Reverse engineering of short-chain fatty acid tolerance and production in Escherichia coli

Product toxicity is a common problem in microbial production of biorenewable fuels and chemicals. Historically, the field of metabolic engineering has relied on enrichment of expression libraries and strain evolution for improving tolerance. Reverse engineering of these strains can aid in the identification and development of rational design strategies for strain improvement, according to Orgel’s Second Rule that “evolution is cleverer than you are”. Here, we investigated the evolved strain with increased short-chain fatty acid tolerance and 5-fold higher fatty acid titer relative to its parent strain to discover and understand the mechanisms of the phenotypic changes. Four mutations were identified in the evolved strains, as well as the chronological order of mutations during adaptive evolution. Then we studied each mutation and their synergistic interaction by characterizing the reconstructed strains, which had single mutation, double mutation or triple mutations replaced into the genome of the parent strain. The waaG mutation contributed to increased C8 tolerance, fatty acid titer, and membrane integrity. The increased fatty acid titer was mainly affected by the mutant rpoC gene. The decreased membrane fluidity and increased cell surface hydrophobicity of evolved strain were caused by the synergistic interaction of waaG, rpoC, and basR mutations. We also noticed each mutation was able to alter the membrane lipid composition differently. The association between mutations and phenotypic changes we identified could be used as rational engineering strategies for improving tolerance and production of microbial biocatalyst. However, the rpoC and basR encode transcriptional regulators, and we were not able to fully understand the mechanisms of these two mutations by only genome-level reverse engineering. Thus, we further investigated these two mutations by transcriptome analysis. Compared evolved strain LAR1 to parent strain ML115 during fatty acid producing, twenty-nine genes made statistically significant changes in transcript abundance, which could be influenced by rpoC mutation. Also, nine genes were identified had differential transcript abundance, which could be impacted by the basR mutation. Characterization of these interesting genes is in progress to discover the mechanisms of increased short-chain fatty acids tolerance and production of evolved strain LAR1. In order to deepen our understanding the association between fatty acid production and cell membrane properties, we characterized a small group of environmental E. coli isolates with significantly higher short-chain fatty acids production in minimal medium relative to lab strain MG1655. Consistent with previous studies, decreased membrane fluidity was associated with increased fatty acid production. The C16:1/C16:0 ratio (mol/mol) was suggested to be one of the important metrics, when rationally engineering membrane lipid composition for improving short-chain fatty acids production in E. coli. We also discovered the direct association between cell surface hydrophobicity and short-chain-fatty acids titer. In short, the findings of our work could provide new insights into the mechanisms of short-chain fatty acid tolerance and production, and rational engineering strategies for improving performance of biocatalyst

Risk factors for acute respiratory infection in the Australian community

OBJECTIVES The objective of this study was to identify the risk factors for ARI in the Australian community. METHODS We used a national survey of 7578 randomly selected respondents in 2008-2009 to identify the risk factors of ARI. A case was defined as a person experiencing cold or flu with one or more symptoms of: fever, chills, sore throat, runny nose, or cough in the previous four weeks. RESULTS There were 19.8% (1505/7578) of respondents who reported ARI in the four weeks prior to the survey. Age was an independent risk factor for ARI, with the risk of acquiring ARI decreasing as age increased. Respondents reporting asthma (OR 1.4, 95%CI: 1.2-1.7) or having someone in their house attending childcare (OR 1.6, 95%CI: 1.2-2.1) were more likely to report ARI. CONCLUSIONS It is important to identify ways of interrupting transmission of ARI amongst children. Improving identification of risk factors will enable targeted interventions for this exceedingly common syndrome.The study was funded by the Commonwealth Department of Health, and study conduct was overseen by a committee comprised of representatives from Australian government agencies and departments

Understanding Gastroenteritis in Middle‐aged and Older Australians

Background: Gastroenteritis is an important cause of morbidity in older adults, resulting in a significant health burden globally. The aims of this thesis were to describe the epidemiology of gastroenteritis in older adults and to investigate factors associated with hospitalisation with all-cause and cause-specific gastroenteritis in a cohort of middle-aged and older Australians. Methods: I used design-based logistic regression and proportional hazards regression to analyse two datasets: (1) a national survey of gastroenteritis in the Australian community conducted in 2008–2009; and (2) a large-scale population-based cohort of middle-aged and older Australians with data linkage to hospitalisations, pharmaceuticals, notifiable diseases and deaths data. Additionally, I conducted a systematic review and meta-analysis of Clostridium difficile infection among people with inflammatory bowel disease. Results: I estimated that 78,356 people aged ≥65 years old visited a doctor due to gastroenteritis in Australia annually, with 157,317 million courses of medication usage in one year from 2008−2009. From population-based cohort data, I demonstrated that the incidence of hospitalisation with gastroenteritis increased with older age; from 2.4 per 1,000 person-years in adults aged 45-54 years old to 9.5 per 1,000 in those aged ≥65 years. Compared to adults aged 45-54 years old, older persons had a higher incidence of hospitalisation with Salmonella infection and C. difficile infection. After adjustment, the risk of hospitalisation with gastroenteritis differed depending on sex and region of residence. Poor self-rated health and use of proton pump inhibitors (PPI) were significantly associated with gastroenteritis hospitalisation. Hospitalisation with C. difficile infection was associated with longer hospital stays, greater in-hospital costs and higher in-hospital deaths compared to hospitalisation without C. difficile infection. In a meta-analysis of six international studies included in the systematic review, C. difficile infection was a significant risk factor for colectomy among patients with inflammatory bowel disease (Odds Ratio: 1.90; 95%CI 1.23-2.93). Conclusions: This thesis demonstrates a significant burden of gastroenteritis in older Australians. Incidence of hospitalisation with all-cause and cause-specific gastroenteritis increases significantly with age. Future efforts should focus on defining and improving preventive measures for gastroenteritis hospitalisation among the elderly. The risk of hospitalisation varies by sex and region of residence, which reflects differences in exposure. PPI use is significantly associated with gastroenteritis hospitalisation. Given the widespread of PPI use, particularly among older people, clinicians should be aware of this potential association when considering PPI therapy. In addition, early recognition and supportive treatment of diarrhoea in older patients with poor self-rated health may prevent subsequent hospitalisation and improve their health outcome

Lessons in Membrane Engineering for Octanoic Acid Production from Environmental Escherichia coli Isolates

Fermentative production of many attractive biorenewable fuels and chemicals is limited by product toxicity in the form of damage to the microbial cell membrane. Metabolic engineering of the production organism can help mitigate this problem, but there is a need for identification and prioritization of the most effective engineering targets. Here, we use a set of previously characterized environmental Escherichia coli isolates with high tolerance and production of octanoic acid, a model membrane-damaging biorenewable product, as a case study for identifying and prioritizing membrane engineering strategies. This characterization identified differences in the membrane lipid composition, fluidity, integrity, and cell surface hydrophobicity from those of the lab strain MG1655. Consistent with previous publications, decreased membrane fluidity was associated with increased fatty acid production ability. Maintenance of high membrane integrity or longer membrane lipids seemed to be of less importance than fluidity. Cell surface hydrophobicity was also directly associated with fatty acid production titers, with the strength of this association demonstrated by plasmid-based expression of the multiple stress resistance outer membrane protein BhsA. This expression of bhsA was effective in altering hydrophobicity, but the direction and magnitude of the change differed between strains. Thus, additional strategies are needed to reliably engineer cell surface hydrophobicity. This work demonstrates the ability of environmental microbiological studies to impact the metabolic engineering design-build-test-learn cycle and possibly increase the economic viability of fermentative bioprocesses

Cardiac arrest and catecholamine cardiomyopathy secondary to a misdiagnosed ectopic pheochromocytoma

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Development and thermal performance of a vapor chamber with multi-artery reentrant microchannels for high-power LED

Abstract(#br)This study developed a vapor chamber (VC) with radial multi-artery reentrant microchannels for thermal management of high-power light emitting diodes (LEDs). It featured Ω-shaped reentrant microchannels inside porous wicks to provide separated flow passages for vapor and liquid flow. It was integrated with a high-power LED module for fast heat dissipation and efficient thermal management. Experiments are systematically conducted to evaluate thermal performance of the VC for a wide range of input power of LEDs, air flow rates and inclination angles of LED module. The VC is compared to a copper plate heat sink in the same operation conditions. Results show that compared to the copper plate, the VC presented a faster temperature rise, and was earlier to reach equilibrium state. The VC reduced the substrate surface temperature of LED module for 7% to 27%, and introduced a reduction in the thermal resistance for 19% to 48%, indicating that the VC enhanced cooling capacity remarkably and yield a notable favorable performance for the heat dissipations of LEDs. The thermal performance of the VC was significantly dependent on the input power of LEDs and air flow rates, whereas the inclination angle of LED module showed negligible effects on thermal performance

SOX2 Gene Regulates the Transcriptional Network of Oncogenes and Affects Tumorigenesis of Human Lung Cancer Cells

Recent studies demonstrated that cancer stem cells (CSCs) have higher tumorigenesis properties than those of differentiated cancer cells and that transcriptional factor-SOX2 plays a vital role in maintaining the unique properties of CSCs; however, the function and underlying mechanism of SOX2 in carcinogenesis of lung cancer are still elusive. This study applied immunohistochemistry to analyze the expression of SOX2 in human lung tissues of normal individuals as well as patients with adenocarcinoma, squamous cell carcinoma, and large cell and small cell carcinoma and demonstrated specific overexpression of SOX2 in all types of lung cancer tissues. This finding supports the notion that SOX2 contributes to the tumorigenesis of lung cancer cells and can be used as a diagnostic probe. In addition, obviously higher expression of oncogenes c-MYC, WNT1, WNT2, and NOTCH1 was detected in side population (SP) cells than in non-side population (NSP) cells of human lung adenocarcinoma cell line-A549, revealing a possible mechanism for the tenacious tumorigenic potential of CSCs. To further elucidate the function of SOX2 in tumorigenesis of cancer cells, A549 cells were established with expression of luciferase and doxycycline-inducible shRNA targeting SOX2. We found silencing of SOX2 gene reduces the tumorigenic property of A549 cells with attenuated expression of c-MYC, WNT1, WNT2, and NOTCH1 in xenografted NOD/SCID mice. By using the RNA-Seq method, an additional 246 target cancer genes of SOX2 were revealed. These results present evidence that SOX2 may regulate the expression of oncogenes in CSCs to promote the development of human lung cancer

A population-based longitudinal study of Clostridium difficile infection-related hospitalization in mid-age and older Australians

Clostridium difficile is the principal cause of infectious diarrhoea in hospitalized patients. We investigated the incidence and risk factors for hospitalization due to C. difficile infection (CDI) in older Australians. We linked data from a population-based prospective cohort study (the 45 and Up Study) of 266 922 adults aged ⩾45 years recruited in New South Wales, Australia to hospitalization and death records for 2006–2012. We estimated the incidence of CDI hospitalization and calculated days in hospital and costs per hospitalization. We also estimated hazard ratios (HR) for CDI hospitalization using Cox regression with age as the underlying time variable. Over a total follow-up of 1 126 708 person-years, 187 adults had an incident CDI hospitalization. The crude incidence of CDI hospitalization was 16·6/100 000 person-years, with a median hospital stay of 6 days, and a median cost of AUD 6102 per admission. Incidence increased with age and year of follow-up, with a threefold increase for 2009–2012. After adjustment, CDI hospitalization rates were significantly lower in males than females (adjusted HR 0·6, 95% confidence interval 0·4–0·7). CDI hospitalization rates increased significantly over 2009–2012. There is a need to better understand the increasing risk of CDI hospitalization in women.Y.C. received the 2014 Prime Minister’s Australia Asia Postgraduate Scholarship from the Australian Government Department of Education and Training (3929_2014). B.L. is funded by an NHMRC Fellowship

Population-Level Human Secretor Status Is Associated With Genogroup 2 Type 4 Norovirus Predominance

Background. Noroviruses are a leading cause of acute gastroenteritis. Genogroup 2 type 4 (GII.4) has been the dominant norovirus genotype worldwide since its emergence in the mid-1990s. Individuals with a functional fucosyltransferase-2 gene, known as secretors, have increased susceptibility to GII.4 noroviruses. We hypothesized that this individual-level trait may drive GII.4 norovirus predominance at the human population level. Methods. We conducted a systematic review for studies reporting norovirus outbreak or sporadic case genotypes and merged this with data on proportions of human secretor status in various countries from a separate systematic review. We used inverse variance-weighted linear regression to estimate magnitude of the population secretor-GII.4 proportion association. Results. Two hundred nineteen genotype and 112 secretor studies with data from 38 countries were included in the analysis. Study-level GII.4 proportion among all noroviruses ranged from 0% to 100%. Country secretor proportion ranged from 43.8% to 93.9%. We observed a 0.69% (95% confidence interval, 0.19-1.18) increase in GII.4 proportion for each percentage increase in human secretor proportion, controlling for Human Development Index. Conclusions. Norovirus evolution and diversity may be driven by local population human host genetics. Our results may have vaccine development implications including whether specific antigenic formulations would be required for different populations.This work was funded by the National Institutes of Health/National Institute of General Medical Sciences (R01GM124280; to B. L.) and the Centers for Disease Control and Prevention (IPA 48195; to B. L.)