The role of embryo contact and focal adhesions during maternal recognition of pregnancy.

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

Aldosterone breakthrough in dogs with naturally occurring myxomatous mitral valve disease.

INTRODUCTION: Aldosterone breakthrough (ABT) is the condition in which angiotensin converting enzyme inhibitors (ACEIs) and/or angiotensin receptor blockers fail to effectively suppress the activity of the renin angiotensin aldosterone system. The objective of this study was to determine if ABT occurs in dogs with naturally occurring myxomatous mitral valve disease receiving an ACEI, using the urine aldosterone to creatinine ratio (UAldo:C) as a measure of renin angiotensin aldosterone system activation. ANIMALS, MATERIALS AND METHODS: This study includes 39 dogs with myxomatous mitral valve disease. A UAldo:C cut-off definition (derived from a normal population of healthy, adult, and client-owned dogs) was used to determine the prevalence of ABT in this population. Spearman analysis and univariate logistic regression were used to evaluate the relationship between UAldo:C and ABT (yes/no) and eight variables (age, serum K+ concentration, serum creatinine concentration, ACEI therapy duration and ACEI dosage, furosemide therapy duration and furosemide dosage, and urine sample storage time). Finally, the UAldo:C in dogs receiving spironolactone, as part congestive heart failure (CHF) therapy, was compared to dogs with CHF that were not receiving spironolactone. RESULTS: The prevalence of ABT was 32% in dogs with CHF and 30% in dogs without CHF. There was no relationship between either the UAldo:C or the likelihood of ABT and the eight variables. Therapy with spironolactone lead to a significant elevation of the UAldo:C. DISCUSSION: Using the UAldo:C and a relatively stringent definition of ABT, it appears that incomplete RAAS blockade is common in dogs with MMVD receiving an ACEI. The prevalence of ABT in this canine population mirrors that reported in humans. While the mechanism of ABT is likely multifactorial and still poorly understood, the proven existence of ABT in dogs offers the potential to improve the prognosis for MMVD with the addition of a mineralocorticoid receptor blocker to current therapeutic regimens. CONCLUSIONS: Approximately 30% of dogs being treated for heart disease and CHF satisfied the definition of ABT. Identifying patient subpopulations experiencing ABT may help guide future study design and clinical decision-making.

Qualitative assessment of bone density at the distal articulating surface of the third metacarpal in Thoroughbred racehorses with and without condylar fracture.

REASONS FOR PERFORMING STUDY: Changes in subchondral bone density, induced by the repetitive cyclical loading of exercise, may potentiate fatigue damage and the risk of fracture. OBJECTIVES: To use computed tomography (CT) to characterise bone density patterns at the articular surface of the third metacarpal bone in racehorses with and without lateral condylar fractures. STUDY DESIGN: Case control METHODS: Computed tomographic images of the distal articulating surface of the third metacarpal bone were obtained from Thoroughbred racehorses subjected to euthanasia in the UK. Third metacarpal bones were divided into 3 groups based on lateral condyle status; fractured (FX, n = 42), nonfractured contralateral condyle (NFX, n = 42) and control condyles from horses subjected to euthanasia for reasons unrelated to the third metacarpal bone (control, n = 94). Colour CT images were generated whereby each colour represented a range of pixel values and thus a relative range of bone density. A density value was calculated qualitatively by estimating the percentage of each colour within a specific region. Subchondral bone density was assessed in 6 regions from dorsal to palmar and 1 mm medial and lateral to the centre of the lateral parasagittal groove in NFX and control condyles and 1 mm medial and lateral to the fracture in FX condyles. RESULTS: Bone density was significantly higher in the FX and NFX condyles compared with control condyles for all 6 regions. A significantly higher bone density was observed in FX condyles relative to NFX condyles in the lateral middle and lateral palmar regions. Fractured condyles had increased heterogeneity in density among the 6 regions of interest compared with control and NFX condyles. CONCLUSIONS: Adjacent to the fracture, a focal increase in bone density and increased heterogeneity of density were characteristic of limbs with lateral condylar fractures compared with control and NFX condyles. These differences may represent pathological changes in bone density that increase the risk for lateral condylar fractures in racehorses.

Effect of packaging during storage time on retail display microbial population of beef strip loins from two different production systems.

Two studies were conducted to evaluate the influence of packaging during storage of strip loins (to simulate export shipment) from steers fattened on intensive grazing systems (Uruguay; UR) or on a high-concentrate diet (United States; US) on retail display life microbial growth. Four or 3 different packaging treatments were applied to UR and US strip loin roasts or steaks during 35 d of storage; treatments were applied 7 d following slaughter. After 35 d of storage, the samples were evaluated during simulated retail display for up to 6 d. In Exp. 1, the treatments were vacuum packaging (VP), low-oxygen modified atmosphere packaging (MAP) with N and CO (MAP/CO), low-oxygen MAP with N plus CO and CO, and VP plus an application of peroxyacetic acid (VP/PAA). In Exp. 2, block 1, the treatments were VP, MAP/CO, and VP with ethyl--lauroyl--arginate HCl incorporated into the film as an antimicrobial agent (VP/AM). In Exp. 2, block 2, the treatments were VP, MAP/CO, MAP/CO, and VP/AM. For retail display, VP treatments were sliced and repackaged in PVC overwrap, and MAP treatments were actually PVC overwrap trays that were removed from a master bag with the prescribed gas treatment. Regardless of production system and packaging treatment, mesophilic and psychrotrophic counts of 6.9 to 7.8 and 6.7 to 7.7 log10 CFU/cm, respectively, were obtained at the end of retail display, except for US samples in Exp. 2 (5.5 to 6.3 log CFU/cm). No differences ( > 0.05) were detected for spp. counts among packaging treatments in US steaks at the end of the display time in Exp.1, whereas, for UR steaks, both MAP treatments had lower ( < 0.05) spp. counts than VP treatments. spp. counts were lower ( < 0.05) in the MAP/CO treatment than in the other 3 treatments in US samples on d 6 of retail display for Exp. 2. At the end of display time and for Exp. 1, US steaks under MAP/CO had greater ( < 0.05) lactic acid bacteria (LAB) counts than samples in both VP treatments; no differences ( > 0.05) among packaging were detected for UR steaks. Both MAP and VP/AM treatments in the US samples for Exp. 2 had lower ( < 0.05) LAB counts on d 6 of display than the VP treatment, but no differences ( > 0.05) were found among packaging treatments for the UR samples. To maximize shelf life (storage and display life) of exported fresh beef, it is critical to minimize bacterial populations during processing and storage.

Effect of packaging during storage time on retail display shelf life of longissimus muscle from two different beef production systems.

Two studies were conducted to evaluate the influence of packaging and production system (PS) on retail display life color (L*, a*, and b*), fatty acid profile (% of total fatty acids), lipid oxidation (thiobarbituric acid reactive substances; mg malondialdehyde/kg of muscle), vitamin E content (µg/g of muscle), and odor (trained panelists) during storage of LM. Four (or 3) different packaging treatments were applied to LM from steers fattened on grazing systems (Uruguayan) or on high-concentrate diets (U.S.). From fabrication to application of treatments, Uruguayan LM were vacuum packaged for air shipment and U.S. LM were also vacuum packaged and kept in a cooler until Uruguayan samples arrived. Treatments were applied 7 d after slaughter. In Exp. 1, treatments were vacuum packaging (VP), low-oxygen (O) modified atmosphere packaging (MAP) with nitrogen (N2) and carbon dioxide (MAP/CO), low-O MAP with N2 plus CO and carbon monoxide (MAP/CO), and VP plus an application of peroxyacetic acid (VP/PAA). In Exp. 2 block 1, treatments were VP, MAP/CO, and VP with ethyl-arginate HCl incorporated into the film as an antimicrobial agent (VP/AM). In Exp. 2 block 2, treatments were VP, MAP/CO, MAP/CO, and VP/AM. After 35 d storage, steaks were evaluated during simulated retail display for up to 6 d. In Exp. 1, Uruguayan steaks under MAP/CO had greater ( < 0.05) a* values than VP/PAA and MAP/CO on d 6 of display. For U.S. beef, the MAP/CO had the reddest lean color ( < 0.05) compared with the other 3 packaging treatments on d 6 of display in Exp. 1. Packaging × PS × time interaction was significant ( < 0.05) in Exp. 1. In Exp. 2, MAP/CO in Uruguayan steaks also had the greatest a* values on d 6 of display, but no differences ( > 0.05) were detected among both VP and MAP/CO in U.S. steaks at this time. No significant ( > 0.05) packaging × PS × time interaction was observed in Exp. 2. Only PS (both experiments) and time (Exp. 1) affected ( < 0.05) L* values. In both experiments, U.S. steaks had greater ( < 0.05) L* values than Uruguayan steaks. Vitamin E content in Uruguayan steaks was greater ( < 0.05) than in U.S. steaks. Packaging × PS, PS × time, and packaging × PS × time interactions were not significant ( > 0.05) for any of the fatty acids. Beef from Uruguayan had lower ( < 0.05) SFA and MUFA and greater ( < 0.05) PUFA and n-6 and n-3 fatty acid percentages than U.S. beef. Complexity of fresh meat postmortem chemistry warrants a more comprehensive approach to maximize shelf life.

Equine endometrial gene expression changes during and after maternal recognition of pregnancy.

The mechanism for maternal recognition of pregnancy (MRP) in horses is unknown. To maintain a pregnancy, a mobile conceptus must be recognized by the uterus before d 14 postovulation (PO). This recognition prevents endometrial secretion of PGF2α on d14 through 16, which would otherwise initiate luteolysis. The objective of this study was to evaluate gene expression in the endometrium of pregnant and nonpregnant mares during and after MRP to identify possible genes involved during this time. Twelve normally cycling mares were used in a crossover design and randomly assigned to a specific collection day. Endometrial samples were collected from a pregnant and nonpregnant (nonmated) mare on cycle d 12, 14, 16, and 18 (n = 3/d) PO. Microarray analysis comparing the endometrial gene expression in pregnant and nonpregnant mares revealed no differences at d 12. Ten genes were identified to have consistently higher or lower expression levels in the endometrium from pregnant versus nonpregnant mares on d 14, 16, and 18 (P < 0.001). The expression of these 10 genes was further analyzed with real-time PCR. d 14, 16, and 18 gene expression patterns were consistent with the microarray analysis, but on d 12, 4 of the 10 were identified as differentially expressed. Endometrial samples were then collected on d 13 PO (n = 3) and processed for western blot and immunohistochemical analysis of 2 proteins due to their reproductive significance. SPLA2 and DKK1 antibody specificity were confirmed via western blot analysis but were not different in samples from pregnant and nonpregnant mares (P = 0.114 and P = 0.514, respectively) and cellular localization was examined by immunohistochemical analysis. This is the first study to describe gene expression and cellular localization in the endometrium at the time of MRP for these genes and suggests that the uterus does not prepare to support a pregnancy until d 14. The function of these genes may be critical in the process of MRP.

Prognostic significance of histologic features in canine renal cell carcinomas: 70 nephrectomies.

The prognostic significance of histologic and clinical features was evaluated in a retrospective study of 70 dogs treated with nephrectomy for renal cell carcinoma. Dogs presenting with hematuria and cachexia had significantly decreased overall and tumor-specific survival. Mitotic index (MI), nuclear size, nuclear pleomorphism, tumor differentiation, invasiveness, Fuhrman nuclear grade, and clear cell morphology were significantly associated with survival times (overall and tumor specific) in univariate analyses. A multivariate Cox proportional hazards model was constructed using stepwise selection to evaluate potential histologic predictor variables. This multivariate analysis revealed MI, defined as the number of mitotic figures in ten 400× fields, as the sole independent prognostic variable. Median survival for dogs with an MI >30 was 187 days compared with 1184 days for dogs with an MI of <10. Dogs with an intermediate MI of 10 to 30 had a median survival of 452 days. Canine renal carcinomas were categorized into the following subtypes based on histologic features and histochemical and immunohistochemical staining: (1) clear cell, (2) chromophobe, (3) papillary, and (4) multilocular cystic renal cell carcinomas. Clear cell carcinoma was diagnosed in 6 of 70 (9%) canine tumors and was associated with a significantly decreased median survival time. Papillary carcinomas were identified in 15 of 70 tumors (21%), chromophobe in 6 of 70 (9%), and the multilocular cystic variant of canine renal cell carcinoma in 3 of 70 tumors (4%). These findings facilitate uniform categorization of canine renal cell carcinoma and provide veterinary pathologists with criteria to determine prognostic information.

MicroRNA levels are modulated in Aedes aegypti after exposure to Dengue-2.

To define microRNA (miRNA) involvement during arbovirus infection of Aedes aegypti, we mined deep sequencing libraries of Dengue type 2 (DENV2)-exposed mosquitoes. Three biological replicates for each timepoint [2, 4 and 9 days post-exposure (dpe)] and treatment group allowed us to remove the outliers associated with sample-to-sample variability. Using edgeR (R Bioconductor), designed for use with replicate deep sequencing data, we determined the log fold-change (logFC) of miRNA levels [18-23 nucleotides (nt)]. The number of significantly modulated miRNAs increased from ≤ 5 at 2 and 4 dpe to 23 unique miRNAs by 9 dpe. Putative miRNA targets were predicted by aligning miRNAs to the transcriptome, and the list was reduced to include the intersection of hits found using the Miranda, PITA, and TargetScan algorithms. To further reduce false-positives, putative targets were validated by cross-checking them with mRNAs reported in recent DENV2 host response transcriptome reports; 4076 targets were identified. Of these, 464 gene targets have predicted miRNA-binding sites in 3' untranslated regions. Context-specific target functional groups include proteins involved in transport, transcriptional regulation, mitochondrial function, chromatin modification and signal transduction processes known to be required for viral replication and dissemination. The miRNA response is placed in context with other vector host response studies by comparing the predicted targets with those of transcriptome studies. Together, these data are consistent with the hypothesis that profound and persistent changes to gene expression occur in DENV2-exposed mosquitoes.

Building an idealized measurement system to improve clinical office practice performance.

The aim of this article is to introduce key concepts and approaches for building a better practice-based measurement. Physicians are being challenged to produce measurably higher quality of services, lower costs, and better clinical outcomes to remain viable. In the absence of provider-driven practice improvements and independent measurement systems, office practices will remain dependent on performance data from external forces as drivers of change. Three key questions will be addressed in our pursuit of better measurement systems for continuous improvement and competitive advantage: (1) What do we want a measurement system to tell us in the first place? (2) What might an idealized measurement system look like if we had one? (3) What are some of the challenges that office practices face in closing the gap between building an idealized measurement system and the current state of office practice measurement?