On the track for an efficient detection of Escherichia coli in water : A review on PCR-based methods

Ensuring water safety is an ongoing challenge to public health providers. Assessing the presence of fecal contamination indicators in water is essential to protect public health from diseases caused by waterborne pathogens. For this purpose, the bacteria Escherichia coli has been used as the most reliable indicator of fecal contamination in water. The methods currently in use for monitoring the microbiological safety of water are based on culturing the microorganisms. However, these methods are not the desirable solution to prevent outbreaks as they provide the results with a considerable delay, lacking on specificity and sensitivity. Moreover, viable but non-culturable microorganisms, which may be present as a result of environmental stress or water treatment processes, are not detected by culture-based methods and, thus, may result in false-negative assessments of E. coli in water samples. These limitations may place public health at significant risk, leading to substantial monetary losses in health care and, additionally, in costs related with a reduced productivity in the area affected by the outbreak, and in costs supported by the water quality control departments involved. Molecular methods, particularly polymerase chain reaction-based methods, have been studied as an alternative technology to overcome the current limitations, as they offer the possibility to reduce the assay time, to improve the detection sensitivity and specificity, and to identify multiple targets and pathogens, including new or emerging strains. The variety of techniques and applications available for PCR-based methods has increased considerably and the costs involved have been substantially reduced, which together have contributed to the potential standardization of these techniques. However, they still require further refinement in order to be standardized and applied to the variety of environmental waters and their specific characteristics. The PCR-based methods under development for monitoring the presence of E. coli in water are here discussed. Special emphasis is given to methodologies that avoid pre-enrichment during the water sample preparation process so that the assay time is reduced and the required legislated sensitivity is achieved. The advantages and limitations of these methods are also reviewed, contributing to a more comprehensive overview toward a more conscious research in identifying E. coli in water.Diana Mendes (SFRH/BDE/33752/2009) was recipient of a fellowship from the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal) and Frilabo, Lda. The authors thank Tatiana Aguiar (Centre of Biological Engineering) for English proofreading, the financial support from the Project "Desenvolvimento de um kit de detecao e quantificacao de E. coli e bacterias coliformes em aguas", Ref. 2009/5787, Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER, the FCT Strategic Project PEst-OE/EQB/LA0023/2013 and the Project "Biolnd-Biotechnology and Bioengineering for improved Industrial and processes", REF. NORTE-07-0124-FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER

VE-statin/egfl7 Expression in Endothelial Cells Is Regulated by a Distal Enhancer and a Proximal Promoter under the Direct Control of Erg and GATA-2

Angiogenesis is the process by which new blood vessels arise from existing ones by the budding out of endothelial cell capillaries from the luminal side of blood vessels. Blood vessel formation is essential for organ development during embryogenesis and is associated with several physiological and pathological processes, such as wound healing and tumor development. The VE-statin/egfl7 gene is specifically expressed in endothelial cells during embryonic development and in the adult. We studied here the regulatory mechanisms that control this tissue-specific expression. RT-qPCR analyses showed that the specificity of expression of VE-statin/egfl7 in endothelial cells is not shared with its closest neighbor genes notch1 and agpat2 on the mouse chromosome 2. Chromatin-immunoprecipitation analysis of histone modifications at the VE-statin/egfl7 locus showed that the chromatin is specifically opened in endothelial cells, but not in fibroblasts at the transcription start sites. A 13 kb genomic fragment of promoter was cloned and analyzed by gene reporter assays which showed that two conserved regions are important for the specific expression of VE-statin/egfl7 in endothelial cells; a −8409/−7563 enhancer and the −252/+38 region encompassing the exon-1b transcription start site. The latter contains essential GATA and ETS-binding sites, as assessed by linker-scanning analysis and site-directed mutagenesis. An analysis of expression of the ETS and GATA transcription factors showed that Erg, Fli-1 and GATA-2 are the most highly expressed factors in endothelial cells. Erg and GATA-2 directly control the expression of the endogenous VE-statin/egfl7 while Fli-1 probably exerts an indirect control, as assessed by RNA interference and chromatin immunoprecipitation. This first detailed analysis of the mechanisms that govern the expression of the VE-statin/egfl7 gene in endothelial cells pinpoints the specific importance of ETS and GATA factors in the specific regulation of genes in this cell lineage

Etude du rôle et de l'expression de la VE-statine (egfl7)

Notre équipe a récemment découvert le gène VE-statine (ou egfl7) et montré qu'il s'exprime spécifiquement dans les cellules endothéliales des vaisseaux sanguins embryonnaires. Ce gène code pour une protéine sécrétée de 30 kDa : la VE-statine ou Vascular Endothelial-statin', qui a peu d'homologie de structure avec d'autres protéines connues, excepté deux domaines Epidermal Growth Factor' (EGF) et un domaine Emilin (EMI). In vitro, la VE-statine est capable d'inhiber la migration des cellules musculaires lisses induite par le PDGF-BB. In vivo, la diminution d'expression d'egfl7 chez le poisson zèbre (danio rerio) provoque des anomalies vasculaires et perturbe la tubulogenèse, avec pour conséquence l'absence de circulation sanguine. Le rôle physiologique de la protéine et ses mécanismes d'action sont encore très peu connus. Afin d'évaluer le rôle de la VE-statine chez l'adulte, nous avons étudié son patron d'expression dans diverses situations normales ou pathologiques, par hybridation in situ et RT-PCR quantitative en temsp réel. L'expression de ce gène est ainsi retrouvée dans tous les tissus adultes analysés et, comme chez l'embryon, exclusivement au niveau des cellules endothéliales. Nous avons mis en évidence une répression de l'expression du gène par les cytokines pro-inflammatoires IL1- , IL1- et TNF- dans les cellules endothéliales. Cet effet implique le facteur NF- B et fait intervenir des phénomènes épigénétiques de régulation de l'acétylation des histones. Nous avons d'autre part cloné l'ADNc de la VE-statine dans un vecteur d'expression et mis au point la production et la purification de la protéine recombinante afin d'étudier la réponse des cellules musculaires lisses aortiques à la stimulation par la VE-statine. Enfin, la recherche de gènes cibles régulés par la VE-statine dans les cellules musculaires lisses stimulées par le PDGF-BB est en cours d'une approche de puces à ADN.VE-statine (or egfl7) is a novel gene recently discovered by our team which is specifically expressed by endothelial cells of embryonic blood vessels. The gene codes for a 30kDa secreted protein, VE-statin (Vascular Endothelial-statin), which shares no clear structural homology with other known proteins, except for two Epidermal Growth Factor (EGF) domains and an Emilin (EMI) domain. In vitro, VE-statin inhibits PDGF-BB-induced smooth muscle cell migration. In vivo, egfl7 knockdown in the zebrafishinduces major vascular defects, with a defective tubulogenesis and a lack of blood flow. Despite these results, the physiological role of the protein and its mechanisms of action remain largely unknown. We have studied the VE-statin expression pattern in various normal and pathological situations in adults using in situ hybridization and real-time quantitative RT-PCR. We have shown that VE-statin is expressed in all tissues analysed, and like in the embryo, exclusively in endothelial cells. Moreover, expression of the gene is down-regulated by pro-inflammatory cytokines IL1- , IL1- and TNF- . The NF- B nuclear factor is involved in this effect, as well as epigenetic machanisms, especially those involving the regulation of histone acetylation. In addition, we have cloned the VE-statin cDNA in an expression vector and designed a protocol for the production and purification of the mouse recombinant protein, with the aim of studying aortic smooth muscle cell response to VE-statin. Finally, we have initiated a micro-array analysis in order to identify VE-statin regulated genes in smooth muscle cells.LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Behaviour of a birch plywood under various experimental conditions

Liquid Natural Gaz -LNG- carriers must provide cryogenic reliability and safety for at least 40 years. Most of the insulation systems belong to the so-called membrane type, in which the “insulation complex” is composed of a metal membrane as a barrier, a plywood panel, insulation foam and a plywood panel glued to the inner hull. During construction and exploitation at cryogenic temperature, an impact of the membrane may occur and could lead to a LNG leakage. In order to evaluate this risk, and the role of the plywood in the perforation process of the membrane, a series of original impact tests are carried out at room and cryogenic temperatures on metal-plywood samples thanks to a specially designed catapult. In order to obtain a reliable numerical simulation of the impact, a series of impact and out of plane compression tests at room and cryogenic temperatures are carried out. Thanks to a design experiment, we identified the influence of the moisture content, from 2% up to 30%, and of the temperature, from − 173 ∘C up to +20 ∘C, on the strain rate sensitivity of the plywood

Iodine deficiency induces a thyroid stimulating hormone-independent early phase of microvascular reshaping in the thyroid

Expansion of the thyroid microvasculature is the earliest event during goiter formation, always occurring before thyrocyte proliferation; however, the precise mechanisms governing this physiological angiogenesis are not well understood. Using reverse transcriptase-polymerase chain reaction and immunohistochemistry to measure gene expression and laser Doppler to measure blood flow in an animal model of goitrogenesis, we show that thyroid angiogenesis occurred into two successive phases. The first phase lasted a week and involved vascular activation; this process was thyroid-stimulating hormone (TSH)-independent and was directly triggered by expression of vascular endothelial growth factor (VEGF) by thyrocytes as soon as the intracellular iodine content decreased. This early reaction was followed by an increase in thyroid blood flow and endothelial cell proliferation, both of which were mediated by VEGF and inhibited by VEGF-blocking antibodies. The second, angiogenic, phase was TSH-dependent and was activated as TSH levels increased. This phase involved substantial up-regulation of the major proangiogenic factors VEGF-A, fibroblast growth factor-2, angiopoietin 1, and NG2 as well as their receptors Flk-1/VEGFR2, Flt-1/VEGFR1, and Tie-2. In conclusion, goiter-associated angiogenesis promotes thyroid adaptation to iodine deficiency Specifically, as soon as the iodine supply is limited, thyrocytes produce proangiogenic signals that elicit early ISH-independent microvascular activation; if iodine deficiency persists, TSH plasma levels increase, triggering the second angtogenic phase dim supports thy rocyte proliferation

Endothelial Cell Activation Is Regulated by Epidermal Growth Factor-like Domain 7 (Egfl7) during Inflammation

International audienceActivation of the blood vessel endothelium is a critical step during inflammation. Endothelial cells stimulated by pro-inflammatory cytokines play an essential part in the adhesion and extravasation of circulating leukocytes into inflamed tissues. The endothelial egfl7 gene (VE-statin) represses endothelial cell activation in tumors, and prior observations suggested that it could also participate in the regulation of endothelial cell activation during inflammation. We show here that Egfl7 expression is strongly repressed in mouse lung endothelial cells during LPS- and TNFα-induced inflammation in vivo LPS have a limited effect on Egfl7 expression by endothelial cells in vitro, whereas the pro-inflammatory cytokine TNFα strongly represses Egfl7 expression in endothelial cells. TNFα regulates the egfl7 gene promoter through regions located between -7585 and -5550 bp ahead of the main transcription start site and via an NF-κB-dependent mechanism. Conversely, Egfl7 regulates the response of endothelial cells to TNFα by restraining the induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, resulting in a decreased adhesion of leukocytes onto endothelial cells stimulated by TNFα. Egfl7 regulates the expression of these adhesion molecules through the NF-κB and MEK/Erk pathways, in particular by preventing the proteasome-mediated degradation of IkBα both in non-activated endothelial cells and during activation. Egfl7 is thus an endogenous and constitutive repressor of blood vessel endothelial cell activation in normal and inflammatory conditions and participates in a loop of regulation of activation of these cells by pro-inflammatory cytokines

Egfl7 Represses the Vasculogenic Potential of Human Endothelial Progenitor Cells

International audienc

The experience of adolescents and parents before, after, and in the process of redirection into the Adapted program of education with lower educational standard

This diploma paper studies the experience of adolescents and their parents where the adolescents were redirected from the regular school program into the Adapted program of education with lower educational standard. Individuals with mild intellectual disability are typically characterised by reduced learning abilities, but in the adapted learning environment they are able to achieve basic school aims. Due to extensive learning difficulties such students are unable to follow the regular school program, which is why they are schooled, or are later redirected into the Adapted program of education with lower educational standard. If the students are redirected later, learning difficulties and failure in the field of education increase, which can cause behavioural and emotional problems, as well as have an important role on students' self-respect. Due to constant failure the students may develop fear of school, and consequently dislike of learning or of school in general. Emotional tension and distress related to school can cause psychosomatic problems or disorders in students. But learning failure extends beyond the scope of student's perception of school alone. It also affects how students spend their free time and their social relations in the broader and narrow environment. This research studies four students, who were redirected into the Adapted program of education with lower educational standard. It is a case study which provides an insight into the experience of parents and their children in the process of redirection into the above mentioned program. To obtain the research data we used the half-structured interview. The interviews were recorded and transcribed. The transcript was then summarized and paraphrased and then analysed in accordance with elements of qualitative text analysis. The research shows that the students felt unsuccessful in the regular school, having difficulties in their relations with peers and teachers, as well as spending a lot of time for their school work. Their parents’ observations were similar, reporting about various behavioural and emotional problems of their children. After the redirection both the students and their parents noted that they had less learning difficulties, the students' well-being improved along with their attitude to school and relations with teachers and students. This research can help understand the problems of students with learning difficulties that are, despite effort and assistance, unable to achieve minimal learning standards. The experience of students and parents is a source of information to those parents and teachers who are dealing with the process of redirection into the Adapted program of education with lower educational standard