Comparison of Three Different Analyzers to Measure Canine Serum Progesterone

The canine estrous cycle is unique compared to other domestic animal species. During estrus, serum progesterone concentrations ([P4]) rise two days prior to ovulation. In fact, the luteinization of pre-ovulatory follicles resulting in this initial increase in [P4] (1.5-2.5 ng/ml) cannot be temporally dissociated from the onset of the surge in luteinizing hormone. For this reason, measuring progesterone from daily blood samples is commonly used to determine the optimal breeding day in female dogs. In addition, the fall in [P4] (<2 ng/ml) prior to parturition can be used for the purposes of determining the timing of an elective C-section in dogs. There are several methods veterinarians can use to measure [P4] but directly comparing results between assays without formulaic adjustments often yields unreliable results. The objective of this research was to compare [P4] measured on three different veterinary analyzers (enzyme linked fluorescent assay (ELFA), colorimetric immunoassay (CIA), and chemiluminescent immunoassay (CLIA)). It was hypothesized that irrespective of analyzer used, the [P4] measurement would be reliable for determining timing for breeding or C-section. Venous blood samples (n=116) were collected from privately-owned female dogs (n=44) at the Waipahu Waikele Pet Hospital in Honolulu, Hawaii. Dogs were fasted 6 to 8 hours prior to each blood collection and blood samples were collected into tubes containing a clot activator but not serum separating gel. Blood samples were allowed to clot for 30 to 60 minutes and then were centrifuged for five minutes at 3,500 rpm to allow for serum removal. In accordance with the manufacturer’s instructions, [P4] was determined from three analyzers. Data were managed in Google Sheets and analyzed using R. Using simple linear regression, the coefficients of determination (R2) between CIA and ELFA, CIA and CLIA, and CLIA and ELFA was 0.88, 0.914, and 0.957, respectively. The simple linear regression was also used to determine the regression equation, which was CLIA = 0.536*CIA+0.688, CLIA = 0.417*ELFA+0.321, and CIA = -0.45*ELFA+0.73. Using a Passing-Bablok regression, the Pearson correlation coefficients were 0.938, 0.956, and 0.978, respectively. However, analysis of the residuals showed an increase in the spread as the [P4] increased. The results from this study show that comparison of [P4] between the analyzers is accurate at lower values (<5 ng/mL) but the variability in [P4] increases as the value increases

Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains

AbstractTau in Alzheimer neurofibrillary tangles has been shown to be hyperphosphorylated and CDK5, GSK3, MAP kinase and SAP kinases are the candidate kinases for the phosphorylation of tau. Recently, it was reported that the conversion of p35, the activator of CDK5, to p25 was upregulated in Alzheimer’s disease (AD) brains, and that p35 is cleaved to yield p25 by calpain. Here we show that p35 is rapidly cleaved to p25 in rat and human brains within a short postmortem delay and that the conversion of p35 to p25 is partially dependent on calpain activity. Immunoblot analysis of brains prepared from patients with AD or age-matched control individuals with a short postmortem delay revealed no specific increase in the levels of p25 in AD brains, whereas the levels of active form of calpain were increased in AD brains compared to the those in controls. These observations suggest that the conversion of p35 to p25 is a postmortem degradation event and may not be upregulated in AD brains

Markedly lower follow-up rate after liver biopsy in patients with non-alcoholic fatty liver diseases than those with viral hepatitis in Japan

<p>Abstract</p> <p>Background</p> <p>Patients with non-alcoholic fatty liver diseases (NAFLD) are recommended to have periodic follow-up exams because these patients are at increased risk of the presence of non-alcoholic steatohepatitis (NASH), which can lead to cirrhosis or hepatocellular carcinoma. We investigated the follow-up status of NAFLD patients after a liver biopsy examination.</p> <p>Methods</p> <p>We compared the follow-up rates of NAFLD patients who had received an ultrasonography-guided liver biopsy and patients who had received a liver biopsy for chronic viral hepatitis (hepatitis B or C).</p> <p>Results</p> <p>The 1- and 3-year follow-up rates after the liver biopsy were 92.7% and 88.3% for patients with chronic HBV infection, and 93.4% and 88.2% for patients with chronic HCV infection, respectively. In contrast, the follow-up rates for NAFLD patients were 77.6% and 49.9%, respectively, which were significantly lower than those of patients with chronic viral hepatitis (<it>p </it>< 0.0001). Among NAFLD patients, the respective 1- and 3-year follow-up rates were 73.0% and 44.6% for patients with simple steatosis and 80.0% and 52.4% for patients with NASH based on a pathologic diagnosis, without significant difference between these two subgroups (<it>p </it>= 0.5202).</p> <p>Conclusions</p> <p>The outpatient-based follow-up rate after a liver biopsy was significantly lower in NAFLD patients compared to patients with chronic viral hepatitis, regardless of the presence of NASH. It is important to determine how to maintain regular hospital visits for NAFLD patients, preventing patient attrition.</p

Identification and functional characterisation of CRK12:CYC9, a novel cyclin-dependent kinase (CDK)-cyclin complex in Trypanosoma brucei

The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes trypanosomiasis in humans and animals. Both the life cycle and cell cycle of the parasite are complex. Trypanosomes have eleven cdc2-related kinases (CRKs) and ten cyclins, an unusually large number for a single celled organism. To date, relatively little is known about the function of many of the CRKs and cyclins, and only CRK3 has previously been shown to be cyclin-dependent in vivo. Here we report the identification of a previously uncharacterised CRK:cyclin complex between CRK12 and the putative transcriptional cyclin, CYC9. CRK12:CYC9 interact to form an active protein kinase complex in procyclic and bloodstream T. brucei. Both CRK12 and CYC9 are essential for the proliferation of bloodstream trypanosomes in vitro, and we show that CRK12 is also essential for survival of T. brucei in a mouse model, providing genetic validation of CRK12:CYC9 as a novel drug target for trypanosomiasis. Further, functional characterisation of CRK12 and CYC9 using RNA interference reveals roles for these proteins in endocytosis and cytokinesis, respectively

Phagocytosis depends on TRPV2-mediated calcium influx and requires TRPV2 in lipids rafts: alteration in macrophages from patients with cystic fibrosis.

Whereas many phagocytosis steps involve ionic fluxes, the underlying ion channels remain poorly defined. As reported in mice, the calcium conducting TRPV2 channel impacts the phagocytic process. Macrophage phagocytosis is critical for defense against pathogens. In cystic fibrosis (CF), macrophages have lost their capacity to act as suppressor cells and thus play a significant role in the initiating stages leading to chronic inflammation/infection. In a previous study, we demonstrated that impaired function of CF macrophages is due to a deficient phagocytosis. The aim of the present study was to investigate TRPV2 role in the phagocytosis capacity of healthy primary human macrophage by studying its activity, its membrane localization and its recruitment in lipid rafts. In primary human macrophages, we showed that P. aeruginosa recruits TRPV2 channels at the cell surface and induced a calcium influx required for bacterial phagocytosis. We presently demonstrate that to be functional and play a role in phagocytosis, TRPV2 might require a preferential localization in lipid rafts. Furthermore, CF macrophage displays a perturbed calcium homeostasis due to a defect in TRPV2. In this context, deregulated TRPV2-signaling in CF macrophages could explain their defective phagocytosis capacity that contribute to the maintenance of chronic infection