Chlorophyllin Supplementation of Medicated or Unmedicated Swine Diets Impact on Fecal Escherichia coli and Enterococci.

Considering that certain catabolic products of anaerobic chlorophyll degradation inhibit efflux pump activity, this study was conducted to test if feeding pigs a water-soluble chlorophyllin product could affect the antibiotic resistance profiles of select wild-type populations of fecal bacteria. Trial 1 evaluated the effects of chlorophyllin supplementation (300 mg/meal) on fecal E. coli and enterococcal populations in pigs fed twice daily diets supplemented without or with ASP 250 (containing chlortetracycline, sulfamethazine and penicillin at 100, 100 and 50 g/ton, respectively). Trial 2, conducted similarly, evaluated chlorophyllin supplementation in pigs fed diets supplemented with or without 100 g tylosin/ton. Each trial lasted 12 days, and fecal samples were collected and selectively cultured at 4-day intervals to enumerate the total numbers of E. coli and enterococci as well as populations of these bacteria phenotypically capable of growing in the presence of the fed antibiotics. Performance results from both studies revealed no adverse effect (p > 0.05) of chlorophyllin, antibiotic or their combined supplementation on average daily feed intake or average daily gain, although the daily fed intake tended to be lower (p = 0.053) for pigs fed diets supplemented with tylosin than those fed diets without tylosin. The results from trial 1 showed that the ASP 250-medicated diets, whether without or with chlorophyllin supplementation, supported higher (p < 0.05) fecal E. coli populations than the non-medicated diets. Enterococcal populations, however, were lower, albeit marginally and not necessarily significantly, in feces from pigs fed the ASP 250-medicated diet than those fed the non-medicated diet. Results from trial 2 likewise revealed an increase (p < 0.05) in E. coli and, to a lesser extent, enterococcal populations in feces collected from pigs fed the tylosin-medicated diet compared with those fed the non-medicated diet. Evidence indicated that the E. coli and enterococcal populations in trial 1 were generally insensitive to penicillin or chlortetracycline, as there were no differences between populations recovered without or with antibiotic selection. Conversely, a treatment x day of treatment interaction observed in trial 2 (p < 0.05) provided evidence, albeit slight, of an enrichment of tylosin-insensitive enterococci in feces from the pigs fed the tylosin-medicated but not the non-medicated diet. Under the conditions of the present study, it is unlikely that chlorophyllin-derived efflux pump inhibitors potentially present in the chlorophyllin-fed pigs were able to enhance the efficacy of the available antibiotics. However, further research specifically designed to optimize chlorophyll administration could potentially lead to practical applications for the swine industry.

Striated muscle: an inadequate soil for cancers.

Many organs of the body are susceptible to cancer development. However, striated muscles-which include skeletal and cardiac muscles-are rarely the sites of primary cancers. Most deaths from cancer arise due to complications associated with the development of secondary metastatic tumours, for which there are few effective therapies. However, as with primary cancers, the establishment of metastatic tumours in striated muscle accounts for a disproportionately small fraction of secondary tumours, relative to the proportion of body composition. Examining why primary and metastatic cancers are comparatively rare in striated muscle presents an opportunity to better understand mechanisms that can influence cancer cell biology. To gain insights into the incidence and distribution of muscle metastases, this review presents a definitive summary of the 210 case studies of metastasis in muscle published since 2010. To examine why metastases rarely form in muscles, this review considers the mechanisms currently proposed to render muscle an inhospitable environment for cancers. The "seed and soil" hypothesis proposes that tissues' differences in susceptibility to metastatic colonization are due to differing host microenvironments that promote or suppress metastatic growth to varying degrees. As such, the "soil" within muscle may not be conducive to cancer growth. Gaining a greater understanding of the mechanisms that underpin the resistance of muscles to cancer may provide new insights into mechanisms of tumour growth and progression, and offer opportunities to leverage insights into the development of interventions with the potential to inhibit metastasis in susceptible tissues.

Selection for high and low antibody responses to sheep red blood cells influences cytokine and chemokine expression in chicken peripheral blood leukocytes and splenic tissue.

White Leghorn chickens from a common founder population have been divergently selected for high (HAS) or low (LAS) antibody responses to sheep red blood cells (SRBC) for 49 generations resulting in 2 diverse lines for this trait. Much has been studied in these two lines; however, the impact of these selection pressures on cytokine and chemokine expression is not fully understood. The purpose of this study is to determine if selection for antibody response to SRBC impacts cytokine and chemokine expression in peripheral blood leukocytes (PBL) and spleen from HAS and LAS chickens. Total RNA was isolated from PBL and spleen after which mRNA expression of cytokines (IL4, IL6, IL10, TGF-ß4) and chemokines (CXCL8, CCL4) were determined by quantitative real-time RT-PCR (qRT-PCR). The data were analyzed using Student's t test comparing HAS and LAS (P < 0.05) and are reported as corrected 40-CT. PBL and spleen samples were analyzed separately. With respect to PBL, expression of IL6 was higher (P < 0.05) in PBL isolated from LAS chickens compared to those from the HAS line whereas there were no differences (P > 0.05) in IL4, IL10, CXCL8, CCL4, or TGF-ß4. The cytokine and chemokine mRNA expression profiles were different in the spleen between the two lines. IL4 and CXCL8 expression were higher (P < 0.05) in spleen samples from HAS chickens than LAS. The expression of IL6, IL10, CCL4, or TGF-ß4 in the spleens did not differ (P > 0.05) between the lines. The data indicate that selection for specific antibody responses to SRBC impacts the cytokine and chemokine expression profile in PBL and spleens but in different ways in HAS and LAS. These studies provide insight into the influence that selection pressures for antibody responses have on different immune response components, specifically cytokines and chemokines typically involved in the innate response.

Ehf controls mammary alveolar lineage differentiation and is a putative suppressor of breast tumorigenesis.

The transcription factor EHF is highly expressed in the lactating mammary gland, but its role in mammary development and tumorigenesis is not fully understood. Utilizing a mouse model of Ehf deletion, herein, we demonstrate that loss of Ehf impairs mammary lobuloalveolar differentiation at late pregnancy, indicated by significantly reduced levels of milk genes and milk lipids, fewer differentiated alveolar cells, and an accumulation of alveolar progenitor cells. Further, deletion of Ehf increased proliferative capacity and attenuated prolactin-induced alveolar differentiation in mammary organoids. Ehf deletion also increased tumor incidence in the MMTV-PyMT mammary tumor model and increased the proliferative capacity of mammary tumor organoids, while low EHF expression was associated with higher tumor grade and poorer outcome in luminal A and basal human breast cancers. Collectively, these findings establish EHF as a non-redundant regulator of mammary alveolar differentiation and a putative suppressor of mammary tumorigenesis.

Loss of tumor-derived SMAD4 enhances primary tumor growth but not metastasis following BMP4 signalling.

BACKGROUND: Bone morphogenetic protein 4 (BMP4) is a potent inhibitor of breast cancer metastasis. However, a tumor-promoting effect of BMP4 is reported in other tumor types, especially when SMAD4 is inactive. METHODS: To assess the requirement for SMAD4 in BMP4-mediated suppression of metastasis, we knocked down SMAD4 in two different breast tumors and enforced SMAD4 expression in a third line with endogenous SMAD4 deletion. In addition, we assessed the requirement for SMAD4 in tumor cell-specific BMP signalling by expression of a constitutively active BMP receptor. Delineation of genes regulated by BMP4 in the presence or absence of SMAD4 was assessed by RNA sequencing and a BMP4-induced gene, MYO1F was assessed for its role in metastasis. Genes regulated by BMP4 and/or SMAD4 were assessed in a publicly available database of gene expression profiles of breast cancer patients. RESULTS: In the absence of SMAD4, BMP4 promotes primary tumor growth that is accompanied by increased expression of genes associated with DNA replication, cell cycle, and MYC signalling pathways. Despite increased primary tumor growth, BMP4 suppresses metastasis in the absence of tumor cell expression of SMAD4. Consistent with the anti-metastatic activity of BMP4, enforced signalling through the constitutively active receptor in SMAD4 positive tumors that lacked BMP4 expression still suppressed metastasis, but in the absence of SMAD4, the suppression of metastasis was largely prevented. Thus BMP4 is required for suppression of metastasis regardless of tumor SMAD4 status. The BMP4 upregulated gene, MYO1F, was shown to be a potent suppressor of breast cancer metastasis. Gene signature upregulated by BMP4 in the absence of SMAD4 was associated with poor prognosis in breast cancer patients, whereas gene signature upregulated by BMP4 in the presence of SMAD4 was associated with improved prognosis. CONCLUSIONS: BMP4 expression is required for suppression of metastasis regardless of the SMAD4 status of the tumor cells. Since BMP4 is a secreted protein, we conclude that it can act both in an autocrine manner in SMAD4-expressing tumor cells and in a paracrine manner on stromal cells to suppress metastasis. Deletion of SMAD4 from tumor cells does not prevent BMP4 from suppressing metastasis via a paracrine mechanism.

Bacterial proximity effects on the transfer of antibiotic resistance genes within the alimentary tract of yellow mealworm larvae.

The arthropod intestinal tract and other anatomical parts naturally carry microorganisms. Some of which are pathogens, secrete toxins, or carry transferable antibiotic-resistance genes. The risks associated with the production and consumption of edible arthropods are dependent on indigenous microbes, as well as microbes introduced during the processes of rearing. This mass arthropod production puts individual arthropods in close proximity, which increases the possibility of their exposure to antibiotic-resistant bacteria carried by bacteria from fellow insects, industry workers, or rearing hardware and substrates. The purpose of this study was to determine if the alimentary tract of the yellow mealworm provided an environment permitting horizontal gene transfer between bacteria. The effect of the concentration of bacterial exposure was also assessed. Antibiotic resistance gene transfer between marker Salmonella Lignières (Enterobacterales: Enterobacteriaceae) and Escherichia coli (Migula) (Enterobacterales: Enterobacteriaceae) introduced into the larval gut demonstrated that the nutrient-rich environment of the yellow mealworm gut provided favorable conditions for the transfer of antibiotic resistance genes. Conjugation frequencies were similar across inoculum concentrations; however, transconjugant production correlated positively to increased exposure concentration. The lowest concentration of bacterial exposure required enrichment to detect and thus may have been approaching a threshold level for the 2 bacteria to colocate within the expanse of the larval gut. While many factors can affect this transfer, the simple factor of the proximity of donor and recipient bacteria, as defined by the concentration of bacteria within the volume of the insect gut, likely primarily contributed to the efficiency of antibiotic gene transfer.

Campylobacter jejuni Response When Inoculated in Bovine In Vitro Fecal Microbial Consortia Incubations in the Presence of Metabolic Inhibitors.

Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h-1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.

Chlortetracycline Concentration Impact on Salmonella Typhimurium Sustainability in the Presence of Porcine Gastrointestinal Tract Bacteria Maintained in Continuous Culture.

Concern exists that the continued use of antibiotics in animal feeds may lead to an increased prevalence of resistant bacteria within the host animal's gastrointestinal tract. To evaluate the effect of chlortetracycline on the persistence of Salmonella enterica serotype Typhimurium within a diverse population of porcine cecal bacteria, we cultured a mixed population of cecal bacteria without or with added chlortetracycline. When grown at a 24 h vessel turnover rate, chlortetracycline-susceptible S. Typhimurium exhibited more than 2.5 times faster (p < 0.05) disappearance rates than theoretically expected (0.301 log10 colony-forming unit/mL per day) but did not differ whether treated or not with 55 mg of chlortetracycline/L. Chlortetracycline-resistant S. Typhimurium was not recovered from any of these cultures. When the mixed cultures were inoculated with a chlortetracycline-resistant S. Typhimurium, rates of disappearance were nearly two times slower (p < 0.05) than those observed earlier with chlortetracycline-susceptible S. Typhimurium, and cultures persisted at >2 log10 colony-forming units/mL for up to 14 days of treatment with 110 mg of chlortetracycline/L. Under the conditions of this study, chlortetracycline-resistant S. Typhimurium was competitively enabled to persist longer within the mixed populations of porcine gut bacteria than chlortetracycline-susceptible S. Typhimurium, regardless of the presence or absence of added chlortetracycline.

Inhibition of EphA3 Expression in Tumour Stromal Cells Suppresses Tumour Growth and Progression.

Tumour progression relies on interactions with untransformed cells in the tumour microenvironment (TME), including cancer-associated fibroblasts (CAFs), which promote blood supply, tumour progression, and immune evasion. Eph receptor tyrosine kinases are cell guidance receptors that are most active during development but re-emerge in cancer and are recognised drug targets. EphA3 is overexpressed in a wide range of tumour types, and we previously found expression particularly in stromal and vascular tissues of the TME. To investigate its role in the TME, we generated transgenic mice with inducible shRNA-mediated knockdown of EphA3 expression. EphA3 knockdown was confirmed in aortic mesenchymal stem cells (MSCs), which displayed reduced angiogenic capacity. In mice with syngeneic lung tumours, EphA3 knockdown reduced vasculature and CAF/MSC-like cells in tumours, and inhibited tumour growth, which was confirmed also in a melanoma model. Single cell RNA sequencing analysis of multiple human tumour types confirmed EphA3 expression in CAFs, including in breast cancer, where EphA3 was particularly prominent in perivascular- and myofibroblast-like CAFs. Our results thus indicate expression of the cell guidance receptor EphA3 in distinct CAF subpopulations is important in supporting tumour angiogenesis and tumour growth, highlighting its potential as a therapeutic target.

Single-cell RNA sequencing captures patient-level heterogeneity and associated molecular phenotypes in breast cancer pleural effusions.

BACKGROUND: Malignant pleural effusions (MPEs) are a common complication of advanced cancers, particularly those adjacent to the pleura, such as lung and breast cancer. The pathophysiology of MPE formation remains poorly understood, and although MPEs are routinely used for the diagnosis of breast cancer patients, their composition and biology are poorly understood. It is difficult to distinguish invading malignant cells from resident mesothelial cells and to identify the directionality of interactions between these populations in the pleura. There is a need to characterize the phenotypic diversity of breast cancer cell populations in the pleural microenvironment, and investigate how this varies across patients. METHODS: Here, we used single-cell RNA-sequencing to study the heterogeneity of 10 MPEs from seven metastatic breast cancer patients, including three Miltenyi-enriched samples using a negative selection approach. This dataset of almost 65 000 cells was analysed using integrative approaches to compare heterogeneous cell populations and phenotypes. RESULTS: We identified substantial inter-patient heterogeneity in the composition of cell types (including malignant, mesothelial and immune cell populations), in expression of subtype-specific gene signatures and in copy number aberration patterns, that captured variability across breast cancer cell populations. Within individual MPEs, we distinguished mesothelial cell populations from malignant cells using key markers, the presence of breast cancer subtype expression patterns and copy number aberration patterns. We also identified pleural mesothelial cells expressing a cancer-associated fibroblast-like transcriptomic program that may support cancer growth. CONCLUSIONS: Our dataset presents the first unbiased assessment of breast cancer-associated MPEs at a single cell resolution, providing the community with a valuable resource for the study of MPEs. Our work highlights the molecular and cellular diversity captured in MPEs and motivates the potential use of these clinically relevant biopsies in the development of targeted therapeutics for patients with advanced breast cancer.

Estrogen receptor beta expression in triple negative breast cancers is not associated with recurrence or survival.

BACKGROUND: Triple negative BCa (TNBC) is defined by a lack of expression of estrogen (ERα), progesterone (PgR) receptors and human epidermal growth factor receptor 2 (HER2) as assessed by protein expression and/or gene amplification. It makes up ~ 15% of all BCa and often has a poor prognosis. TNBC is not treated with endocrine therapies as ERα and PR negative tumors in general do not show benefit. However, a small fraction of the true TNBC tumors do show tamoxifen sensitivity, with those expressing the most common isoform of ERß1 having the most benefit. Recently, the antibodies commonly used to assess ERß1 in TNBC have been found to lack specificity, which calls into question available data regarding the proportion of TNBC that express ERß1 and any relationship to clinical outcome. METHODS: To confirm the true frequency of ERß1 in TNBC we performed robust ERß1 immunohistochemistry using the specific antibody CWK-F12 ERß1 on 156 primary TNBC cancers from patients with a median of 78 months (range 0.2-155 months) follow up. RESULTS: We found that high expression of ERß1 was not associated with increased recurrence or survival when assessed as percentage of ERß1 positive tumor cells or as Allred > 5. In contrast, the non-specific PPG5-10 antibody did show an association with recurrence and survival. CONCLUSIONS: Our data indicate that ERß1 expression in TNBC tumours does not associate with prognosis.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer.

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Effects of Hops Treatment on Nitrogen Retention, Volatile Fatty Acid Accumulations, and Select Microbial Populations of Composting Poultry Litter Intended for Use as a Ruminant Feedstuff.

Poultry litter is a valuable crude protein feedstuff for ruminants, but it must be treated to kill pathogens before feeding. Composting effectively kills pathogens, but it risks losing ammonia to volatilization or leaching during degradation of uric acid and urea. Hops bitter acids also exert antimicrobial activity against certain pathogenic and nitrogen-degrading microbes. Consequently, the present studies were conducted to test if adding bitter acid-rich hop preparations to simulated poultry litter composts may improve nitrogen retention while simultaneously improving pathogen killing. Results from an initial study, testing doses of Chinook or Galena hops preparations designed to each deliver 79 ppm hops ß-acid, revealed that, after nine days simulated composting of wood chip litter, ammonia concentrations were 14% lower (p < 0.05) in Chinook-treated composts than untreated composts (13.4 ± 1.06 µmol/g). Conversely, urea concentrations were 55% lower (p < 0.05) in Galena-treated than untreated composts (6.2 ± 1.72 µmol/g). Uric acid accumulations were unaffected by hops treatments in this study but were higher (p < 0.05) after three days than after zero, six, or nine days of composting. In follow-up studies, Chinook or Galena hops treatments (delivering 2042 or 6126 ppm of ß-acid, respectively) for simulated composts (14 days) of wood chip litter alone or mixed 3:1 with ground Bluestem hay (Andropogon gerardii) revealed that these higher dosages had little effect on ammonia, urea, or uric acid accumulations when compared to untreated composts. Volatile fatty acid accumulations measured in these later studies were affected by the hops treatments, with butyrate accumulations being lower after 14 days in hops-treated composts than in untreated compost. In all studies, beneficial effects of Galena or Chinook hops treatments were not observed on the antimicrobial activity of the simulated composts, with composting by itself decreasing (p < 0.05) counts of select microbial populations by more than 2.5 log10 colony forming units/g compost dry matter. Thus, while hops treatments had little effect on pathogen control or nitrogen retention within the composted litter, they did lessen accumulations of butyrate, which may prevent adverse effects of this fatty acid on palatability of litter fed to ruminants.

COMMD3 loss drives invasive breast cancer growth by modulating copper homeostasis.

BACKGROUND: Despite overall improvement in breast cancer patient outcomes from earlier diagnosis and personalised treatment approaches, some patients continue to experience recurrence and incurable metastases. It is therefore imperative to understand the molecular changes that allow transition from a non-aggressive state to a more aggressive phenotype. This transition is governed by a number of factors. METHODS: As crosstalk with extracellular matrix (ECM) is critical for tumour cell growth and survival, we applied high throughput shRNA screening on a validated '3D on-top cellular assay' to identify novel growth suppressive mechanisms. RESULTS: A number of novel candidate genes were identified. We focused on COMMD3, a previously poorly characterised gene that suppressed invasive growth of ER + breast cancer cells in the cellular assay. Analysis of published expression data suggested that COMMD3 is normally expressed in the mammary ducts and lobules, that expression is lost in some tumours and that loss is associated with lower survival probability. We performed immunohistochemical analysis of an independent tumour cohort to investigate relationships between COMMD3 protein expression, phenotypic markers and disease-specific survival. This revealed an association between COMMD3 loss and shorter survival in hormone-dependent breast cancers and in particularly luminal-A-like tumours (ER+/Ki67-low; 10-year survival probability 0.83 vs. 0.73 for COMMD3-positive and -negative cases, respectively). Expression of COMMD3 in luminal-A-like tumours was directly associated with markers of luminal differentiation: c-KIT, ELF5, androgen receptor and tubule formation (the extent of normal glandular architecture; p < 0.05). Consistent with this, depletion of COMMD3 induced invasive spheroid growth in ER + breast cancer cell lines in vitro, while Commd3 depletion in the relatively indolent 4T07 TNBC mouse cell line promoted tumour expansion in syngeneic Balb/c hosts. Notably, RNA sequencing revealed a role for COMMD3 in copper signalling, via regulation of the Na+/K+-ATPase subunit, ATP1B1. Treatment of COMMD3-depleted cells with the copper chelator, tetrathiomolybdate, significantly reduced invasive spheroid growth via induction of apoptosis. CONCLUSION: Overall, we found that COMMD3 loss promoted aggressive behaviour in breast cancer cells.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Effects in air-exposed corn silage of medium chain fatty acids on select spoilage microbes, zoonotic pathogens, and in vitro rumen fermentation.

Medium chain fatty acid (MCFA) treatment (0.75% C6, hexanoic; C8, octanoic; C10, decanoic; or equal proportion mixtures of C6:C8:C10:C12 or C8:C10/g; C12 = dodecanoic acid) of aerobically-exposed corn silage on spoilage and pathogenic microbes and rumen fermentation were evaluated in vitro. After 24 h aerobic incubation (37 °C), microbial enumeration revealed 3 log10 colony-forming units (CFU)/g fewer (P = 0.03) wild-type yeast and molds in C8:C10-treated silage than controls. Compared with controls, wild-type enterococci decreased (P < 0.01) in all treatments except the C6:C8:C10:C12 mixture; lactic acid bacteria were decreased (P < 0.01) in all treatments except C6 and the C6:C8:C10:C12 mixture. Total aerobes and inoculated Staphylococcus aureus or Listeria monocytogenes were unaffected by treatment (P > 0.05). Anaerobic incubation (24 h at 39 °C) of ruminal fluid (10 mL) with 0.02 g overnight air-exposed MCFA-treated corn silage revealed higher hydrogen accumulations (P = 0.03) with the C8:C10 mixture than controls. Methane, acetate, propionate, butyrate, or estimates of fermented hexose were unaffected. Acetate:propionate ratios were higher (P < 0.01) and fermentation efficiencies were marginally lower (P < 0.01) with C8- or C8:C10-treated silage than controls. Further research is warranted to optimize treatments to target unwanted microbes without adversely affecting beneficial microbes.

The bacterial and archaeal communities of flies, manure, lagoons, and troughs at a working dairy.

Background: Fundamental investigations into the location, load, and persistence of microbes, whether beneficial or detrimental, are scarce. Many questions about the retention and survival of microbes on various surfaces, as well as the load necessary for spread, exist. To answer these questions, we must know more about where to find various microbes and in what concentrations, the composition of the microbial communities, and the extent of dissemination between various elements. This study investigated the diversity, composition, and relative abundance of the communities associated with manure, lagoons, troughs, house flies, and stable flies present at a dairy, implementing two different free-stall management systems: flow-through and cross-vent. Shotgun metagenomics at the community level was used to compare the microbiomes within the dairy, allowing confident interpretation at the species level. Results: The results showed that there were significant difference in microbial composition between not only each of the dairy elements but also management styles. The primary exceptions were the microbiomes of the house fly and the stable fly. Their compositions heavily overlapped with one another, but interestingly, not with the other components sampled. Additionally, both species of flies carried more pathogens than the other elements of the dairy, indicating that they may not share these organisms with the other components, or that the environments offered by the other components are unsatisfactory for the survival of some pathogens.. Conclusion: The lack of overlapping pathogen profiles suggests a lack of transfer from flies to other dairy elements. Dairy health data, showing a low incidence of disease, suggests minimal sharing of bacteria by the flies at a level required for infection, given the health program of this dairy. While flies did carry a multitude of pathogenic bacteria, the mere presence of the bacteria associated with the flies did not necessarily translate into high risk leading to morbidity and mortality at this dairy. Thus, using flies as the sole sentinel of dairy health may not be appropriate for all bacterial pathogens or dairies.

Assessment of Potential Anti-Methanogenic and Antimicrobial Activity of Ethyl Nitroacetate, α-Lipoic Acid, Taurine and L-Cysteinesulfinic Acid In Vitro.

Livestock producers need new technologies to maintain the optimal health and well-being of their animals while minimizing the risks of propagating and disseminating pathogenic and antimicrobial-resistant bacteria to humans or other animals. Where possible, these interventions should contribute to the efficiency and profitability of animal production to avoid passing costs on to consumers. In this study, we examined the potential of nitroethane, 3-nitro-1-propionate, ethyl nitroacetate, taurine and L-cysteinesulfinic acid to modulate rumen methane production, a digestive inefficiency that results in the loss of up to 12% of the host's dietary energy intake and a major contributor of methane as a greenhouse gas to the atmosphere. The potential for these compounds to inhibit the foodborne pathogens, Escherichia coli O157:H7 and Salmonella Typhimurium DT104, was also tested. The results from the present study revealed that anaerobically grown O157:H7 and DT104 treated with the methanogenic inhibitor, ethyl nitroacetate, at concentrations of 3 and 9 mM had decreased (p < 0.05) mean specific growth rates of O157:H7 (by 22 to 36%) and of DT104 (by 16 to 26%) when compared to controls (0.823 and 0.886 h-1, respectively). The growth rates of O157:H7 and DT104 were decreased (p < 0.05) from controls by 31 to 73% and by 41 to 78% by α-lipoic acid, which we also found to inhibit in vitro rumen methanogenesis up to 66% (p < 0.05). Ethyl nitroacetate was mainly bacteriostatic, whereas 9 mM α-lipoic acid decreased (p < 0.05) maximal optical densities (measured at 600 nm) of O157:H7 and DT104 by 25 and 42% compared to controls (0.448 and 0.451, respectively). In the present study, the other oxidized nitro and organosulfur compounds were neither antimicrobial nor anti-methanogenic.

Inhibitory Effect of Select Nitrocompounds and Chlorate against Yersinia ruckeri and Yersinia aleksiciae In Vitro.

Yersinia ruckeri is an important fish pathogen causing enteric redmouth disease. Antibiotics have traditionally been used to control this pathogen, but concerns of antibiotic resistance have created a need for alternative interventions. Presently, chlorate and certain nitrocompounds were tested against Y. ruckeri as well as a related species within the genus, Y. aleksiciae, to assess the effects of these inhibitors. The results reveal that 9 mM chlorate had no inhibitory effect against Y. ruckeri, but inhibited growth rates and maximum optical densities of Y. aleksciciae by 20-25% from those of untreated controls (0.46 h-1 and 0.29 maximum optical density, respectively). The results further reveal that 2-nitropropanol and 2-nitroethanol (9 mM) eliminated the growth of both Y. ruckeri and Y. aleksiciae during anaerobic or aerobic culture. Nitroethane, ethyl nitroacetate and ethyl-2-nitropropionate (9 mM) were less inhibitory when tested similarly. Results from a mixed culture of Y. ruckeri with fish tank microbes and of Y. aleksiciae with porcine fecal microbes reveal that the anti-Yersinia activity of the tested nitrocompounds was bactericidal, with 2-nitropropanol and 2-nitroethanol being more potent than the other tested nitrocompounds. The anti-Yersinia activity observed with these tested compounds warrants further study to elucidate the mechanisms of action and strategies for their practical application.