (1.066-1.088) and 1.384 (1.068-1.792). Diabetes duration became a significant factor when those who died of colon cancer within 5 years of diabetes diagnosis were excluded to minimize the possible contamination of diabetes caused by incipient colon cancer, with an adjusted hazard ratio of 1.021 (1.007-1.034). Sex, diabetes type, insulin use, body mass index and area of residence were not significant predictors for colon cancer mortality in the diabetic patients. Although insulin use was categorized into subgroups of duration of use (non-users and users < 5 years, 5-9 years and ≥ 10 years), none of the HRs for colon cancer mortality was significant with regards to different durations of insulin use.CONCLUSION: Colon cancer mortality is increasing in Taiwan. A higher risk is observed in diabetic patients. Smoking, but not insulin use, is a modifiable risk factor.