In situ continuous monitoring of dissolved gases (N2, O2, CO2, H2) prior to H2 injection in an aquifer (Catenoy, France) by on-site Raman and infrared spectroscopies: instrumental assessment and geochemical baseline establishment.

The establishment of a baseline of gases from an aquifer appears to be an essential prerequisite for monitoring and securing underground storage operations such as the storage of carbon dioxide (carbon capture and storage: CCS), methane or hydrogen. This study describes an innovative metrological technique dedicated to the in situ and continuous quantification of dissolved gases (CO2, O2, N2, CH4 and H2) in a shallow aquifer, on the site of Catenoy (Paris Basin) with a water table at a depth of 13 m. Monitoring was carried out from May 7, 2019 to November 19, 2019, before the simulation of H2 injection. Gases as vapors were collected from the aquifer through a nine-meter long, half-permeable polymer membrane positioned below a packer in a 25-meter deep well. Collected gases were analyzed simultaneously at the surface by fiber Raman (CO2, O2, N2, CH4 and H2) and infrared sensors (CO2). Gas concentrations were determined from Raman and infrared data, and then converted into dissolved concentrations using Henry's law. The dissolved gas concentrations were about constant over the 6 months period with average values of 31-40 mg L-1 (CO2), 8 mg L-1 (O2), 17 mg L-1 (N2), and 0 mg L-1 (H2, CH4) indicating a very low variability in the aquifer. This is believed to allow for rapid detection of any possible abnormal concentration variation, in particular linked to an accidental arrival of gases such as hydrogen. Such an online gas measurement system can be deployed as is on any site type of underground storage without any need for adaptation.

Children and screens: Groupe de Pédiatrie Générale (Société française de pédiatrie) guidelines for pediatricians and families.

The Groupe de Pédiatrie Générale (General Pediatrics Group), a member of the Société française de pédiatrie (French Pediatrics Society), has proposed guidelines for families and doctors regarding children's use of digital screens. A number of guidelines have already been published, in particular by the French Academy of Sciences in 2013 and the American Academy of Pediatrics in 2016. These new guidelines were preceded by an investigation into the location of digital screen use by young children in France, a survey of medical concerns on the misuse of digital devices, and a review of their documented benefits. The Conseil Supérieur de l'Audiovisuel (Higher Council on Audiovisual Technology) and the Union Nationale de Associations Familiales (National Union of Family Associations) have taken part in the preparation of this document. Five simple messages are proposed: understanding without demonizing; screen use in common living areas, but not in bedrooms; preserve time with no digital devices (morning, meals, sleep, etc.); provide parental guidance for screen use; and prevent social isolation.

Fecal sterols, seasonal variability, and probable sources along the ring of cenotes, Yucatan, Mexico.

Rapid development in Yucatan has had a dramatic impact on the environment, especially the water supply. Groundwater is the only source of water in Yucatan, since surface water is virtually absent due to the karstic nature of the soil. The ring of cenotes (RC) is a geological feature which functions as a source of water and as nodes in the underground river system that canalizes water towards the coast. Numerous productive and domestic activities take place around the RC in the absence of wastewater treatment or sewage systems. Consequently, a number of researchers have hypothesized that pollutants could migrate from the land surface to the underlying aquifer and, eventually, to the coast. Therefore, the present study investigates the relationship among sources of fecal sterols and their levels in cenotes, using the expected levels of fecal sterols obtained by a spatial analysis of the sources and a Pollution Source Index. Accordingly, expected levels are compared with the detected levels of fecal sterols in 5 areas around the RC. Regarding levels, observed during a sampling campaign carried out along the RC during September 2011 (rainy season) and May 2012 (dry season), varied from low to high concentrations of sterols (0.5-2396.42 µg g(-1)) and fecal sterols (0.3-1690.18 µg g(-1)). These concentrations showed no relationship between neighboring cenotes, where similar fecal sterol concentrations or gradients were expected. When comparing expected fecal sterols levels with the detected ones, only two of the five analyzed areas concur, suggesting that no clear relationship exists among sources and fecal sterols levels at the regional scale. Multivariate analysis showed that fecal sterols were associated with sterols and fine grain particulates during the rainy season, which suggests co-transport. During the dry season, fecal sterols associated with fine grain particulate and organic matter, which indicates a change to a deposition phenomenon. These findings indicate that defining a relationship among sources and fecal sterols levels is highly difficult and this could be the result of the absorption or migration through an intricate conduit, crack, or fracture karst system. Nevertheless, the "source-levels approach", used in this study, was consistent for the northeast edge and the middle western part of the RC. New and more extensive research should be done to assess the environmental fate of fecal sterols, especially considering the intricate karstic system and its compound retention capacity.

Transient inflammatory-like state and microbial dysbiosis are pivotal in establishment of mucosal homeostasis during colonisation of germ-free mice.

The gut microbiota is increasingly recognised as a key-player in defining the health status of the gastrointestinal tract. Recently, we demonstrated that colonisation of healthy germfree mice with a conventional microbiota (conventionalisation) elicits temporal and region specific host-microbe communication responses that lead to the establishment of a microbiota-accommodating homeostatic state within 30 days. Here, the microbiota composition profiles, mucosal transcriptomes and plasma-analytes in germ-free and conventionalised C57/BL 6 J mice were assessed to decipher the features of the distinctive and pivotal events occurring four days after initiation of the conventionalisation process. The dominance of the microbial genera Helicobacter, Sphingomonas and Mucispirillum in the gut microbiota coincided with the transient mounting of proinflammatory responses in the mucosa and the transiently elevated levels of specific (inflammatory) cytokines and amines in plasma. The overrepresented microbes have previously been associated with the potential to cause disease under certain conditions, illustrating that conventionalisation proceeds through a transient state that resembles situations associated with dysbiosis. However, no overt mucosal inflammation was observed, suggesting a pivotal role of the overrepresented bacterial groups in priming and maturation of the immune system during the process of conventionalisation. These findings imply that the transiently elevated relative overgrowth of particular microbial genera functions as pivotal adjuvants to elicit the corresponding proinflammatory cascades, which precede the full maturation of the different arms of the immune system following these events and is required to achieve a microbiota-accommodating homeostasis in healthy animals.

Origin of fecal contamination in waters from contrasted areas: stanols as Microbial Source Tracking markers.

Improving the microbiological quality of coastal and river waters relies on the development of reliable markers that are capable of determining sources of fecal pollution. Recently, a principal component analysis (PCA) method based on six stanol compounds (i.e. 5ß-cholestan-3ß-ol (coprostanol), 5ß-cholestan-3α-ol (epicoprostanol), 24-methyl-5α-cholestan-3ß-ol (campestanol), 24-ethyl-5α-cholestan-3ß-ol (sitostanol), 24-ethyl-5ß-cholestan-3ß-ol (24-ethylcoprostanol) and 24-ethyl-5ß-cholestan-3α-ol (24-ethylepicoprostanol)) was shown to be suitable for distinguishing between porcine and bovine feces. In this study, we tested if this PCA method, using the above six stanols, could be used as a tool in "Microbial Source Tracking (MST)" methods in water from areas of intensive agriculture where diffuse fecal contamination is often marked by the co-existence of human and animal sources. In particular, well-defined and stable clusters were found in PCA score plots clustering samples of "pure" human, bovine and porcine feces along with runoff and diluted waters in which the source of contamination is known. A good consistency was also observed between the source assignments made by the 6-stanol-based PCA method and the microbial markers for river waters contaminated by fecal matter of unknown origin. More generally, the tests conducted in this study argue for the addition of the PCA method based on six stanols in the MST toolbox to help identify fecal contamination sources. The data presented in this study show that this addition would improve the determination of fecal contamination sources when the contamination levels are low to moderate.

Development of microbial and chemical MST tools to identify the origin of the faecal pollution in bathing and shellfish harvesting waters in France.

The microbiological quality of coastal or river waters can be affected by faecal pollution from human or animal sources. An efficient MST (Microbial Source Tracking) toolbox consisting of several host-specific markers would therefore be valuable for identifying the origin of the faecal pollution in the environment and thus for effective resource management and remediation. In this multidisciplinary study, after having tested some MST markers on faecal samples, we compared a selection of 17 parameters corresponding to chemical (steroid ratios, caffeine, and synthetic compounds), bacterial (host-specific Bacteroidales, Lactobacillus amylovorus and Bifidobacterium adolescentis) and viral (genotypes I-IV of F-specific bacteriophages, FRNAPH) markers on environmental water samples (n = 33; wastewater, runoff and river waters) with variable Escherichia coli concentrations. Eleven microbial and chemical parameters were finally chosen for our MST toolbox, based on their specificity for particular pollution sources represented by our samples and their detection in river waters impacted by human or animal pollution; these were: the human-specific chemical compounds caffeine, TCEP (tri(2-chloroethyl)phosphate) and benzophenone; the ratios of sitostanol/coprostanol and coprostanol/(coprostanol+24-ethylcopstanol); real-time PCR (Polymerase Chain Reaction) human-specific (HF183 and B. adolescentis), pig-specific (Pig-2-Bac and L. amylovorus) and ruminant-specific (Rum-2-Bac) markers; and human FRNAPH genogroup II.

HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgG-seronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4(+) cell infection: an IgA gene and functional analysis.

AIDS is mainly a sexually transmitted disease, and accordingly, mucosal tissues are the primary sites of natural human immunodeficiency virus type-1 (HIV-1) transmission. Mucosal immunoglobulin A (IgA) antibody specific for HIV-1 envelope gp41 subunit is one correlate of protection in individuals who are highly sexually exposed to HIV-1 but remain persistently IgG seronegative (HEPS). Understanding these peculiar IgAs at the gene and functional level is possible only with monoclonal IgAs. We have constructed a mucosal Fab IgA library from HEPS and have characterized a series of HIV-1 IgAs specific for gp41 that, in vitro, are transcytosis-blocking and infection-neutralizing. Characterization of their IgA genes shows that Fab specific for the gp41 membrane-proximal region harbors a long heavy-chain CDR3 loop (CDRH3) similar to the two broadly neutralizing IgG monoclonal antibodies, 2F5 and 4E10. Furthermore, the selected Fab IgA shows extensive somatic mutations that cluster in the CDR regions, indicating that affinity maturation due to an antigen-driven process had occurred in HEPS individuals, presumably upon multiple exposures to HIV. This analysis of HEPS monoclonal IgA gives a unique opportunity to correlate an antibody function (resistance to a pathogen in vivo) with an antibody gene. Such neutralizing monoclonal IgAs could be used in microbicide formulation.

Characterization of the main placental cytokine profiles from HIV-1-infected pregnant women treated with anti-retroviral drugs in France.

Cytokines are involved in regulating HIV-1 infection. They are also placental environment major components. We assessed the potential impact of HIV-1 infection and/or anti-retroviral drugs on the placental cytokine profiles that may be involved in controlling HIV-1 placental dissemination. Placental explants were obtained after elective caesarean section from anti-retroviral-treated HIV-1-infected pregnant women and from HIV-1 non-infected pregnant women. The main placental cytokines were assessed for protein secretion in the supernatants of 24-h placental culture explants and/or in uncultured placental explants for mRNA expression levels. The cytokine profiles were different between the HIV-1-infected and the non-infected groups. Higher medians of leukaemia inhibiting factor (LIF), tumour necrosis factor (TNF)-alpha and interleukin (IL)-8 secretion were found in the 24-h culture supernatant of term placenta from HIV-1-infected women. High median levels of IL-16 and regulated upon activation normal T cell expressed and secreted (RANTES) levels were found in both groups. The mRNA expression medians were lower for TNF-alpha and IL-8 and higher for stromal cell-derived factor-1 (SDF-1) in uncultured placental explants from HIV-1-infected women. In the HIV-1-infected group, but not in the non-infected group, the secretion levels of TNF-alpha and IL-8, as well as their mRNA expression levels, were highly positively correlated; furthermore, their secretion levels were correlated positively with LIF and IL-10 secretion levels. We found no correlation between the cytokine levels and the immunovirological status of the HIV-1-infected mothers or the type or duration of treatment. These results highlight the potential impact of HIV-1 and of the anti-retroviral treatments on the placental cytokines pattern, independently of their anti-viral activity.

Evaluation of the placental environment with a new in vitro model of histocultures of early and term placentae: determination of cytokine and chemokine expression profiles.

We aimed to set up and validate a new in vitro model of placental histocultures, for the evaluation of cytokine and chemokine profiles of the placental environment, over a long culture period. Micro-explant cultures from 6 early and 6 term placentae were set up on collagen sponge gel supports at a liquid/air interface. At various times during culture, we analyzed tissue morphology and cell death by microscopy and quantified beta-hCG production and mRNA levels for beta-hCG and insulin-like 4 (INSL4). Levels of IL-6, LIF, TNF alpha, IL-10, IFN-gamma, IL-16 and RANTES in the medium were measured by ELISA on days 1, 4 and 7 of culture. SDF-1 mRNA expression was determined by real-time PCR at the same time points. Histocultures from early and term placentae remained viable until day 10. High levels of IL-6 and LIF production, low levels of TNF alpha, IL-10 and IFN-gamma production and significant SDF-1 expression were observed. These data indicate that placental histoculture is a suitable and reliable in vitro model for studying the placental environment.

Cell-free HIV type 1 infection is restricted in the human trophoblast choriocarcinoma BeWo cell line, even with expression of CD4, CXCR4 and CCR5.

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

Anti-interleukin-3 and anti-nerve growth factor increase neonatal mice survival to reovirus type 3 clone 9 per oral challenge.

Reovirus type 3 clone 9 (T3C9) induces lethal encephalitis in neonatal, but not adult mice. Whether host factors that promote the development and/or functioning of nervous and gastrointestinal tissues could modulate the pathogenesis of this enteric virus was examined. The results showed that antibody specific for interleukin-3 or nerve growth factor antiserum, but not anti-interleukin-6 or anti-tumor necrosis factor-alpha/beta increased mice survival to T3C9 and decreased viral titers in nervous tissues early after infection. These data suggest that IL-3 and NGF are involved in the pathogenesis of T3C9 infection in neonatal mice.

Tyrosine phosphorylation of A17 during vaccinia virus infection: involvement of the H1 phosphatase and the F10 kinase.

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specificity phosphatase (VH1), suggesting that phosphorylation and dephosphorylation of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vindH1), we have previously demonstrated that repression of H1 leads to the maturation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demonstrate that among these is a 25-kDa protein that is phosphorylated on tyrosine residues in H1-deficient virions and can be dephosphorylated by recombinant H1. We demonstrate that the 25-kDa phosphoprotein represents the product of the A17 gene and that A17 is phosphorylated on serine, threonine, and tyrosine residues during infection. Detection of phosphotyrosine within A17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutated to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during nonpermissive infections performed with temperature-sensitive mutants defective in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specificity. This hypothesis is strengthened by the observation that Escherichia coli induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence for phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.

Reovirus type 3 clone 9 increases interleukin-1alpha level in the brain of neonatal, but not adult, mice.

Reovirus Type 3 clone 9 (T3C9)-induced lethal encephalitis is age dependent. We examined the effects of T3C9 inoculated into neonatal and adult mice by intracerebral, intramuscular, or peroral routes and the effect of lipopolysaccharide (LPS) on IL-1alpha levels in the blood and the brain. In parallel, we measured mice survival to T3C9 challenge, primary replication, and growth in and spread to the brain. The results show that T3C9 infection increased IL-1alpha only in the brain of neonatal mice, whereas LPS enhanced IL-1alpha in the brain and in the blood in both neonatal and adult mice. In neonatal mice, a T3C9-induced IL-1alpha increase coincided with viral replication-induced nervous tissue injury and preceded death. Anti-IL-1alpha antibody partially protected neonatal mice against T3C9 peroral challenge, further suggesting that this cytokine is involved in the mechanisms leading to lethal encephalitis. In adult mice, T3C9 was not lethal and did not modify IL-1alpha levels although it slowly replicated in nervous tissues when inoculated directly into the brain. Together, these results suggest that differences in nervous tissue response to T3C9 replication between newborn and adult mice could account in part for the age-dependent susceptibility to T3C9-induced lethal encephalitis.

The selective CCK-B agonist, BC 264, impairs socially reinforced memory in the three-panel runway test in rats.

The role of CCK-B receptor activation in memory processes has been reassessed using the three-panel runway task, under conditions which avoid the effects of CCK on satiety and reduce emotional responses. For this purpose the food reinforcement usually used was replaced by a social reinforcement. The results show that learning and memory can be assessed using this procedure. Moreover, under these experimental conditions, drugs such as scopolamine, amphetamine or kinurenic acid injected into the nucleus accumbens produced behavioral deficits. BC 264, a highly selective CCK-B agonist, peripherally administered or infused into the anterolateral part of the nucleus accumbens also impaired memory. These effects were suppressed by L-365,260 supporting the involvement of CCK-B receptors and of the nucleus accumbens in memory processes.

Heterogeneity of CCK-B receptors involved in animal models of anxiety.

The effects of the selective CCK-B agonists, BC 264 and BC 197, and the nonselective CCK agonist BDNL were investigated in the elevated plus-maze in rats. BDNL and BC 197 induced anxiogeniclike effects, in contrast to BC 264, which had no effect. The behavioral responses induced by BDNL were not significantly blocked by L-365,260, but were suppressed by CI-988, another selective CCK-B antagonist, and by high doses of L-364,718, a selective CCK-A antagonist. BC 197-induced effects were also blocked by CI-988. Competition experiments performed with [3H]pBC 264 using brain membranes of guinea pig, mouse, and rat were significantly better fitted when analyzed by a two site model than by a one site model with BC 197 but not with BC 264. Moreover, BC 264 produced anxiogeniclike effects when administered with increasing doses of L-365,260 and opposing effects with increasing doses of CI-988. Together these results give pharmacological and behavioral evidence for the existence of CCK-B receptor subtypes.

Effects induced by BC 264, a selective agonist of CCK-B receptors, on morphine-dependent rats.

The aim of this study was to investigate the possible interaction between neuronal cholecystokinin (CCK) and opiate dependence. Rats were made dependent to morphine and the ability of cholecystokinin-octapeptide (CCK-8) and Tyr(SO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2 (BC 264), a selective agonist of CCK-B receptors, to induce signs of morphine withdrawal after ICV injection was tested. Behavioral responses were compared to those occurring during the naloxone-precipitated morphine withdrawal syndrome. In contrast to naloxone, CCK-8 (0.1, 1, and 10 micrograms, ICV) did not precipitate any sign of withdrawal. BC 264 (0.1, 1, and 10 micrograms, ICV) induced a strong hyperlocomotion and wet dog shakes in morphine-dependent rats, the latter effect also observed in nondependent animals. In rats receiving acute morphine, BC 264 induced an opposite effect (i.e., blockade of morphine-induced hyperactivity). Taken together, these results suggest that CCK plays only a minor role in the expression of morphine physical dependence.

Antidepressant-like effects of CCKB antagonists in mice: antagonism by naltrindole.

1. The effects of selective CCKB agonists, BC 264 and BC 197 were investigated in the conditioned suppression of motility test in mice, an animal model used to select antidepressant drugs. The results showed that both CCKB agonists at doses of 3 and 30 micrograms kg-1, accentuated the suppression of motility in shocked mice and did not modify the behaviour of non-shocked mice. The effects of BC 264 were suppressed by L-365,260. 2. L-365,260 alone, at doses of 0.2 and 2 mg kg-1 decreased motor inhibition in shocked mice and had no effect in non-shocked mice. 3. The effects of L-365,260 observed in shocked mice were suppressed by naltrindole, a selective antagonist for delta-opioid receptors, suggesting the occurrence of physiological adverse interactions between CCK and opioid systems. 4. Together, these results suggest that CCKB antagonists could block centrally located CCKB receptors to produce antidepressant-like effects which could indirectly involve delta-opioid receptor stimulation.