Development and assessment of herpes simplex virus type 1 (HSV-1) amplicon vectors with sensory neuron-selective promoters

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters’ candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for ND

Ring-Like Distribution of Constitutive Heterochromatin in Bovine Senescent Cells

Background: Cells that reach ‘‘Hayflick limit’ ’ of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. Methods: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Results: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli

Воздействие высокой концентрации оксида азота на оксигенаторы аппаратов искусственного кровообращения (экспериментальное исследование)

The aim of the study. To study the effect of high nitric oxide concentrations on hollow polypropylene fibers of oxygenators.Materials and methods. The study was conducted in two stages. At the first stage, we evaluated the stability of oxygenator membrane made of hollow polypropylene fibers after six hours of exposure to air-oxygen mixture containing NO at 500 parts per million, or 500 pro pro mille (ppm) concentration, using mass spectrometry and infrared spectroscopy. At the second stage, an experiment with cardiopulmonary bypass (CPB) was conducted on 10 pigs. In the study group (n=5) animals sweep gas was supplied to the oxygenator as an air-oxygen mixture with NO at 100 ppm. In the control group animals (n=5) an air-oxygen mixture was used without NO. The CPB lasted for 4 hours, followed by observation for 12 hours. NO, NO2 (at the inlet and outlet of the oxygenator), and the dynamics of methemoglobin were evaluated. After weaning of animals from CPB, the oxygenators were tested for leakproofness, and scanning electron microscopy (SEM) was performed.Results. The oxygenator made of polypropylene hollow fibers retained its gas transfer parameters after six hours of exposure to air-oxygen mixture containing NO at 500 ppm. Based on IR-Fourier spectroscopy findings, NO did not affect structural integrity of polypropylene membranes. NO added to gas mixture at 100 ppm did not increase NO2 to toxic level of 2 ppm in 91% of control tests during 4 hours CPB in pigs; mean value was 1.58 ± 0.28 ppm. Methemoglobin concentration did not exceed the upper limit of permissible level (3%), and there were no statistically significant differences with the control group. All tested oxygenators have passed the leakproofness test. According to SEM findings, larger amounts of fibrin deposits were found in the control group oxygenators vs study group.Conclusion. There were no negative effects of NO at 500 ppm concentration on the oxygenator membrane made of hollow polypropylene fibers. NO at 100 ppm in a gas-mixture supplied to oxygenators did not lead to an exceedance of safe NO2 and methemoglobin concentrations in an animal model. Reduced fibrin deposits on hollow fibers of polypropylene oxygenator membranes were observed when with NO at a level of 100 ppm was added to a gas mixture.  Цель исследования. Изучить воздействие высоких концентраций оксида азота на полипропиленовые полые волокна оксигенаторов.Материалы и методы. Исследование провели в два этапа. На первом этапе с помощью масс-спектрометрии и инфракрасной спектроскопии выполнили оценку стабильности мембраны оксигенатора из полых волокон полипропилена после шестичасового воздействия воздушно-кислородной смеси, содержащей NO в концентрации 500 пропромилле, или 500 частей на миллион – parts per million (ppm). На втором этапе провели эксперимент на 10 свиньях с подключением аппарата искусственного кровообращения (ИК). Животным основной группы (n=5) в оксигенатор подавали воздушно-кислородную смесь, содержащую NO в концентрации 100 ppm. Животным контрольной группы (n=5) в оксигенатор подавали воздушно-кислородную смесь без NO. Процедура ИК длилась 4 часа, затем следовало наблюдение в течение 12 часов. Оценивали NO, NO2 (на входе и выходе из оксигенатора), динамику метгемоглобина. После отключения от ИК оксигенаторы тестировали на герметичность, а также выполняли сканирующую электронную микроскопию (СЭМ).Результаты. Оксигенатор из полипропиленовых полых волокон сохранял свои газотранспортные характеристики после шестичасового воздействия воздушно-кислородной смеси с добавлением NO в концентрации 500 ppm. По данным ИК-Фурье спектроскопии показали, что NO не влияет на структуру мембран из полипропилена. Добавление NO в дозировке 100 ppm во время 4 часов ИК у свиней не сопровождалось повышением концентрации NO2 до токсичного уровня 2 ppm в 91% измерений: среднее значение составило 1,58 ± 0,28 ppm. Концентрация метгемоглобина не превышала верхнего  предела  допустимых  значений  (3%),  не  обнаружили  каких-либо статистически значимых различий при сравнении с группой контроля. Все исследуемые оксигенаторы выдержали тестирование на герметичность. По результатам СЭМ оксигенаторы группы контроля характеризовались большим количеством отложений фибрина, чем оксигенаторы основной группы.Заключение. Негативного воздействия NO в концентрации 500 ppm на мембраны оксигенаторов из полых волокон полипропилена не обнаружили. Подача в оксигенатор NO в концентрации 100 ppm NO2 не приводила к превышению безопасного содержания NO2 и метгемоглобина в эксперименте на животных. Выявили снижение образования отложений фибрина на полых волокнах мембран оксигенаторов из полипропилена при подаче NO в концентрации 100 ppm

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

L'architecture nucléaire chez les embryons bovins produits par fécondation in vitro et clonage (la dynamique de l' hétérochromatine)

Developmental reprogramming during mammalian fertilization and pre-implantation development in in vitro fertilized (IVF) embryos is a complex process that allows the highly differentiated gametes to revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in chromatin structure, in global epigenetic modifications and in nuclear architecture of embryonic nuclei. The objective of the present study was to develop approaches for understanding these series of phenomena in early embryos. We used the possibility to compare normal (IVF) and nuclear tranfer (NT) embryos in bovine species where the pre-implantation development is long enough to assess the nuclear dynamics of reprogramming events before major embryonic genome activation. In a first approach, we studied the establishment of heterochromatin pattern specific for embryo during the reprogramming period in IVF and NT bovine embryos. We applied two markers of pericentric heterochromatin: heterochromatin protein 1 (HP1beta) and tri methylated histone H3 (H3K9me3); and a marker of centromeres (CENPA/B), in order to characterize structural parameters of constitutive heterochromatin in early stages of development. Using immunofluorescence technique, we were able to observe dynamic changes in heterochromatin organization in connection with embryos origin. In IVF and some NT embryos, heterochromatin was observed in dispersed state up to the 8-cell stage, and then in a condensed pattern corresponding to the well characterized chromocenters constituted by blocks of HP1beta and H3K9me3 associated with centromeres. However, a significant part of NT embryos underwent an altered dynamics of heterochromatinization characterized by a precocious heterochromatin condensation as soon as the 2-cell stage. In a second approach, we used senescent somatic cells as donors for nuclear transfer experiments in order to assess the influence of structural organization of donor cell nucleus on structural organization of nuclei in cloned embryos. Surprisingly, the large-scale three-dimensional arrangement of heterochromatin within the senescent-NT nucleus recapitulated the dynamics observed in IVF or somatic non-senescent NT embryos, with comparable percentages of embryos with dispersed and precociously condensed heterochromatin similar to those observed in the previous investigation. These results suggest that a robust process of epigenetic reprogramming is piloted by bovine oocyte in early embryos. The altered kinetics in genome restructuring in some NT embryos might be linked to the precocious transcriptional activation and precocious increase of DNA methylation level reported in other studies. The present work allowed us to point out the distinction between NT embryos with correct developmental reprogramming and abnormal, at least temporally, epigenetic reprogramming, as well as to demonstrate that the impact of nuclear organization of heterochromatin in the donor cell on structural organization of nucleus in NT embryos is relatively low. This may account for the relatively high development efficiency in bovine cloning as compared to other species.La reprogrammation au cours de la fécondation et du développement pré-implantatoire de l embryon fécondé in vitro (IVF) chez les mammifères est un processus complexe qui permet le retour à un état indifférencié des cellules hautement spécialisées que sont les gamètes lors de la syngamie, puis leur différenciation progressive en différent lignages cellulaires dans l embryon. Ces processus comprennent des changements dans la structure de la chromatine, dans les modifications épigénétiques et dans l architecture nucléaire des noyaux embryonnaires. L objectif de cette étude était de mettre en place des approches pour la compréhension de ces processus dans les embryons précoces. Nous avons tiré parti de la comparaison entre embryons normaux (IVF) et issus de transfert de noyau (NT) dans l espèce bovine où le développement pré-implantatoire s étant sur une période suffisamment longue pour caractériser la dynamique des événements de reprogrammation nucléaire avant la mise en route de l activation majeure du génome embryonnaire. Dans une première approche, nous avons étudié la mise en place de l hétérochromatine spécifique à l embryon au cours de la période de reprogrammation dans les embryons bovins issus de fécondation et de NT. Nous avons utilisé deux marqueurs de l hétérochromatine péricentrique : la protéine hétérochromatine 1 (HP1-beta) et l histone H3 tri-méthylée (H3K9me3) ; et un marqueur des centromères (CENP A/B), de façon à caractériser les paramètres structuraux de l hétérochromatine constitutive dans les premiers stades de développement embryonnaire. En utilisant des techniques d immunofluorescence, nous avons pu observer les changements dynamiques dans l organisation de l hétrochromatine en relation avec l origine des embryons. Dans les embryons IVF et une partie des embryons NT, l hétérochromatine apparaît dans un état dispersé, et ceci jusqu au stade 8-cellule ; puis sous une forme condensée qui correspond à l association bien caractéristique de blocs d hétérochromatine et d H3K9me3 avec les centromères, connues sous le nom de chromocentres. Cependant, une partie significative des embryons NT montrent une altération de la dynamique d hétérochromatinisation révélée par une condensation précoce, dès le stade 2-cellule, de HP1 et H3K9-me3. Dans une seconde approche, nous avons utilisé des cellules somatiques au stade de la sénescence comm cellules donneuses de noyau de façon à tester l influence de l organisation structurale du noyau donneur sur l organisation structurale des noyaux dans les embryons clonés. Les arrangements tridimensionnels à large échelle de l hétérochromatine dans les noyaux sénescents NT récapitulent la dynamique observée dans les embryons IVF ou NT issus de noyau de cellules non sénescentes. Notamment, les pourcentages d embryons avec une hétérochromatine dispersée ou condensée prématurément sont comparables avec les études précédentes.Ces résultats suggèrent l existence d un processus de reprogrammation épigénétique robuste piloté par l ovocyte dans les embryons précoces de bovins. Les cinétiques de restructuration du génome altérées dans une partie des embryons NT pourraient être liées à l activation précoce de la transcription et l augmentation du taux de méthylation qui ont été rapportées dans d autres études. Le présent travail nous a permis de mettre en évidence une distinction entre des embryons NT avec une reprogrammation normale et des embryons NT avec une anomalie, au moins temporelle, de la reprogrammtion épigénétique. Egalement, nous avons démontré que l impact de l organisation nucléaire de la cellule donneuse au niveau de l hétérochromatine sur l organisation structurale du noyau dans l embryon NT est relativement modéré.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF

Restructuring the genome during early development is correlated with the onset of transcription and involves large-scale chromatin movements

National audienc

Sterilization influence on PET track membrane properties

Polyethylene terephthalate (PET) track membrane (TM) has a great opportunity to use as a bio implant in ophthalmology's surgery due to its physical and chemical properties and biological comparability. Sterilization of medical implants can change its properties and can influence on regeneration process and success of surgical treatment. We researched influence on the PET track membrane of two sterilization methods wide used in medicine. The first sterilization method was steam sterilization. The second method was γ-irradiation of Co{60} radionuclide sterilization. The sterilization processes influence on the track membrane properties was assessed by surface topography analysis, IR Fourier spectroscopy, wettingangle and surface energy of TM surface measurement

Dynamics of constitutive heterochromatin: two contrasted kinetics of genome restructuring in early cloned bovine embryos

International audienc

Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

International audienceIt becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP)

Development and Assessment of Herpes Simplex Virus Type 1 (HSV-1) Amplicon Vectors with Sensory Neuron-Selective Promoters

Background: Neurogenic detrusor overactivity (NDO) is a severe pathological condition characterized by involuntary detrusor contractions leading to urine leakage. This condition is frequent after spinal cord injury (SCI). Gene therapy for NDO requires the development of vectors that express therapeutic transgenes driven by sensory neuron-specific promoters. The aim of this study was to develop and assess tools for the characterization of sensory neuron-specific promoters in dorsal root ganglia (DRG) neurons after transduction with herpes simplex virus type 1 (HSV-1)-based amplicon defective vectors. Methods: The HSV-1 vector genome encoded two independent transcription cassettes: one expressed firefly luciferase (FLuc) driven by different promoters’ candidates (rTRPV1, rASIC3, rCGRP, or hCGRP), and the other expressed a reporter gene driven by an invariable promoter. The strength and selectivity of promoters was assessed in organotypic cultures of explanted adult DRG, or sympathetic and parasympathetic ganglia from control and SCI rats. Results: The rCGRP promoter induced selective expression in the DRG of normal rats. The rTRPV-1 promoter, which did not display selective activity in control rats, induced selective expression in DRG explanted from SCI rats. Conclusions: This study provides a methodology to assess sensory neuron-specific promoters, opening new perspectives for future gene therapy for NDO