Evaluation of mericon E. coli O157 Screen Plus and mericon E. coli STEC O-Type Pathogen Detection Assays in Select Foods: Collaborative Study, First Action 2017.05.

QIAGEN mericon Escherichia coli O157 Screen Plus and mericon E. coli Shiga toxin-producing E. coli (STEC) O-Type Pathogen Detection Assays use Real-Time PCR technology for the rapid, accurate detection of E. coli O157 and the "big six" (O26, O45, O103, O111, O121, O145) (non-O157 STEC) in select food types. Using a paired study design, the assays were compared with the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook Chapter 5.09 reference method for the detection of E. coli O157:H7 in raw ground beef. Both mericon assays were evaluated using the manual and an automated DNA extraction method. Thirteen technicians from five laboratories located within the continental United States participated in the collaborative study. Three levels of contamination were evaluated. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference between laboratories POD (dLPOD) value with a 95% confidence interval of 0.00 (-0.12, 0.12) for the mericon E. coli O157 Screen Plus with manual and automated extraction and mericon E. coli STEC O-Type with manual extraction and -0.01 (-0.13, 0.10) for the mericon E. coli STEC O-Type with automated extraction. The dLPOD results indicate equivalence between the candidate methods and the reference method.

A Novel von Hippel Lindau Gene Intronic Variant and Its Reclassification from VUS to Pathogenic: the Impact on a Large Family.

We present a case where a variant of uncertain significance in the von Hippel Lindau syndrome gene (VHL) was identified in a proband with haemangioblastoma, and in a second degree relative with phaeochromocytoma. Initial uncertainty due to the unclear nature of the variant created psychosocial challenges for this family, in which four other genetic conditions were also present. Subsequent RNA studies confirmed this as a novel pathogenic mutation affecting splicing of exon 2. A third relative has since been diagnosed with haemangioblastoma. We suggest that this mutation possibly has reduced penetrance as there was no history of haemangioblastoma, renal tumours (apart from small cysts) or other VHL tumours among five mutation positive and seven untested adult relatives at 50 % risk of the VHL mutation (average age 46 years, range 18-70 years). This case presents a novel VHL splicing mutation and highlights the psychosocial and medical value of additional laboratory studies on uncertain variants for individuals, their families and for the health professionals providing advice and counseling.

An attenuated total reflection (ATR) and Raman spectroscopic investigation into the effects of chloroquine on Plasmodium falciparum-infected red blood cells.

Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Raman spectroscopy were used to compare chloroquine (CQ)-treated and untreated cultured Plasmodium falciparum-infected human red blood cells (iRBCs). The studies were carried out in parallel from the same starting cultures using both spectroscopic techniques, in duplicate. ATR FTIR spectra showed modifications in the heme vibrational bands as well as increases in the CH2/CH3 stretching bands in the 3100-2800 cm(-1) region of CQ-treated iRBCs consistent with an increase in lipid content. Other changes consisted of secondary structural variations including shifts in the amide I and II modes, along with changes in RNA and carbohydrate bands. Raman microspectroscopy of single red blood cells using 532 nm revealed subtle changes in the positions and intensity of ν37 of the core size region marker band and ν4 in the pyrrole ring-stretching region between untreated and CQ-treated iRBCs. Similar patterns in the corresponding relations were also observed in the non-fundamental (overtone region) between the control and treated cells. These differences were consistent with higher levels of oxygenated hemoglobin (oxyHb) in the treated cells as shown in a Principle Component Analysis (PCA) loadings plot. The results obtained demonstrate that vibrational spectroscopic techniques can provide insight into the effect of quinolines on iRBCs and thus may assist understanding the sensitivity and resistance of new and existing anti-malarial drugs.

IFPA meeting 2014 workshop report: Animal models to study pregnancy pathologies; new approaches to study human placental exposure to xenobiotics; biomarkers of pregnancy pathologies; placental genetics and epigenetics; the placenta and stillbirth and fetal growth restriction.

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2014 there were six themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of animal models, xenobiotics, pathological biomarkers, genetics and epigenetics, and stillbirth and fetal growth restriction.

Predictive genetic testing of a bone marrow recipient-ethical issues involving unexpected results, gender issues, test accuracy, and implications for the donor.

We present a case where an apparently straightforward Lynch syndrome predictive genetic test of DNA from a blood sample from a woman yielded an unexpected result of X/Y chromosome imbalance. Furthermore, it demonstrates the complexities of genetic testing in people who have had bone marrow transplants. This highlights the potential for multiple ethical and counselling challenges, including the inadvertent testing of the donor. Good communication between clinics and laboratories is essential to overcome such challenges and to minimise the provision of false results.

A model for peer experiential and reciprocal supervision (PEERS) for genetic counselors: development and preliminary evaluation within clinical practice.

A model for practising genetic counselors to obtain clinical supervision via reciprocal peer observation and feedback was developed and trialled. The model was developed in response to a perceived lack of opportunity for immediate observational feedback for practising genetic counselors. The aims reached by consensus were to facilitate learning new approaches and skills, to revitalise current ways of practising, and to enhance supervision skills in a two-way process, where the observer learnt from the counselor, and vice-versa. The genetic counselors agreed on a process of paired reciprocal observation whereby the observer was present in the room during the counseling session, and a reflective feedback discussion was arranged within 24 h of the session. Four main themes emerged from analysis of the recorded discussions were (i) "I wasn't sure if I-": voicing of doubts or internal questions that occurred during session for the counselor conducting the session, (ii) "I really liked that": positive feedback and validation from the observer, (iii) "I wonder whether-": offering of alternative views, insights and strategies by the observer, and (iv) "That's a real thing for me to take away and think about": evidence of learning by both observers and counselors.

Impact of a genetic diagnosis of a mitochondrial disorder 5-17 years after the death of an affected child.

This study used in-depth interviews to explore the experiences of parents who were re-contacted with new genetic results many years after the death of a child with a mitochondrial disorder. At the time of their child's illness, parents had consented to a tissue sample being taken to help with diagnosis of a suspected mitochondrial disorder, and subsequently further DNA testing identified the genetic cause. Parents did not express negative feelings about being re-contacted with new information, and hoped that continuing research might help other families. Positive aspects included relief from feelings of guilt over the cause of the child's disorder, and having accurate genetic information available for surviving children. Difficult emotional and psychosocial implications included contradictions to previous beliefs about inheritance, deciding how and when to communicate information to surviving children, and coping with new fears for the mother's health if a gene located in the mitochondrial DNA was identified. In half of the families the new results significantly altered the parents' understanding of the inheritance pattern. This study highlights the impact of new genetic information offered after a delay of several years, which has the potential to re-open feelings of grief and uncertainty and can present a new inheritance scenario for which research participants or their families are unprepared. Health professionals involved in conveying genetic research results can help to support families through this process.

Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans.

A gene encoding a cyanide hydratase was cloned from an aggressive isolate of Leptosphaeria maculans, the fungus which causes blackleg disease of oilseed Brassica spp. This enzyme catalyses the breakdown of hydrogen cyanide to a less toxic compound, formamide. The predicted amino acid sequence of cyanide hydratase in L. maculans is 77% and 82% identical to cyanide hydratases from two other ascomycetes, Gloeocercospora sorghi and Fusarium lateritium, respectively. The gene is present as a single copy in the L. maculans genome, in both aggressive and non-aggressive isolates, although there is a restriction fragment length polymorphism between these two isolate groups for this gene. The cyanide hydratase promoter contains four putative target sites for GATA transcription factors, proteins that regulate nitrogen metabolism and other processes. Transcription of cyanide hydratase in an aggressive L. maculans isolate is induced strongly by potassium cyanide. Transcription of the gene is detectable in cotyledons of Brassica juncea and B. napus during infection. L. maculans can utilise the reaction product, formamide, as a sole source of nitrogen.

Cloning, characterization and chromosomal location of three genes encoding host-cell-wall-degrading enzymes in Leptosphaeria maculans, a fungal pathogen of Brassica spp.

The ascomycete, Leptosphaeria maculans, causes blackleg disease of oilseed Brassica spp. such as canola (Brassica napus). We have cloned a gene encoding endopolygalacturonase, pg1, and two genes encoding cellulases, cel1 and cel2, in L. maculans. These genes are not clustered in the genome, as they are located on different chromosomes. The deduced amino acid sequences of all three genes predict an N-terminal signal sequence, as is common for secreted fungal enzymes that degrade plant cell walls. The endopolygalacturonase encoded by pg1 shows the highest similarity (54% amino acid identity) to endopolygalacturonase 4 from Botrytis cinerea. Both cel1 and cel2 appear to encode cellobiohydrolase, and neither gene encodes a recognizable cellulose-binding domain or linker region. Transcription of pg1 is induced in cultures containing 1% polygalacturonic acid or pectin, and cel1 is induced in 1% cellulose or carboxymethylcellulose, as shown by Northern analysis. Glucose represses the induction of cel1 caused by cellulose and carboxymethylcellulose, but does affect transcription of pg1. Transcription of cel2 (but not cel1 or pg1) is detectable during infection of B. napus and B. juncea cotyledons and leaves using reverse transcription-PCR.

Pharmacological and histochemical evidence for P2X receptors in human umbilical vessels.

The presence of P2X purinoceptors in human umbilical vessels were studied with organ bath recording, radioligand binding assays, autoradiography, and immunohistochemistry. In isolated umbilical arteries and veins from normal term pregnancy, both ATP and alpha,beta-methylene ATP caused concentration-dependent contractions. ATP-induced responses were blocked by desensitisation with alpha,betamethylene ATP. However, both the ATP- and alpha,beta-methylene ATP-induced responses were not antagonised by suramin. No significant difference in responses was observed in the vessels with or without endothelial cells. Radioligand binding assays using [3H]alpha,beta-methylene ATP showed the presence of a population of high-affinity binding sites in both the arteries and veins. The Kd values of the binding sites were 2.77 + 1.10 nM for the arteries, and 3.23+/-1.22 nM for the veins. The maximum binding site densities were 634+/-237 and 947+/-308 fmol/mg protein for the arteries and the veins, respectively. Autoradiographic localisation with [3H]alpha,beta-methylene ATP demonstrated that the specific binding sites were only distributed over the smooth muscle cells of the vessels. Immunohistochemical studies with specific polyclonal antibodies against P2X1-6 receptors showed that positive immunostaining was also restricted to smooth muscle cells. Antibodies against P2X1 receptors produced the strongest signals, while antibodies against the other five P2X subtypes produced much weaker signals. The results in the present study indicate the existence of P2X purinoceptors in the smooth muscle of human umbilical vessels. Their physiological functions remain to be studied.

Electron-microscopic immunolabelling of vasoactive substances in human umbilical endothelial cells and their actions in early and late pregnancy.

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of feto-placental blood flow. The pharmacological effects of 5-hydroxytryptamine, histamine and endothelin were examined in umbilical arteries and veins from legal terminations (gestational age 8-17 weeks, n=12) and normal term vaginal deliveries (gestational age 38-41, n=12). Immunocytochemistry of human unbilical vessels indicated that 5-hydroxytryptamine, histamine and endothelin were localised in subpopulations of endothelial cells of both artery and vein in late, but not early, pregnancy. 5-Hydroxytryptamine (10 nM-30 microM) caused sustained concentration-dependent contractions in all vessels from early and late pregnancy. Histamine (0.1 microM-30 mM) also caused sustained contractions in all vessels from late pregnancy but only 27% of arteries and 41% of veins from early pregnancy responded. Endothelin (10 pM-30 nM) caused slow long-lasting contractions in all vessels from early and late pregnancy. Atrial natriuretic peptide and neuropeptide Y did not alter vascular tone. The endothelium may thus play an autocrine/paracrine role, by synthesizing and releasing the above reactive substances in late pregnancy to influence feto-placental blood flow.

A study of the ultrastructure of developing human umbilical vessels.

Electron microscopic techniques were used to examine the ultrastructure of developing human umbilical arteries and vein (8-12, 13-17 and 37-40 wk gestational age). These showed that with increasing age there is (1) an increase in the size of the lumen and the thickness of the media; (2) an increase in the ratio of contractile smooth muscle phenotypic cells; (3) an increase in the myofilament content of the smooth muscle cells and the number of Weibel-Palade bodies; (4) a decrease in the glycogen content; (5) an appearance of microvilli on the luminal surface of the endothelium. Lipid vesicles, nerves and vasa vasorum were not observed in any region of the umbilical vein or arteries.

Nitric oxide and human umbilical vessels: pharmacological and immunohistochemical studies.

Human umbilical vessels are devoid of nerves and therefore endothelial cells may play an important role in the control of fetoplacental blood flow. In this study we examined the pharmacological effects of various substances, known to produce endothelial-mediated vasodilation in many blood vessels, on the human umbilical artery and vein from legal terminations [mean gestational age, 15 (8-17) weeks; n = 12] and normal term vaginal deliveries [mean gestational age, 39 (38-41) weeks; n = 12]. Acetylcholine, adenosine 5'-triphosphate, the calcium ionophore A23187 and substance P had no effect on raised vascular tone, whereas sodium nitroprusside relaxed 5-hydroxytryptamine (5-HT) preconstricted, umbilical artery and vein from both early and late pregnancy. L-NG-Nitroarginine methyl ester (L-NAME) had no effect on basal tone or on high tone, after it was raised by 5-HT. Localization of nitric oxide synthase [NOS, type I (neuronal)] was examined in the same umbilical vessels using electron immunocytochemistry. No NOS-immunoreactive endothelial cells were observed in the umbilical vessels taken during early pregnancy. However, the percentage of NOS-immunoreactive endothelial cells in umbilical artery and vein from late pregnancy was 3 and 10 per cent, respectively. These results suggest that nitric oxide contributes little, if any, to the local control of umbilical blood flow throughout pregnancy, despite the presence of NOS-immunoreactivity in a subpopulation of endothelial cells in late pregnancy.

Endothelium of human umbilical blood vessels: ultrastructural immunolocalization of neuropeptides.

Endothelial cells of human umbilical vein and artery, both in situ and in culture, were examined ultrastructurally and at the light-microscope level using either the pre-embedding peroxidase-antiperoxidase or avidin-biotin peroxidase complex immunostaining techniques. Vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and arginine-vasopressin (AVP) immunoreactivity were localized in subpopulations of endothelial cells of the term umbilical vein and artery. The percentage of VIP-, SP-, CGRP- and AVP-immunoreactive cells in the umbilical vein was 12, 10, 11, and 7.5%, respectively, out of a total of 5,364 cells (from 15 umbilical cords) examined. The artery contained fewer VIP-, SP-, and CGRP-immunoreactive cells, but more AVP-immunoreactive cells, than the vein. In conclusion, subpopulations of endothelial cells in the human umbilical vein and artery contain the neuropeptides VIP, SP, CGRP and AVP, although their physiological roles are not yet known.

Localization of neuropeptide Y and atrial natriuretic peptide in the endothelial cells of human umbilical blood vessels.

The localization of neuropeptide Y (NPY) and atrial natriuretic peptide (ANP) in the endothelial cells of human umbilical blood vessels was studied using the pre-embedding peroxidase-antiperoxidase (PAP) technique for electron microscopy and avidin-biotin-complex (ABC) immunostaining for endothelial cells cultured from umbilical vein. Subpopulations of NPY- and ANP-immunoreactive endothelial cells were present in term umbilical vein and artery. The umbilical vein contained more positive cells than the artery. The percentage of NPY- and ANP-immunoreactive umbilical vein cells in culture was 32% and 44%, respectively, out of a total of 3013 cells examined. The possibility that these potent vasoactive substances located in the endothelial cells of the non-innervated umbilical vessels are involved in the local regulation of blood flow is discussed.