Development and evaluation of a novel fast broad-range PCR and sequencing assay (FBR-PCR/S) using dual priming oligonucleotides targeting the ITS/LSU gene regions for rapid diagnosis of invasive fungal diseases: multi-year experience in a large Canadian healthcare zone.

BACKGROUND: This study evaluated the performance of a novel fast broad range PCR and sequencing (FBR-PCR/S) assay for the improved diagnosis of invasive fungal disease (IFD) in high-risk patients in a large Canadian healthcare region. METHODS: A total of 114 clinical specimens (CS) including bronchoalveolar lavages (BALs) were prospectively tested from 107 patients over a 2-year period. Contrived BALs (n = 33) inoculated with known fungi pathogens were also tested to increase diversity. Patient characteristics, fungal stain and culture results were collected from the laboratory information system. Dual-priming oligonucleotide (DPO) primers targeted to the internal transcribed spacer (ITS) (~ 350 bp) and large subunit (LSU) (~ 550 bp) gene regions were used to perform FBR-PCR/S assays on extracted BALs/CS. The performance of the molecular test was evaluated against standard microbiological methods and clinical review for the presence of IFD. RESULTS: The 107 patients were predominantly male (67, 62.6%) with a mean age of 59 years (range = 0-85 years): 74 (69.2%) patients had at least one underlying comorbidity: 19 (34.5%) had confirmed and 12 (21.8%) had probable IFD. Culture recovered 66 fungal isolates from 55 BALs/CS with Candida spp. and Aspergillus spp. being most common. For BALs, the molecular assay vs. standard methods had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and efficiency of 88.5% vs.100%, 100% vs. 61.1%, 100% vs. 88.5%, 61.1% vs. 100%, and 90.2% for both. For other CS, the molecular assay had similar performance to standard methods with sensitivity, specificity, PPV, NPV and efficiency of 66.7%, 87.0%, 66.7%, 87.0% and 81.3% for both methods. Both methods also performed similarly, regardless of whether CS stain/microscopy showed yeast/fungal elements. FBR-PCR/S assays results were reported in ~ 8 h compared to fungal cultures that took between 4 and 6 weeks. CONCLUSIONS: Rapid molecular testing compared to standard methods have equivalent diagnostic efficiency but improves clinical utility by reporting a rapid species-level identification the same dayshift (~ 8 h).

Role of secretin peptide family and their receptors in the hypothalamic control of energy homeostasis.

Secretin family of peptide hormones is a group of structurally related brain-gut peptides that exert their functions via interactions with the class B1 G protein-coupled receptors (GPCRs). Recent researches of these peptides and receptors in metabolism have been an area of intense focus for the development of promising drug targets as therapeutic potentials for metabolic disorders. The fact that agonists of GLP-1, a member in the family, have already started being used as therapeutics clearly indicates the importance and relevance of further research on the clinical applications of these peptides. This review aims to provide an overview of the current understanding regarding the importance of this family of peptides as well as their receptors in metabolism with special focus on their actions in the hypothalamus.

Prognostication of cardiac CT angiography.

The rapidly emerging technique of cardiac computed tomography angiography (CTA) has enabled the anatomical assessment of coronary artery disease. CTA has very good diagnostic accuracy with the ability to detect nonobstructive from obstructive coronary artery disease and provides information on the presence of coronary artery calcification as well as on left ventricular function. Over the last few years, many prognostic studies have reviewed the outcome benefit of different scoring indices in predicting hard cardiac events. The following article will review the most recent literature available on the use of CTA in measuring luminal stenoses, identifying high-risk obstructive CAD, calcium plaque score, and LV function all in different models with their impact on the estimation of clinical risk. More recent data from a large multicenter registry supports the incremental benefit of CAD severity and LVEF as independent predictors of prognosis. Future directions and emerging applications such as the utility of CTA combined with perfusion analysis may lead to a new anatomical-functional diagnostic test that may provide optimal noninvasive assessment of coronary artery anatomy and be superior to invasive coronary angiography.

[Partially French Canadians are susceptible to increased cardiovascular risk factors: A population-based retrospective cohort study]. / Les Canadiens partiellement Français sont susceptibles à une augmentation des facteurs de risque cardiovasculaire : une étude de cohorte rétrospective basée sur la population.

BACKGROUND: Through various research lead in the past, it has been made evident that Quebec is home to higher rates of acute myocardial infarction (AMI) and higher prevalence of cardiovascular risk factors than other Canadian provinces. This proposed study will perform a retrospective analysis on Caucasian populations in order to analyze the cardiovascular risk factors in partially francophone populations in comparison to French and Non-French Canadians. Furthermore, we will closely analyze both genders of aforementioned populations. METHODS: This population-based retrospective cohort study was achieved using the University of Ottawa Heart Institute CCTA registry. Included are Caucasian patients of all ages who came to UOHI for a CCTA between 2006 and 2018 and provided written informed consent. SPSS was used to compare the different populations (French Canadian, partially French Canadian and non-French Canadian) and sex. RESULTS: The PFC population more closely resembles FC, having higher incidence of cardiovascular risk factors such as smoking, dyslipidemia and type 2 diabetes. INTERPRETATION: Our results suggest that PFC, like FC, may benefit from more intensive education and lifestyle modification techniques.

Latent membrane protein 1 suppresses RASSF1A expression, disrupts microtubule structures and induces chromosomal aberrations in human epithelial cells.

Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC) and can be detected in early premalignant lesions of nasopharyngeal epithelium. The latent membrane protein 1 (LMP1) is an oncoprotein encoded by the EBV and is believed to play a role in transforming premalignant nasopharyngeal epithelial cells into cancer cells. RASSF1A is a tumor-suppressor gene commonly inactivated in many types of human cancer including NPC. In this study, we report a novel function of LMP1, in down-regulating RASSF1A expression in human epithelial cells. Downregulation of RASSF1A expression by LMP1 is dependent on the activation of intracellular signaling of NF-kappaB involving the C-terminal activating regions (CTARs) of LMP1. LMP1 expression also suppresses the transcriptional activity of the RASSF1A core promoter. RASSF1A stabilizes microtubules and regulates mitotic events. Aberrant mitotic spindles and chromosome aberrations are reported phenotypes in RASSF1A inactivated cells. In this study, we observed that LMP1 expression in human epithelial cells could induce aberrant mitotic spindles, disorganized interphase microtubules and aneuploidy. LMP1 expression could also suppress microtubule dynamics as exemplified by tracking movements of the growing tips of microtubules in live cells by transfecting EGFP-tagged EB1 into cells. The aberrant mitotic spindles and interphase microtubule organization induced by LMP1 could be rescued by transfecting RASSF1A expression plasmid into cells. Downregulation of RASSF1A expression by LMP1 may facilitate its role in transformation of premalignant nasopharyngeal epithelial cells into cancer cells.

CCS/CAR/CANM/CNCS/CanSCMR joint position statement on advanced noninvasive cardiac imaging using positron emission tomography, magnetic resonance imaging and multidetector computed tomographic angiography in the diagnosis and evaluation of ischemic heart disease--executive summary.

BACKGROUND: Over the past few decades, advanced imaging modalities with excellent diagnostic capabilities have emerged. The aim of the present position statement was to systematically review existing literature to define Canadian recommendations for their clinical use. METHODS: A systematic literature review to 2005 was conducted for positron emission tomography (PET), multidetector computed tomographic angiography and magnetic resonance imaging (MRI) in ischemic heart disease. Papers that met the criteria were reviewed for accuracy, prognosis data and study quality. Recommendations were presented to primary and secondary panels of experts, and consensus was achieved. RESULTS: Indications for PET include detection of coronary artery disease (CAD) with perfusion imaging, and defining viability using fluorodeoxyglucose to determine left ventricular function recovery and/or prognosis after revascularization (class I). Detection of CAD in patients, vessel segments and grafts using computed tomographic angiography was considered class IIa at the time of the literature review. Dobutamine MRI is class I for CAD detection and, along with late gadolinium enhancement MRI, class I for viability detection to predict left ventricular function recovery. Imaging must be performed at institutions and interpreted by physicians with adequate experience and training. CONCLUSIONS: Cardiac imaging using advanced modalities (PET, multidetector computed tomographic angiography and MRI) is useful for CAD detection, viability definition and, in some cases, prognosis. These modalities complement the more widespread single photon emission computed tomography and echocardiography. Given the rapid evolution of technology, initial guidelines for clinical use will require regular updates. Evaluation of their integration in clinical practice should be ongoing; optimal use will require proper training. A joint effort among specialties is recommended to achieve these goals.

Secretin: a pleiotrophic hormone.

Secretin holds a unique place in the history of endocrinology and gastrointestinal physiology, as it is the first peptide designated as a hormone. During the last century since its first discovery, the hormonal effects of secretin in the gastrointestinal tract were extensively studied, and its principal role in the periphery was found to stimulate exocrine secretion from the pancreas. Recently, a functional role of secretin in the brain has also been substantiated, with evidence suggesting a possible role of secretin in embryonic brain development. Given that secretin and its receptors are widely expressed in multiple tissues, this peptide should therefore exhibit pleiotrophic functions throughout the body. The present article reviews the current knowledge on the central and peripheral effects of secretin as well as its therapeutic uses.

Endogenous release of secretin from the hypothalamus.

Previous studies demonstrated that secretin could be released from the cerebellum, where it exerts a facilitatory action on the GABAergic inputs into the Purkinje neurons. In the present article, we provide evidence of the endogenous release of secretin in the hypothalamus and the mechanisms underlying this release. Incubation of the hypothalamic explants with KCl induces the release of secretin to 4.35 +/- 0.45-fold of the basal level. This K+-induced release was tetrodotoxin and cadmium sensitive, suggesting the involvement of voltage-gated sodium and calcium channels. The use of specific blockers further revealed the involvement of L-, N-, and P-type high voltage-activated (HVA) calcium channels. Results present in the current article provide further and more solid evidence of the role of secretin as a neuropeptide in the mammalian central nervous system.

Pharmacological Actions of Glucagon-Like Peptide-1, Gastric Inhibitory Polypeptide, and Glucagon.

Glucagon family of peptide hormones is a group of structurally related brain-gut peptides that exert their pleiotropic actions through interactions with unique members of class B1 G protein-coupled receptors (GPCRs). They are key regulators of hormonal homeostasis and are important drug targets for metabolic disorders such as type-2 diabetes mellitus (T2DM), obesity, and dysregulations of the nervous systems such as migraine, anxiety, depression, neurodegeneration, psychiatric disorders, and cardiovascular diseases. The current review aims to provide a detailed overview of the current understanding of the pharmacological actions and therapeutic advances of three members within this family including glucagon-like peptide-1 (GLP-1), gastric inhibitory polypeptide (GIP), and glucagon.

Association of biliary glycoprotein with protein tyrosine phosphatase SHP-1 in malignant colon epithelial cells.

Biliary glycoprotein (Bgp) is a member of the immunoglobulin superfamily and the carcinoembryonic antigen family. Previous studies have shown that Bgp functions as an intercellular adhesion molecule and a canalicular bile salt transporter. Moreover, we and others demonstrated that Bgp can inhibit colonic and prostatic tumor cell growth in vivo, through a mechanism which depends on sequences present in its cytoplasmic domain. In this study, we have examined the possibility that the cytoplasmic domain of Bgp can interact with signal transduction molecules. We showed that tyrosine phosphorylated Bgp, expressed in mouse colon carcinoma CT51 cells, could reversibly associate with protein tyrosine phosphatase SHP-1. Mutation of either of two tyrosine residues present in the cytoplasmic domain of Bgp abrogated SHP-1 binding, suggesting that this association was mediated by both tyrosine residues. Similarly, we noted that either of the two SH2 domains of SHP-1 could bind tyrosine phosphorylated Bgp in vitro. It is therefore conceivable that some of the functions of Bgp are mediated through its ability to induce intracellular protein tyrosine dephosphorylation.

Secretin facilitates GABA transmission in the cerebellum.

Secretin was the first hormone discovered in human history, and yet, its function as a neuropeptide has been overlooked in the past. The recent discovery of the potential use of secretin in treating autistic patients, together with the conflicting reports on its effectiveness, urges an in-depth investigation of this issue. We show here that in the rat cerebellar cortex, mRNAs encoding secretin are localized in the Purkinje cells, whereas those of its receptor are found in both Purkinje cells and GABAergic interneurons. Immunoreactivity for secretin is localized in the soma and dendrites of Purkinje cells. In addition, secretin facilitates evoked, spontaneous, and miniature IPSCs recorded from Purkinje cells. We propose that secretin is released from the somatodendritic region of Purkinje cells and serves as a retrograde messenger modulating GABAergic afferent activity.

The nucleotide sequence of rabbit liver transferrin cDNA.

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.

Radiation-induced apoptosis in the adult central nervous system is p53-dependent.

Oligodendrocytes and subependymal cells in the adult CNS have been shown to undergo radiation-induced apoptosis. Here, we examined the role of p53 in radiation-induced apoptosis in the adult mouse CNS. In the spinal cord of p53+/+ mice, apoptotic glial cells were observed within 24 h after irradiation, and the apoptotic response peaked at 8 h. These apoptotic cells demonstrated the immunohistochemical phenotype of oligodendrocytes, and decreased oligodendrocyte density was observed at 24 h after 22 Gy. A similar time course of radiation-induced apoptosis was seen in subependymal cells in the adult mouse brain. Radiation-induced apoptosis was preceded by an increase in nuclear p53 expression in glial cells of the spinal cord and subependymal cells of the brain. There was no evidence of radiation-induced apoptosis in the spinal cord and subependymal region of p53-/- animals. We conclude that the p53 pathway may be a mechanism through which DNA damage induces apoptosis in the adult CNS.

Design and baseline characteristics of the Veterans Affairs Non-Q-Wave Infarction Strategies In-Hospital (VANQWISH) trial. VANQWISH Trial Research Investigators.

OBJECTIVES: The Veterans Affairs Non-Q-Wave Infarction Strategies In-Hospital (VANQWISH) trial was designed to compare outcomes of patients with a non-Q wave myocardial infarction (NQMI) who were randomized prospectively to an early "invasive" strategy versus an early "conservative" strategy. The primary objective was to compare early and late outcomes between the two strategies using a combined trial end point (all-cause mortality or nonfatal infarction) during at least 1 year of follow-up. BACKGROUND: Because of the widely held view that survivors of NQMI are at high risk for subsequent cardiac events, management of these patients has become more aggressive during the last decade. There is a paucity of data from controlled trials to support such an approach, however. METHODS: Appropriate patients with a new NQMI were randomized to an early "invasive" strategy (routine coronary angiography followed by myocardial revascularization, if feasible) versus an early "conservative" strategy (noninvasive, predischarge stress testing with planar thallium scintigraphy and radionuclide ventriculography), where the use of coronary angiography and myocardial revascularization was guided by the development of ischemia (clinical course or results of noninvasive tests, or both). RESULTS: A total of 920 patients were randomized (mean follow-up 23 months, range 12 to 44). The mean patient age was 61 +/- 10 years; 97% were male; 38% had ST segment depression at study entry; 30% had an anterior NQMI; 54% were hypertensive; 26% had diabetes requiring insulin; 43% were current smokers; 43% had a previous acute myocardial infarction; and 45% had antecedent angina within 3 weeks of the index NQMI. CONCLUSIONS: Baseline characteristics were compatible with a moderate to high risk group of patients with an NQMI.

Endogenous release and multiple actions of secretin in the rat cerebellum.

Previous studies demonstrated that secretin could modulate synaptic transmission in the rat cerebellum. In the present report, we provide evidence for the endogenous release of secretin in the cerebellum and further characterize the actions of secretin in this brain area. First, to show that secretin is released endogenously, blocks of freshly dissected cerebella were challenged with a high concentration of KCl. Incubation with KCl almost doubled the rate of secretin release. This KCl-induced release was sensitive to tetrodotoxin and cadmium suggesting the involvement of voltage-gated sodium and calcium channels. The use of specific channel blockers further revealed that L-type and P/Q-type calcium channels underlie both basal and KCl-evoked secretin release. In support of this, depolarization of Purkinje neurons in the presence of NMDA, group II mGluR and cannabinoid CB1 receptor blockers resulted in increased inhibitory postsynaptic current frequency. Second, we found that the previously reported facilitatory action of secretin on GABAergic inputs to Purkinje neurons is partly dependent on the release of endogenous glutamate. In the presence of CNQX, an AMPA/kainate receptor antagonist, the facilitatory effect of secretin on GABA release was significantly reduced. In support of this idea, application of AMPA, but not kainate receptor agonist, facilitated GABA release from inhibitory terminals, an action that was sensitive to AMPA receptor antagonists. These data indicate that a direct and an indirect pathway mediate the action of secretin in the basket cell-Purkinje neuron synapse. The results provide further and more solid evidence for the role of secretin as a neuropeptide in the mammalian CNS.

The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways.

The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

Expression and spatial distribution of secretin and secretin receptor in human cerebellum.

The expression and spatial distribution of secretin and its receptor in human cerebellum were investigated by in situ hybridization and immunohistochemical techniques. Secretin mRNAs are found in Purkinje cells whereas secretin receptor transcripts are present in Purkinje cells and basket cells in the molecular cell layer. In addition, secretin-immunoreactivities are localized in both the soma and dendrites of Purkinje cells. These data are the first demonstration of the spatial distribution of secretin and its receptor in distinct neurons within the human cerebellum. The cellular localizations of this ligand-receptor pair are consistent with the proposed actions of secretin in the cerebellum of rodents and hence suggest that secretin also serves specific neural functions in the human cerebellum.