The pseudokinase TRIB3 controls adipocyte lipid homeostasis and proliferation in vitro and in vivo.

OBJECTIVE: In vivo studies in humans and mice have implicated the pseudokinase Tribbles 3 (TRIB3) in various aspects of energy metabolism. Whilst cell-based studies indicate a role for TRIB3 in adipocyte differentiation and function, it is unclear if and how these cellular functions may contribute to overall metabolic health. METHODS: We investigated the metabolic phenotype of whole-body Trib3 knockout (Trib3KO) mice, focusing on adipocyte and adipose tissue functions. In addition, we combined lipidomics, transcriptomics, interactomics and phosphoproteomics analyses to elucidate cell-intrinsic functions of TRIB3 in pre- and mature adipocytes. RESULTS: Trib3KO mice display increased adiposity, but their insulin sensitivity remains unaltered. Trib3KO adipocytes are smaller and display higher Proliferating Cell Nuclear Antigen (PCNA) levels, indicating potential alterations in either i) proliferation-differentiation balance, ii) impaired expansion after cell division, or iii) an altered balance between lipid storage and release, or a combination thereof. Lipidome analyses suggest TRIB3 involvement in the latter two processes, as triglyceride storage is reduced and membrane composition, which can restrain cellular expansion, is altered. Integrated interactome, phosphoproteome and transcriptome analyses support a role for TRIB3 in all three cellular processes through multiple cellular pathways, including Mitogen Activated Protein Kinase- (MAPK/ERK), Protein Kinase A (PKA)-mediated signaling and Transcription Factor 7 like 2 (TCF7L2) and Beta Catenin-mediated gene expression. CONCLUSIONS: Our findings support TRIB3 playing multiple distinct regulatory roles in the cytoplasm, nucleus and mitochondria, ultimately controlling adipose tissue homeostasis, rather than affecting a single cellular pathway.

Open fire ovens and effects of in-home lavash bread baking on carbon monoxide exposure and carboxyhemoglobin levels among women in rural Armenia.

Lavash is a traditional flatbread commonly baked at home by women in Armenia and other Middle Eastern and Caucasus countries. The baking process follows centuries' old recipes and is done primarily in open fire ovens. Data are limited regarding the impact of baking on indoor air quality and health outcomes. This study aimed at assessing the effects of lavash baking on household air pollution and cardiovascular outcomes among women who bake lavash in rural Armenia. A convenience sample of 98 bakers, all women, never-smokers, representing 36 households were enrolled. Carbon monoxide (CO) concentrations and carboxyhemoglobin (COHb) levels were monitored before, during, and/or after baking. As expected, exposure to concentrations of CO peaking at/or above 35-ppm during baking was more likely to occur in homes with fully enclosed and poorly ventilated baking rooms, compared to those with three or fewer walls and/or one or more windows. Bakers in homes where CO concentrations peaked at/or above 35-ppm were more likely to have an increase in post-baking COHb levels compared to those in homes with lower CO concentrations.

Exposure to Household Air Pollution from Biomass Cookstoves and Levels of Fractional Exhaled Nitric Oxide (FeNO) among Honduran Women.

Household air pollution is estimated to be responsible for nearly three million premature deaths annually. Measuring fractional exhaled nitric oxide (FeNO) may improve the limited understanding of the association of household air pollution and airway inflammation. We evaluated the cross-sectional association of FeNO with exposure to household air pollution (24-h average kitchen and personal fine particulate matter and black carbon; stove type) among 139 women in rural Honduras using traditional stoves or cleaner-burning Justa stoves. We additionally evaluated interaction by age. Results were generally consistent with a null association; we did not observe a consistent pattern for interaction by age. Evidence from ambient and household air pollution regarding FeNO is inconsistent, and may be attributable to differing study populations, exposures, and FeNO measurement procedures (e.g., the flow rate used to measure FeNO).

Influenza A (H1N1pdm09)-Related Critical Illness and Mortality in Mexico and Canada, 2014.

OBJECTIVES: The 2009-2010 influenza A (H1N1pdm09) pandemic caused substantial morbidity and mortality among young patients; however, mortality estimates have been confounded by regional differences in eligibility criteria and inclusion of selected populations. In 2013-2014, H1N1pdm09 became North America's dominant seasonal influenza strain. Our objective was to compare the baseline characteristics, resources, and treatments with outcomes among critically ill patients with influenza A (H1N1pdm09) in Mexican and Canadian hospitals in 2014 using consistent eligibility criteria. DESIGN: Observational study and a survey of available healthcare setting resources. SETTING: Twenty-one hospitals, 13 in Mexico and eight in Canada. PATIENTS: Critically ill patients with confirmed H1N1pdm09 during 2013-2014 influenza season. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: The main outcome measures were 90-day mortality and independent predictors of mortality. Among 165 adult patients with H1N1pdm09-related critical illness between September 2013 and March 2014, mean age was 48.3 years, 64% were males, and nearly all influenza was community acquired. Patients were severely hypoxic (median PaO2-to-FIO2 ratio, 83 mm Hg), 97% received mechanical ventilation, with mean positive end-expiratory pressure of 14 cm H2O at the onset of critical illness and 26.7% received rescue oxygenation therapy with prone ventilation, extracorporeal life support, high-frequency oscillatory ventilation, or inhaled nitric oxide. At 90 days, mortality was 34.6% (13.9% in Canada vs 50.5% in Mexico, p < 0.0001). Independent predictors of mortality included lower presenting PaO2-to-FIO2 ratio (odds ratio, 0.89 per 10-point increase [95% CI, 0.80-0.99]), age (odds ratio, 1.49 per 10 yr increment [95% CI, 1.10-2.02]), and requiring critical care in Mexico (odds ratio, 7.76 [95% CI, 2.02-27.35]). ICUs in Canada generally had more beds, ventilators, healthcare personnel, and rescue oxygenation therapies. CONCLUSIONS: Influenza A (H1N1pdm09)-related critical illness still predominantly affects relatively young to middle-aged patients and is associated with severe hypoxemic respiratory failure. The local critical care system and available resources may be influential determinants of patient outcome.

Duration of Antimicrobial Treatment for Bacteremia in Canadian Critically Ill Patients.

OBJECTIVES: The optimum duration of antimicrobial treatment for patients with bacteremia is unknown. Our objectives were to determine duration of antimicrobial treatment provided to patients who have bacteremia in ICUs, to assess pathogen/patient factors related to treatment duration, and to assess the relationship between treatment duration and survival. DESIGN: Retrospective cohort study. SETTINGS: Fourteen ICUs across Canada. PATIENTS: Patients with bacteremia and were present in the ICU at the time culture reported positive. INTERVENTIONS: Duration of antimicrobial treatment for patients who had bacteremia in ICU. MEASUREMENTS AND MAIN RESULTS: Among 1,202 ICU patients with bacteremia, the median duration of treatment was 14 days, but with wide variability (interquartile range, 9-17.5). Most patient characteristics were not associated with treatment duration. Coagulase-negative staphylococci were the only pathogens associated with shorter treatment (odds ratio, 2.82; 95% CI, 1.51-5.26). The urinary tract was the only source of infection associated with a trend toward lower likelihood of shorter treatment (odds ratio, 0.67; 95% CI, 0.42-1.08); an unknown source of infection was associated with a greater likelihood of shorter treatment (odds ratio, 2.14; 95% CI, 1.17-3.91). The association of treatment duration and survival was unstable when analyzed based on timing of death. CONCLUSIONS: Critically ill patients who have bacteremia typically receive long courses of antimicrobials. Most patient/pathogen characteristics are not associated with treatment duration; survivor bias precludes a valid assessment of the association between treatment duration and survival. A definitive randomized controlled trial is needed to compare shorter versus longer antimicrobial treatment in patients who have bacteremia.

Association between optic nerve sheath diameter and mortality in patients with severe traumatic brain injury.

PURPOSE: Increased intracranial pressure (ICP) is associated with worse outcomes following traumatic brain injury (TBI). Studies have confirmed that ICP is correlated with optic nerve sheath diameter (ONSD) on ultrasound. The aim of our study was to assess the independent relationship between ONSD measured using CT and mortality in a population of patients admitted with severe TBI. METHODS: We conducted a retrospective cohort study of patients with a TBI requiring ICP monitoring admitted to the ICU between April 2006 and May 2012 to two neurotrauma centers. ONSD was independently measured by two physicians blinded to patient outcomes. Multivariable logistic regression modeling was used to assess an association between ONSD and hospital mortality. RESULTS: A total of 220 patients were included in the analysis. Overall, the cohort had a mean age of 35 (SD 17) years and 171 of 220 (79 %) were male. The median admission GCS was 6 (IQR 3-8). Intra-class correlation coefficient between raters for ONSD measurements was 0.92 (95 % CI 0.90-0.94, P < 0.0001). On multivariable analysis, each 1 mm increase in ONSD was associated with a twofold increase in hospital mortality (OR 2.0, 95 % CI 1.2-3.2, P = 0.007). Using linear regression, ONSD was independently associated with increased ICP in the first 48 h after admission (ß = 4.4, 95 % CI 2.5-6.3, P < 0.0001). CONCLUSIONS: In patients with TBI, ONSD measured on CT scanning was independently associated with ICP and mortality.

Coagulase-negative Staphylococcus, catheter-related, bloodstream infections and their association with acute phase markers of inflammation in the intensive care unit: An observational study.

OBJECTIVE: To examine the relationship between the isolation of coagulase-negative Staphylococcus in blood cultures and acute phase markers of inflammation. METHODS: The present study was a prospective observational analysis conducted at three medical/surgical intensive care units (ICUs) involving adult patients with an expected ICU stay of more than 24 h duration. RESULTS: Of the 598 patients enrolled, 573 developed suspected bloodstream infection and 434 (72.6%) had blood cultures sent 24 h after ICU admission; 142 were excluded due to positive cultures from other sites. Of the remaining 292 patients, 31 (10.7%) grew coagulase-negative Staphylococcus, 59 (20.2%) grew known pathogenic organisms and 202 (69.2%) did not grow any organisms in their blood cultures. Twenty-five patients without suspicion of infection served as the control group. Interleukin (IL)-6, procalcitonin (PCT) and C-reactive protein (CRP) levels were highest among the known pathogen group (IL-6 271.8 U/L, PCT 4.6 U/L and CRP 164 mg/L), were similar between the coagulase-negative Staphylococcus and negative culture groups (IL-6 67.0 U/L versus 61.4 U/L [P=1.00]; PCT 1.0 U/L versus 0.9 U/L [P=0.80]; and CRP 110 mg/L versus 103 mg/L [P=0.75]), and were lowest in the control group (IL-6 31.0 U/L, PCT 0.2 U/L and CRP 41.0 mg/L). In the coagulase-negative Staphylococcus group, patients who died by day 28 had increased inflammatory bio-marker levels compared with survivors, although the differences were not statistically significant. CONCLUSIONS: Coagulase-negative Staphylococcus isolated from blood cultures were associated with lower levels of inflammation compared with bloodstream infections due to known pathogens and were comparable with levels in patients with negative cultures.

OBJECTIF: Examiner la relation entre l'isolement de staphylocoque à coagulase négative dans les hémocultures et les marqueurs d'inflammation en phase aiguë. MÉTHODOLOGIE: Étude d'observation prospective. LIEU: Trois unités de soins intensifs (USI) médicaux et chirurgicaux. PATIENTS: Tous les patients dont le séjour prévu à l'USI pour adultes dépassait 24 heures. RÉSULTATS: Sur les 598 patients participants, 573 ont présenté une présomption d'infection du sang, et une hémoculture a été envoyée en laboratoire 24 heures après l'admission à l'USI de 434 (72,6 %) d'entre eux; 142 ont été exclus en raison de cultures positives d'autres foyers. Sur les 292 patients restants, 31 (10,7 %) ont développé un staphylocoque à coagulase négative, 59 (20,2 %), des organismes pathogènes connus et 202 (69,2 %) ne présentaient aucun organisme dans le sang. Vingt-cinq patients sans présomption d'infection ont servi de groupe témoin. Les taux d'interleukine (IL)-6, de procalcitonine (PCT) et de protéine C réactive (CRP) étaient les plus élevés dans le groupe infecté par des pathogènes connus (IL-6 271,8 U/L, PCT 4,6 U/L et CRP 164 mg/L), étaient similaires dans les groupes de staphylocoque à coagulase négative et aux cultures négatives (IL-6 67,0 U/L par rapport à 61,4 U/L [P=1,00]; PCT 1,0 U/L par rapport à 0,9 U/L [P=0,80] et CRP 110 mg/L par rapport à 103 mg/L [P=0,75]) et étaient les plus faibles dans le groupe témoin (IL-6 31,0 U/L, PCT 0,2 U/L et CRP 41,0 mg/L). Dans le groupe ayant un staphylocoque à coagulase négative, les patients qui étaient décédés le 28e jour présentaient des taux de biomarqueurs inflammatoires plus élevés que les survivants, même si les différences n'étaient pas statistiquement significatives. CONCLUSIONS: Le staphylocoque à coagulase négative isolé dans les hémocultures s'associait à des taux plus faibles d'inflammation que les infections du sang causées par des pathogènes connus et était comparable aux taux observés chez les patients dont les cultures étaient négatives.

Community-associated CMRSA-10 (USA-300) is the predominant strain among methicillin-resistant Staphylococcus aureus strains causing skin and soft tissue infections in patients presenting to the emergency department of a Canadian tertiary care hospital.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging pathogen first described among individuals with no contact with health care facilities. The purpose of this study was to determine the proportion of CA-MRSA, defined by pulsed field gel electrophoresis (PFGE), in MRSA skin and soft tissue infections presenting to the Emergency Department (ED). We also aimed to describe the laboratory and clinical characteristics of CA-MRSA infections. From June 1, 2001 to May 30, 2005, MRSA isolates from skin and soft tissue infections presenting to the ED were reviewed. They were characterized by antibiotic susceptibilities and PFGE, and the presence of staphylococcal cassette chromosome (SCC) mec type IVa and Panton-Valentine leukocidin (PVL) genes was assessed on representative isolates. The medical records were reviewed to define risk factors. There were 95 isolates available for analysis, of which 58 (61%) were CMRSA-10 (USA-300), the predominant clone from 2003 onward. All representative isolates (24%) tested in this group had PVL genes and SCCmec type IVa. Their antibiogram showed 100% susceptibility to trimethoprim-sulfamethoxazole, rifampin, and fusidic acid, and 79% to clindamycin. Clinical comparison of CMRSA-10 vs. hospital PFGE type strains showed 22% vs. 60%, respectively, for recent antibiotic use (p < 0.0001), 26% vs. 6%, respectively, for intravenous drug use (p < 0.05), and 57% vs. 6%, respectively, for soft tissue abscess (p < 0.001). CMRSA-10 is a major pathogen in skin and soft tissue abscesses in our ED. It has a characteristic susceptibility, and was associated with intravenous drug use, but not with recent antibiotic usage.

The role of p53 in silica-induced cellular and molecular responses associated with carcinogenesis.

Crystalline silica (silica), a suspected human carcinogen, produces an increase in reactive oxygen species (ROS) when fractured using mechanical tools used in several occupations. Although ROS has been linked to apoptosis, DNA damage, and carcinogenesis, the role of enhanced ROS production by silica in silica-induced carcinogenesis is not completely understood. The goal of this study was to compare freshly fractured and aged silica-induced molecular alterations in human immortalized/transformed bronchial epithelial cells (BEAS-IIB) and lung cancer cells with altered (H460) or deficient (H1299) p53 expression. Exposure to freshly fractured or aged silica produced divergent cellular responses in certain downstream cellular events, including ROS production, apoptosis, cell cycle and chromosomal changes, and gene expression. ROS production increased significantly following exposure to freshly fractured silica compared to aged silica in BEAS-IIB and H460 cells. Apoptosis showed a comparable enhanced level of induction with freshly fractured or aged silica in both cancer lines with p53 functional changes. p53 protein was present in the BEAS-IIB and was absent in cancer cell lines after silica exposure. Exposure to freshly fractured silica also resulted in a rise in aneuploidy in cancer cells with a significantly greater increase in p53-deficient cells. Cytogenetic analysis demonstrated increased metaphase spreads, chromosome breakage, rearrangements, and endoreduplication in both cancer cells. These results suggest that altered and deficient p53 affects the cellular response to freshly fractured silica exposure, and thereby enhances susceptibility and augments cell proliferation and lung cancer development.

Loss of P53 heterozygosity is not responsible for the small colony thymidine kinase mutant phenotype in L5178Y mouse lymphoma cells.

The mouse lymphoma L5178Y Tk+/- 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk-/- mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/- cells or Tk-/- mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/- 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.

Remodeling of DNA methylation and phenotypic and transcriptional changes in synthetic Arabidopsis allotetraploids.

The joining of different genomes in allotetraploids played a major role in plant evolution, but the molecular implications of this event are poorly understood. In synthetic allotetraploids of Arabidopsis and Cardaminopsis arenosa, we previously demonstrated the occurrence of frequent gene silencing. To explore the involvement of epigenetic phenomena, we investigated the occurrence and effects of DNA methylation changes. Changes in DNA methylation patterns were more frequent in synthetic allotetraploids than in the parents. Treatment with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in the development of altered morphologies in the synthetic allotetraploids, but not in the parents. We profiled mRNAs in control and 5-aza-2'-deoxycytidine-treated parents and allotetraploids by amplified fragment length polymorphism-cDNA. We show that DNA demethylation induced and repressed two different transcriptomes. Our results are consistent with the hypothesis that synthetic allotetraploids have compromised mechanisms of epigenetic gene regulation.

Synergistic effects of dust and ammonia on the occupational health effects of poultry production workers.

OBJECTIVE: As production methods for livestock and poultry moved towards large industrial-scale confinement facilities, the occupational health community reported risks for respiratory illnesses in workers. Likely, greater risks for respiratory disease will occur with the continuing trend towards full-time confinement workers, who inspire a combination of bioaerosols, particulates, and gases. Although there have been numerous studies on the individual health effects of air contaminants inside confined animal production facilities, there have been no reports on the effects of combined exposures. The objective of this study was to investigate the combined health effects of air contaminants on poultry production workers. SAMPLE POPULATION: Two hundred and fifty-seven poultry production workers participated in this study. The workers represented various areas of the poultry industry, including turkey growing, broiler production, egg laying, and unloading/shakeling in poultry processing. Worker procedures pulmonary function testing was conducted before and after a four-hour work shift. The work environment was assessed for total and respirable dust, ammonia, endotoxin and CO2. The relationship of simultaneous total dust and ammonia exposures was examined by correlation, logistic modeling, and synergy index calculations. RESULTS: Synergy between ammonia levels and airborne dust explained up to 43% and 63% of the decline (respectively for Forced Expiratory Volume (FEV) in one second and Forced Expiratory Flow (FEF25-75) in pulmonary function over the work shift. Furthermore, assessing the synergy index indicated the combined effect of dust and ammonia is from 53 to 156% (greater combined than individually). The proportion of health effect due to synergy is 35%-61%. CONCLUSIONS: Synergy of simultaneous dust and ammonia exposures in a working environment raises the question of redefining exposure limits for organic dust and ammonia when workers are exposed simultaneously to these substances. CLINICAL RELEVANCE: Control of both dust and ammonia in livestock facilities is extremely important. Lack of control of both these contaminants will increase the risk of respiratory dysfunction to all exposed to this environment, including workers and veterinarians.