Xenohormetic Phytochemicals Inhibit Neovascularization in Microphysiological Models of Vasculogenesis and Tumor Angiogenesis.

Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit-derived Naringenin (Nar) and soybean-derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti-vasculogenic effects of Glyceollin are significantly more potent in sex-matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health-promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations.

Methods for studying microbial acid stress responses: from molecules to populations.

The study of how micro-organisms detect and respond to different stresses has a long history of producing fundamental biological insights while being simultaneously of significance in many applied microbiological fields including infection, food and drink manufacture, and industrial and environmental biotechnology. This is well illustrated by the large body of work on acid stress. Numerous different methods have been used to understand the impacts of low pH on growth and survival of micro-organisms, ranging from studies of single cells to large and heterogeneous populations, from the molecular or biophysical to the computational, and from well-understood model organisms to poorly defined and complex microbial consortia. Much is to be gained from an increased general awareness of these methods, and so the present review looks at examples of the different methods that have been used to study acid resistance, acid tolerance, and acid stress responses, and the insights they can lead to, as well as some of the problems involved in using them. We hope this will be of interest both within and well beyond the acid stress research community.

What makes an athlete? A scoping review: Assessing the use of the word athlete with anterior cruciate ligament rehabilitation review studies. Is there a standard?

The term athlete does not currently have an agreed definition or standardized use across the literature. We analyzed the use of the term "athlete" amongst review studies specific to Anterior Cruciate Ligament (ACL) rehabilitation to investigate if the term was justified in its use. A comprehensive review of a database was performed to identify review papers which used the term "athlete" in the title, and which were related to ACL rehabilitation and surveillance. These papers were analyzed and their source papers were extracted for review. Twenty-eight review papers were identified. Source studies were extracted and analyzed. After removal of duplicates 223 source papers were identified. Despite using the term "athlete" in the review study titles only 5/17 (10.7%) sufficiently justified the use of this term. The term athlete was used in 117/223 (52.5%) of the source studies. Of those, 78/117 source studies (66.7%) justified the term athlete. The remaining 39/117 (33.3%) papers where participants were stated to be athletes, gave no justification. The ambiguous use of the term athlete amongst published studies highlights the need for a definition or justification of the term to be used in studies. The lack of a standard definition leads to the potential for studies to dilute high quality data by the potentially differing rehabilitation requirements and access to resources available to those with varying exercise levels. The indiscriminate use of the term athlete could lead to participants with widely ranging physical activity levels being included in the same study, and being used to create clinical advice for all. Advice could potentially vary across those of differing physical activity levels.

17†Development of a questionnaire to study fear and anxiety factors affecting patients and their families undergoing strabismus surgery.

Patients and their Families undergoing Strabismus Surgery. The aim of this study was to develop a questionnaire to identify perioperative fear and anxiety factors affecting pediatric strabismus surgery patients.First, we reviewed the literature to determine precipitants of fears and anxieties experienced by pediatric patients. Subsequently, we developed a questionnaire for pediatric patients undergoing strabismus surgery. This was a two part questionnaire, consisting of a 16-piece section for patients and a 22-piece section for parents. Finally, we piloted this questionnaire to validate its clinical use.Common anxiety factors for children include pain, minor clinical procedures requiring needles, separation from parents and engaging with medical professionals. We used this information to develop a two part questionnaire for patients and parents. The questionnaire elicited positive and negative aspects of the patient journey, corroborated fears reported in the literature, and identified anxiety inducing factors specific to strabismus patients.There is a lack of evidence regarding fear and anxiety specific to pediatric ophthalmology surgeries. Strabismus surgery carries unique fear inducing factors. Interventions which may alleviate the stress of pediatric surgery, therefore greatly benefit patient experience and surgical outcomes, and should be considered in the care of pediatric patients. Patient educational material is known to provide a sense of control to patients, helping to alleviate such fear.Evidenced by the literature and the pilot questionnaire, there still exists anxiety inducing factors in pediatric surgery. Investigation into patient fears regarding pediatric strabismus surgery is needed to better understand how clinical staff can support patients perioperatively.

Review: Role of early life nutrition in regulating sexual development in bulls.

The availability of high-quality semen from genetically elite bulls is essential to support continued genetic gain and the sustainability of cattle production worldwide. While reducing the age at which usable semen is available also reduces the generation interval, it is dependent on timely onset of puberty in young bulls. There is now good evidence that hastened sexual development in bulls is achieved through enhancing nutrition in early life. This review will cover the physiological and molecular-based response to prevailing diet in key organs that orchestrate the ontogeny of sexual development in the bull calf. Given the central importance of the interaction between metabolic status and neuronal function to the progression of sexual development, we will discuss how communication between metabolic organs, reproductive organs and the brain are mediated via molecular and physiological processes. The availability of high-throughput nucleic acid and protein sequencing technologies and innovative data analytics have allowed us to improve our understanding of molecular regulation of puberty and sexual development. Analysing data from a number of organs, simultaneously, allows for a better understanding of the underlying biology and biochemical interactions that are influencing sexual development. Specifically, we can determine how early life nutritional interventions augment changes in potential key molecules regulating sexual development. Ultimately, a greater understanding of the inherent regulation of postnatal sexual development in the bull calf and how strategically targeted nutritional management can advance the ontogeny of this process, will facilitate the timely availability of high-quality semen from genetically elite animals, thus supporting more economically and environmentally sustainable beef and dairy production systems.

Enhancing early life nutrition alters the hepatic transcriptome of Angus × Holstein-Friesian heifer calves.

Early life nutrition has a major influence on subsequent lifetime performance in cattle. The aim of this experiment was to investigate the effect of plane of nutrition from 3 to 21 weeks of age on the liver transcriptome. Holstein-Friesian × Angus heifer calves with a mean (±SD) age and BW of 19 (±5) days and 51.2 (±7.8) kg, respectively, were assigned to either a high-energy diet to support a mean average daily gain (ADG) of 1.2 kg/day (HI; n = 15) or a moderate diet (MOD; n = 15) to support a mean ADG of 0.5 kg/day. At 145 ±â€¯3 days of age, all calves were euthanised, liver tissue samples collected and flash-frozen in liquid nitrogen. Following RNA sequence analysis, the total number of differentially expressed genes (DEGs) (at false discovery rate (FDR) > 0.05) was 537; 308 upregulated and 229 downregulated in HI compared to MOD. The number of DEGs mapped to IPA (at FDR > 0.05) was 460; 264 upregulated and 196 downregulated. There was greater expression of genes associated with cellular development and metabolism in heifers on the HI compared to the MOD diet. The genes (fold change) of the somatotrophic axis; IGF1 (3.7), IGFALS (2.6) and GHR (1.5) were upregulated in the HI compared to MOD diet. The cytokine receptor genes, IL17RB (1.7) and IL20RA (3.3), were upregulated in the HI heifers, which were detected in a network interacting with metabolically regulated genes. The potential enhanced cell-to-cell communication evident from DEGs would increase the calves' ability to combat health challenges. The findings of this study indicate that enhancing the early life plane of nutrition in heifer calves results in the upregulation of genes that are associated with increased metabolic activity and thus metabolic capacity. Moreover, the interaction between metabolic and immune communication genes indicates that enhanced nutrition has the potential to improve the immune response in the liver which will play a central role in ensuring optimal lifetime performance.

Liver Kinase B1 Regulates Remodeling of the Tumor Microenvironment in Triple-Negative Breast Cancer.

Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1's role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis.

Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling.

Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.

Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility.

Younger bulls typically produce lower volumes of semen per ejaculate with a lower sperm concentration than older more mature, bulls and often fail to meet semen demand using standard collection frequency schedules. The objective of this study was to assess the effect of ejaculate collection frequency on semen output, sperm quality and field fertility in young bulls under commercial conditions. Holstein Friesian bulls aged 366 ± 8 days (mean ± SEM) were assigned one of two ejaculate collection frequencies: (i) HF (n = 14 bulls), where ejaculates were collected twice a day, five days in each two-week period or (ii) LF (n = 12 bulls), where ejaculates were collected once a day, two days per week. The trial period continued until each bull reached both 20 ejaculates and 1000 marketable frozen semen straws. Subjective motility was assessed on all ejaculates pre-freeze and post-thaw (at 0 and 2 h). A subset of ejaculates were assessed post-thaw by computer-assisted sperm analysis for motility, kinematics and morphological defects and by flow cytometry for viability, membrane fluidity, acrosome integrity, reactive oxygen species and DNA fragmentation. A total of 13,846 inseminations (9,541 for HF and 4,305 for LF) were carried out on dairy cows and heifers. HF reached the 1000 straw threshold 41 days earlier than LF (P < 0.01) with the same number of ejaculates. Ejaculate volume and sperm concentration were not affected by treatment but the first ejaculate of the day (HF only) had a greater volume (P < 0.001) and sperm concentration (P < 0.05) than the second ejaculate. HF had higher pre-freeze total (P < 0.01) and gross (P < 0.05) motility than LF. HF had higher post-thaw (2 h) total and gross motility than LF (P < 0.05). Ejaculate rejection rates did not differ between treatments. There was no effect of treatment, week or ejaculate number of the day (HF only) on post-thaw motility and kinematic parameters or sperm viability, membrane fluidity, acrosome integrity and DNA fragmentation. However, HF had lower superoxide production than LF (P < 0.05). Pregnancy per artificial insemination was 64.5 ± 1.0% and 59.9 ± 1.1% for the HF and LF bulls, respectively (mean ± SEM; P = 0.05). In conclusion, collecting ejaculates more frequently from young bulls significantly reduced the number of days required to obtain 1000 straws, increased semen quality in terms of lower superoxide production and increased field fertility.

Physical activity recommendations pre and post abdominal wall reconstruction: a scoping review of the evidence.

PURPOSE: There are no universally agreed guidelines regarding which types of physical activity are safe and/or recommended in the perioperative period for patients undergoing ventral hernia repair or abdominal wall reconstruction (AWR). This study is intended to identify and summarise the literature on this topic. METHODS: Database searches of PubMed, CINAHL, Allied & Complementary medicine database, PEDro and Web of Science were performed followed by a snowballing search using two papers identified by the database search and four hand-selected papers of the authors' choosing. Inclusion-cohort studies, randomized controlled trials, prospective or retrospective. Studies concerning complex incisional hernia repairs and AWRs including a "prehabilitation" and/or "rehabilitation" program targeting the abdominal wall muscles in which the interventions were of a physical exercise nature. RoB2 and Robins-I were used to assess risk of bias. Prospero CRD42021236745. No external funding. Data from the included studies were extracted using a table based on the Cochrane Consumers and Communication Review Group's data extraction template. RESULTS: The database search yielded 5423 records. After screening two titles were selected for inclusion in our study. The snowballing search identified 49 records. After screening one title was selected for inclusion in our study. Three total papers were included-two randomised studies and one cohort study (combined 423 patients). All three studies subjected their patients to varying types of physical activity preoperatively, one study also prescribed these activities postoperatively. The outcomes differed between the studies therefore meta-analysis was impossible-two studies measured hernia recurrence, one measured peak torque. All three studies showed improved outcomes in their study groups compared to controls however significant methodological flaws and confounding factors existed in all three studies. No adverse events were reported. CONCLUSIONS: The literature supporting the advice given to patients regarding recommended physical activity levels in the perioperative period for AWR patients is sparse. Further research is urgently required on this subject.

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

Quantifying Breast Cancer-Driven Fiber Alignment and Collagen Deposition in Primary Human Breast Tissue.

Solid tumor progression is significantly influenced by interactions between cancer cells and the surrounding extracellular matrix (ECM). Specifically, the cancer cell-driven changes to ECM fiber alignment and collagen deposition impact tumor growth and metastasis. Current methods of quantifying these processes are incomplete, require simple or artificial matrixes, rely on uncommon imaging techniques, preclude the use of biological and technical replicates, require destruction of the tissue, or are prone to segmentation errors. We present a set of methodological solutions to these shortcomings that were developed to quantify these processes in cultured, ex vivo human breast tissue under the influence of breast cancer cells and allow for the study of ECM in primary breast tumors. Herein, we describe a method of quantifying fiber alignment that can analyze complex native ECM from scanning electron micrographs that does not preclude the use of replicates and a high-throughput mechanism of quantifying collagen content that is non-destructive. The use of these methods accurately recapitulated cancer cell-driven changes in fiber alignment and collagen deposition observed by visual inspection. Additionally, these methods successfully identified increased fiber alignment in primary human breast tumors when compared to human breast tissue and increased collagen deposition in lobular breast cancer when compared to ductal breast cancer. The successful quantification of fiber alignment and collagen deposition using these methods encourages their use for future studies of ECM dysregulation in human solid tumors.

Fatty acid concentration of plasma, muscle, adipose and liver from beef heifers fed an encapsulated n-3 polyunsaturated fatty acid supplement.

Increasing the content of polyunsaturated fat in the human diet is a priority for reducing cardiovascular disease and cancer risks. Beef has the potential to contribute to the polyunsaturated fat content in the human diet; however, ruminants cannot synthesise many long-chain fatty acids de novo; they require dietary supplementation. The objectives of the current study were to evaluate (i) the effect of a partially rumen protected n-3 long-chain polyunsaturated fatty acid (LC-PUFA) dietary supplement on the fatty acid composition of muscle (Longissimus dorsi), adipose and liver tissues of beef heifers and (ii) the usefulness of blood plasma as a predictor of tissue concentrations of specific fatty acids. Charolais crossbred heifers (n = 20) were assigned to one of two isolipid dietary treatments namely palmitic acid (control) or an n-3 LC-PUFA supplement for a 91-day period. Blood plasma and adipose tissue samples were taken to determine the temporal effect of these diets on fatty acid composition (days 0, 10, 35 and 91), while liver and muscle samples were taken following slaughter. Dietary lipid source did not influence animal growth rate or body condition score. At day 91, the percentage differences between control and n-3 LC-PUFA heifers in concentrations of eicosapentaenoic acid were +61, +176 and +133 % in liver, muscle and adipose, respectively. For docosahexaenoic acid, at the same time point, the percentage differences were +57, +73 and +138 % for liver, muscle and adipose, respectively. Medium-to-strong positive correlation coefficients were evident for liver and plasma fatty acids, in particular, there were positive relationships with concentrations of total saturated fatty acid (SFA), total n-6 PUFA and total n-3 PUFA. This trend also extended to both the ratio of PUFA to SFA (slope (ß1) = 0.56 ±â€¯0.167, intercept (ß0) = 0.56, R2 = 0.61, P < 0.05) and the ratio of n-6 to n-3 PUFA (ß1 = 0.15 ±â€¯0.054, ß0 = 0.24, R2 = 0.52, P < 0.05). A strong correlation was also detected in the ratio of n-6 to n-3 in plasma and muscle tissue of heifers fed the n-3 LC-PUFA diet (ß1 = 0.53 ±â€¯0.089, ß0 = -0.31, R2 = 0.83, P < 0.001). The results of this study show that the n-3 LC-PUFA can be readily increased through targeted supplementation and that plasma concentrations of n-3 LC-PUFA are useful predictors of their concentrations in a number of economically important tissues.

Association between increased plasma ceramides and chronic kidney disease in patients with and without ischemic heart disease.

AIM: Plasma levels of certain ceramides are increased in patients with ischemic heart disease (IHD). Many risk factors for IHD are also risk factors for chronic kidney disease (CKD), but it is currently uncertain whether plasma ceramide levels are increased in patients with CKD. METHODS: We measured six previously identified high-risk plasma ceramide concentrations [Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:0) and Cer(d18:1/24:1)] in 415 middle-aged individuals who attended our clinical Cardiology and Diabetes services over a period of 9 months. RESULTS: A total of 97 patients had CKD (defined as e-GFRCKD-EPI<60ml/min/1.73m2 and/or urinary albumin-to-creatinine ratio≥30mg/g), 117 had established IHD and 242 had type 2 diabetes. Patients with CKD had significantly (P=0.005 or less) higher levels of plasma Cer(d18:1/16:0), Cer(d18:1/18:0), Cer(d18:1/20:0), Cer(d18:1/22:0), Cer(d18:1/24:0), and Cer(d18:1/24:1) compared to those without CKD. The presence of CKD remained significantly associated with higher levels of plasma ceramides (standardized beta coefficients ranging from 0.124 to 0.227, P<0.001) even after adjustment for body mass index, smoking, hypertension, diabetes, prior IHD, plasma LDL-cholesterol, hs-C-reactive protein levels and use of any lipid-lowering medications. Notably, more advanced stages of CKD and abnormal albuminuria were both associated (independently of each other) with increased levels of plasma ceramides. These results were consistent in all subgroups considered, including patients with and without established IHD or those with and without diabetes. CONCLUSION: Increased levels of plasma ceramides are associated with CKD independently of pre-existing IHD, diabetes and other established cardiovascular risk factors.

Ewe breed differences in cervical anatomy and cervicovaginal mucus properties: An international study.

In sheep, cervical artificial insemination (AI) involves depositing semen at the cervical opening, as it is not possible to traverse the cervix due to its complex anatomy. However, internationally this method yields low pregnancy rates when frozen-thawed semen is used. An exception to this is in Norway, in which vaginal deposition of frozen-thawed semen to a natural estrus yields pregnancy rates around 70%. As the cervix and its secretions are the principal factors influencing sperm transport to the site of fertilization the aim of this study was to characterise the differences in the cervical anatomy as well as the cervicovaginal mucus properties of six European ewe breeds across three countries known to have differences in pregnancy rates following cervical AI with frozen-thawed semen. These were Suffolk and Belclare in Ireland, Fur and Norwegian White Sheep (NWS) in Norway and Ile de France and Romanov in France (n = 28-30 ewes/breed). Cervicovaginal mucus was collected at the follicular and luteal phases of both a synchronized and natural cycle and assessed for mucus weight, viscosity and colour. The anatomical characteristics of the cervix (length of the cervix, number of cervical rings and the appearance of the external os) were assessed post-mortem. There was a type of the cycle by ewe breed interaction represented by no differences in mucus production between ewe breeds at the natural cycle for both the follicular and luteal phases of the cycle. However, there were differences between ewe breeds at the synchronized cycle (P < 0.05). Belclare had the lowest mucus production at the follicular phase while NWS had the lowest amount of mucus at the luteal phase of the synchronized cycle. Overall, across all ewe breeds, mucus production was higher at the follicular than at the luteal phase (P < 0.05). Despite reports of Suffolk and NWS having the most divergent pregnancy rates following cervical AI with frozen-thawed semen, both breeds had the lowest overall mucus viscosity at the follicular phase of both types of cycle with no differences between both ewe breeds (P > 0.05). The length of the cervix, number of cervical rings and the external os type were affected by ewe breed (P < 0.05). Suffolk ewes had longer cervices but lower number of cervical rings than NWS and Fur ewes (both with higher pregnancy rates). In conclusion, while mucus production and mucus viscosity was affected by breed, these changes are not consistent with the known differences between ewe breeds in their pregnancy rates following cervical AI with frozen-thawed semen.

Evaluation of intercellular communication between breast cancer cells and adipose-derived stem cells via passive diffusion in a two-layer microfluidic device.

Breast cancer tumorigenesis and response to therapy is regulated by cancer cell interactions with the tumor microenvironment (TME). Breast cancer signaling to the surrounding TME results in a heterogeneous and diverse tumor microenvironment, which includes the production of cancer-associated fibroblasts, macrophages, adipocytes, and stem cells. The secretory profile of these cancer-associated cell types results in elevated chemokines and growth factors that promote cell survival and proliferation within the tumor. Current co-culture approaches mostly rely on transwell chambers to study intercellular signaling between adipose-derived stem cells (ASCs) and cancer cells; however, these methods are limited to endpoint measurements and lack dynamic control. In this study, a 4-channel, "flow-free" microfluidic device was developed to co-culture triple-negative MDA-MB-231 breast cancer cells and ASCs to study intercellular communication between two distinct cell types found in the TME. The device consists of two layers: a top PDMS layer with four imprinted channels coupled with a bottom agarose slab enclosed in a Plexiglas chamber. For dynamic co-culture, the device geometry contained two centered, flow-free channels, which were supplied with media from two outer flow channels via orthogonal diffusion through the agarose. Continuous fresh media was provided to the cell culture channel via passive diffusion without creating any shearing effect on the cells. The device geometry also allowed for the passive diffusion of cytokines and growth factors between the two cell types cultured in parallel channels to initiate cell-to-cell crosstalk. The device was used to show that MDA-MB-231 cells co-cultured with ASCs exhibited enhanced growth, a more aggressive morphology, and polarization toward the ASCs. The MDA-MB-231 cells were found to exhibit a greater degree of resistance to the drug paclitaxel when co-cultured with ASCs when compared to single culture studies. This microfluidic device is an ideal platform to study intercellular communication for many types of cells during co-culture experiments and allows for new investigations into stromal cell-mediated drug resistance in the tumor microenvironment.

The Role of DNMT and HDACs in the Fetal Programming of Hypertension by Glucocorticoids.

The causes of hypertension are complex and involve both genetic and environmental factors. Environment changes during fetal development have been linked to adult diseases including hypertension. Studies show that timed in utero exposure to the synthetic glucocorticoid (GC) dexamethasone (Dex) results in the development of hypertension in adult rats. Evidence suggests that in utero stress can alter patterns of gene expression, possibly a result of alterations in the topology of the genome by epigenetic markers such as DNA methyltransferases (DNMTs) and histone deacetylases (HDACs). The objective of this study was to determine the effects of epigenetic regulators in the fetal programming and the development of adult hypertension. Specifically, this research examined the effects of the HDAC inhibitor valproic acid (VPA) and the DNMT inhibitor 5-aza-2'-deoxycytidine (5aza2DC) on blood pressure (BP) and gene expression in prenatal Dex-programmed rats. Data suggest that both VPA and 5aza2DC attenuated the Dex-mediated development of hypertension and restored BP to control levels. Epigenetic DNMT inhibition (DNMTi) or HDAC inhibition (HDACi) also successfully attenuated elevations in the majority of altered catecholamine (CA) enzyme expression, phenylethanolamine N-methyltransferase (PNMT) protein, and elevated epinephrine (Epi) levels in males. Although females responded to HDACi similar to males, DNMTi drove increased glucocorticoid receptor (GR) and PNMT expression and elevations in circulating Epi in females despite showing normotensive BP.