Epigenetic silencing of CREB3L1 by DNA methylation is associated with high-grade metastatic breast cancers with poor prognosis and is prevalent in triple negative breast cancers.

BACKGROUND: CREB3L1 (cAMP-responsive element-binding protein 3-like protein 1), a member of the unfolded protein response, has recently been identified as a metastasis suppressor in both breast and bladder cancer. METHODS: Quantitative real time PCR (qPCR) and immunoblotting were used to determine the impact of histone deacetylation and DNA methylation inhibitors on CREB3L1 expression in breast cancer cell lines. Breast cancer cell lines and tumor samples were analyzed similarly, and CREB3L1 gene methylation was determined using sodium bisulfite conversion and DNA sequencing. Immunohistochemistry was used to determine nuclear versus cytoplasmic CREB3L1 protein. Large breast cancer database analyses were carried out to examine relationships between CREB3L1 gene methylation and mRNA expression in addition to CREB3L1 mRNA expression and prognosis. RESULTS: This study demonstrates that the low CREB3L1 expression previously seen in highly metastatic breast cancer cell lines is caused in part by epigenetic silencing. Treatment of several highly metastatic breast cancer cell lines that had low CREB3L1 expression with DNA methyltransferase and histone deacetylase inhibitors induced expression of CREB3L1, both mRNA and protein. In human breast tumors, CREB3L1 mRNA expression was upregulated in low and medium-grade tumors, most frequently of the luminal and HER2 amplified subtypes. In contrast, CREB3L1 expression was repressed in high-grade tumors, and its loss was most frequently associated with triple negative breast cancers (TNBCs). Importantly, bioinformatics analyses of tumor databases support these findings, with methylation of the CREB3L1 gene associated with TNBCs, and strongly negatively correlated with CREB3L1 mRNA expression. Decreased CREB3L1 mRNA expression was associated with increased tumor grade and reduced progression-free survival. An immunohistochemistry analysis revealed that low-grade breast tumors frequently had nuclear CREB3L1 protein, in contrast to the high-grade breast tumors in which CREB3L1 was cytoplasmic, suggesting that differential localization may also regulate CREB3L1 effectiveness in metastasis suppression. CONCLUSIONS: Our data further strengthens the role for CREB3L1 as a metastasis suppressor in breast cancer and demonstrates that epigenetic silencing is a major regulator of the loss of CREB3L1 expression. We also highlight that CREB3L1 expression is frequently altered in many cancer types suggesting that it could have a broader role in cancer progression and metastasis.

Archaeal characterization of bioaerosols from cage-housed and floor-housed poultry operations.

Although bioaerosols from both cage-housed (CH) and floor-housed (FH) poultry operations are highly concentrated, the concentrations of dust, endotoxin, and bacteria are significantly higher in FH bioaerosols. Workers from CH operations have reported a greater prevalence of respiratory symptoms. To date, archaea have been examined in swine and dairy bioaerosols but not in poultry bioaerosols. The objective of this study was to directly compare methanogenic archaea concentrations in bioaerosols from CH and FH poultry facilities. Bioaerosols were collected from 15 CH and 15 FH poultry operations, using stationary area samplers as well as personal sampling devices. Archaea were quantified and their diversity was investigated using polymerase chain reaction (PCR) followed by denaturing gradient gel electrophoresis (DGGE) and band sequencing. Archaea were significantly higher in area and personal bioaerosols of CH poultry operations than in those from FH poultry operations (p < 0.001 and p < 0.05, respectively) and did not differ significantly between area and personal samples within each barn type. Sequences matching Methanobrevibacter woesei, an archaea previously found in poultry samples, were detected in bioaerosol samples from CH operations. Methanogenic archaea concentrations are significantly different between bioaerosols from CH and FH poultry operations.

Potentially pathogenic bacteria and antimicrobial resistance in bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND: Antibiotics are used in animal confinement buildings, such as cage-housed (CH) and floor-housed (FH) poultry operations, to lower the likeliness of disease transmission. In FH facilities, antibiotics may also be used at sub-therapeutic levels for growth promotion. Low levels of antibiotic create a selective pressure toward antimicrobial resistance (AMR) in chicken fecal bacteria. OBJECTIVE: The objective of this study was to compare bacteria and AMR genes in bioaerosols from CH and FH poultry facilities. METHODS: Bioaerosols were collected from 15 CH and 15 FH poultry operations, using stationary area samplers as well as personal sampling devices. Bacteria concentrations were determined by genus- or species-specific quantitative polymerase chain reaction (PCR) and AMR genes were detected using endpoint PCR. RESULTS: Enterococcus spp., Escherichia coli, and Staphylococcus spp. were significantly higher in bioaerosols of FH poultry operations than CH bioaerosols (P < 0.001) while Clostridium perfringens was significantly higher in area bioaerosols of CH operations than FH area bioaerosols (P < 0.05). Campylobacter spp. were detected only in bioaerosols of FH facilities. Zinc bacitracin resistance gene, bcrR, erythromycin resistance gene, ermA, and tetracycline resistance gene, tetA/C, were more prevalent in bioaerosols of FH facilities than CH bioaerosols (P < 0.01, P < 0.01, and P < 0.05, respectively). CONCLUSIONS: Most bacteria are more concentrated and most AMR genes are more prevalent in bioaerosols of FH poultry operations, where growth-promoting antibiotics may be used.

Bacterial diversity characterization of bioaerosols from cage-housed and floor-housed poultry operations.

BACKGROUND: Although bioaerosols from both cage-housed (CH) and floor-housed (FH) poultry operations are highly concentrated, workers from CH operations have reported a greater prevalence of respiratory symptoms. OBJECTIVE: The objective of this study was to directly compare bacteria, both quantitatively and qualitatively, in bioaerosols from CH and FH poultry facilities. METHODS: Bioaerosols were collected from fifteen CH and fifteen FH poultry operations, using stationary area samplers as well as personal sampling devices. Dust, endotoxin and bacteria were quantified and bacterial diversity was investigated using PCR followed by denaturing gradient gel electrophoresis (DGGE). RESULTS: Dust (p<0.001), endotoxin (p<0.05) and bacteria (p<0.05) were significantly higher in personal bioaerosols of FH poultry operations than CH bioaerosols. Although dust and endotoxin did not differ significantly between area and personal samples within each barn type, clustering analysis of DGGE profiles of bacteria revealed that area and personal samples shared less than 10% similarity. These data suggest that area samples are not representative of personal bacteria exposures, which may be affected by worker movement, bacteria carried on the worker and worker location. Personal DGGE profiles from CH and FH operations shared less than 20% similarity and composite analysis showed that bacteria were more prevalent in personal samples from CH bioaerosols than FH bioaerosols. CONCLUSIONS: Bacteria concentration and diversity are significantly different between bioaerosols from CH and FH poultry operations.

Endotoxin and dust at respirable and nonrespirable particle sizes are not consistent between cage- and floor-housed poultry operations.

BACKGROUND: Individuals engaged in work in intensive animal houses experience some of the highest rates of occupationally related respiratory symptoms. Organic dust and in particular endotoxin has been most closely associated with respiratory symptoms and lung function changes in workers. It has previously been shown that for intensive poultry operations, type of poultry housing [cage-housed (CH) versus floor-housed (FH)] can influence the levels of environmental contaminants. The goal of the study was to determine the differences in endotoxin and dust levels at different size fractions between CH and FH poultry operations. METHODS: Fifteen CH and 15 FH poultry operations were sampled for stationary measurements (area) of dust and associated endotoxin. Fractioned samples were collected utilizing Marple cascade impactors. Gravimetric and endotoxin analysis were conducted on each of the filters. RESULTS: When assessed by individual Marple stage, there was significantly greater airborne endotoxin concentration (endotoxin units per cubic meter) in the size fraction >9.8 µm for the FH operations whereas at the size fraction 1.6-3.5 µm, the CH operations had significantly greater airborne endotoxin concentration than the FH operations. Endotoxin concentration in the dust mass (endotoxin units per milligram) was significantly greater in the CH operations as compared to the FH operations for all size fractions >1.6 µm. As such, endotoxin in the respirable fraction accounted for 24% of the total endotoxin in the CH operations whereas it accounted for only 11% in the FH operations. There was significantly more dust in all size fractions in the FH operations as compared to the CH poultry operations. CONCLUSIONS: There is more endotoxin in the presence of significantly lower dust levels in the respirable particle size fractions in CH poultry operations as compared to the FH poultry operations. This difference in respirable endotoxin may be important in relation to the differential respiratory response experienced by CH and FH poultry operation workers.

An aerobiological perspective of dust in cage-housed and floor-housed poultry operations.

The Canadian poultry production industry contributes nearly $10 billion to the Canadian economy and employs nearly 50,000 workers. However, modern poultry facilities are highly contaminated with airborne dust. Although there are many bioaerosols in the poultry barn environment, endotoxin is typically attributed with the negative respiratory symptoms observed in workers. These adverse respiratory symptoms have a higher prevalence in poultry workers compared to workers from other animal confinement buildings. Workers in cage-housed operations compared to floor-housed facilities report a higher prevalence of some respiratory symptoms. We review the current state of knowledge on airborne dust in poultry barns and respiratory dysfunction in poultry workers while highlighting the areas that need further investigation. Our review focuses on the aerobiological pathway of poultry dust including the source and aerosolization of dust and worker exposure and response. Further understanding of the source and aerosolization of dust in poultry operations will aid in the development of management practices to reduce worker exposure and response.