Pyrethroid Resistance Intensity in Anopheles gambiae s.l. from Different Agricultural Production Zones in Benin, West Africa.

Agricultural production activities usually occur in Benin with the use of a huge amount of insecticides including pyrethroids for pest control. It is therefore important to regularly monitor pyrethroid resistance intensity in Anopheles gambiae s.l., the main malaria vector. This study was conducted in cereal, cotton, rice growing, and urban market gardening areas throughout the country in 2018 and 2019. Females An. gambiae s.l. field-collected as larvae were exposed to deltamethrin 1 × (0.05%), 2 × (0.1%), 5 × (0.25%), and 10 × (0.5%) and permethrin 1 × (0.75%), 2 × (1.5%), 5 × (3.75%), and 10 × (7.5%). Synergist assays were also performed using World Health Organization articles combining piperonyl butoxide (PBO) (4%) + deltamethrin 1 × and, PBO (4%) + Permethrin 1 × . Molecular species and L1014F kdr mutation were identified using PCR. Expression of metabolic enzymes was also assessed through biochemical tests. After exposure to permethrin and deltamethrin 10 × , An. gambiae s.l. displayed mortality rates <98%. Synergist assays induced significantly higher mortality rates than pyrethroids alone (p < 0.05). An. gambiae s.l. complex was composed of An. gambiae s.s., Anopheles coluzzii, and Anopheles arabiensis, with mean frequency of the L1014F kdr mutation >75%. Overexpression of nonspecific α and ß esterases was observed in the cereal, cotton, and urban market gardening areas, while an overexpression of mixed function oxidases was observed in the cotton and rice growing areas. Overall, An. gambiae s.l. showed high resistance intensity to both deltamethrin and permethrin. The synergist and biochemical tests performed suggest that PBO long-lasting insecticidal nets may provide a greater control of pyrethroid-resistant mosquitoes.

Characterization of resistance profile (intensity and mechanisms) of Anopheles gambiae in three communes of northern Benin, West Africa.

BACKGROUND: The selection and the spread of insecticide resistance in malaria vectors to the main classes of insecticides used in vector control tools are a major and ongoing challenge to malaria vector control programmes. This study aimed to determine the intensity of vector resistance to insecticides in three regions of Benin with different agro-ecological characteristics. METHODS: Larvae of Anopheles gambiae sensu lato (s.l.) were collected from September to November 2017 in different larval sites in three northern Benin communes: Parakou, Kandi and Malanville. Two to five-day-old, non-blood-fed, female mosquitoes were exposed to papers impregnated with deltamethrin, permethrin and bendiocarb at dosages of 1 × the diagnostic dose, 5 × and 10 × to determine the intensity of resistance in these vectors. Molecular frequencies of the kdr L1014F and ace-1R G119S insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled at each study site. RESULTS: Resistance to pyrethroids (permethrin and deltamethrin) was recorded in all three communes with mortality rates below 60% using the diagnostic dose (1x). The results obtained after exposure of An. gambiae to permethrin 10 × were 99% in Kandi, 98% in Malanville and 99% in Parakou. With deltamethrin 10x, mortality rates were 100% in Kandi, 96% in Malanville and 73% in Parakou. For the diagnostic dose of bendiocarb, suspected resistance was recorded in the communes of Malanville (97%) and Kandi (94%) while sensitivity was observed in Parakou (98%).Using the 10 × dose, mortality was 98% in Kandi, 100% in Malanville and 99% in Parakou. The frequencies of the kdr L1014F allele varied between 59 and 83% depending on the sites and species of the An. gambiae complex, while the frequency of the ace-1R G119S gene varied between 0 and 5%. Biochemical tests showed high levels of oxidase and esterase activity compared to the susceptible colony strain of An. gambiae sensu stricto (Kisumu strain). CONCLUSION: Anopheles gambiae showed a generalized loss of susceptibility to permethrin and deltamethrin but also showed moderate to high intensity of resistance in different regions of Benin. This high intensity of resistance is a potential threat to the effectiveness of vector control.

Intensity and mechanisms of deltamethrin and permethrin resistance in Anopheles gambiae s.l. populations in southern Benin.

BACKGROUND: Insecticide resistance is threatening the effectiveness of efforts to control malaria vectors in Benin. This study explores the levels and mechanisms of insecticide resistance in An. gambiae s.l. to pyrethroids. METHODS: Larvae were collected from August 2017 to July 2018 in five communes in southern Benin (Adjohoun, Allada, Bohicon, Cotonou, and Porto-Novo) representing diverse ecological regions, and were reared in Benin's insectary. Two- to five-day-old female mosquitoes from each district were exposed to multiple doses of deltamethrin and permethrin (1×, 2×, 5×, and 10×) using the WHO insecticide resistance intensity bioassay. The effect of pre-exposure to the synergist, piperonyl butoxide (PBO), was also tested at different pyrethroid doses. Molecular allele frequencies of kdr (1014F) and ace-1R (119S) insecticide resistance mutations and levels of detoxification enzymes were determined for mosquitoes sampled from each study area. RESULTS: An. gambiae s.l. were resistant to pyrethroid-only exposure up to 10× the diagnostic doses in all the study sites for both deltamethrin and permethrin. Mortality was significantly higher in An. gambiae s.l. pre-exposed to PBO followed by exposure to deltamethrin or permethrin compared to mosquitoes exposed to deltamethrin or permethrin only (p < 0.001). The difference in mortality between deltamethrin only and PBO plus deltamethrin was the smallest in Cotonou (16-64%) and the greatest in Bohicon (12-93%). The mortality difference between permethrin only and PBO plus permethrin was the smallest in Cotonou (44-75%) and the greatest in Bohicon (22-72%). In all the study sites, the kdr resistance allele (1014F) frequency was high (75-100%), while the ace-1 resistance allele (G119S) frequency was low (0-3%). Analysis of the metabolic enzymatic activity of An. gambiae s.l. showed overexpression of nonspecific esterases and glutathione S-transferases (GST) in all study sites. In contrast to the PBO results, oxidase expression was low and was similar to the susceptible An. gambiae s.s. Kisumu strain in all sites. CONCLUSION: There is high-intensity resistance to pyrethroids in southern Benin. However, pre-exposure to PBO significantly increased susceptibility to the pyrethroids in the different An. gambiae s.l. populations sampled. The use of PBO insecticide-treated bed nets may help maintain the gains in An. gambiae (s.l.) control in southern Benin.

Experimental analysis of the shear flow effect on tidal turbine blade root force from three-dimensional mean flow reconstruction.

In the main tidal energy sites like Alderney Race, turbulence intensity is high and velocity fluctuations may have a significant impact on marine turbines. To understand such phenomena better, a three-bladed turbine model is positioned in the wake of a generic wall-mounted obstacle, representative of in situ bathymetric variation. From two-dimensional Particle Image Velocimetry planes, the time-averaged velocity in the wake of the obstacle is reconstructed in the three-dimensional space. The reconstruction method is based on Proper Orthogonal Decomposition and enables access to a representation of the mean flow field and the associated shear. Then, the effect of the velocity gradient is observed on the turbine blade root force, for four turbine locations in the wake of the obstacle. The blade root force average decreases whereas its standard deviation increases when the distance to the obstacle increases. The angular distribution of this phase-averaged force is shown to be non-homogeneous, with variation of about 20% of its time-average during a turbine rotation cycle. Such force variations due to velocity shear will have significant consequences in terms of blade fatigue. This article is part of the theme issue 'New insights on tidal dynamics and tidal energy harvesting in the Alderney Race'.

Lessons learned, challenges and outlooks for decision-making after a decade of experience monitoring the impact of indoor residual spraying in Benin, West Africa.

BACKGROUND: Since 2008, Indoor Residual Spraying (IRS) has been performed in Benin in 19 districts, including 4 in southern Benin, 9 in Atacora, and 8 in Atacora, Alibori and Donga in northern Benin. However, Benin still struggles with questions about IRS cost-benefit and epidemiological impact. Lessons learned and challenges from 10 years of IRS in Benin to be shared with the stakeholders involved in vector control implementation for decision-making. METHODS: Entomological parameters have been assessed entomological parameters in IRS communes since 2008. In all IRS intervention communes, decreases in human biting rate (HBR) of Anopheles gambiae, blood feeding inhibition and entomological inoculation rate (EIR) as compared to control district have been measured. RESULTS: EIR was reduced by 80-90%, which is encouraging, but should be observed with caution because: (i) the reduction may be insufficient to decrease epidemiological indicators given that the residual EIR in IRS districts is still higher than it is in some regions of stable malaria; (ii) the reduction in EIR is based on comparisons with control communes, but it is difficult to select control areas with the same environmental characteristics as intervention areas; (iii) despite the reduction, half of all mosquitoes that entered IRS-treated houses succeeded in taking human blood meals. Further, there are behaviours among Benin's population that limit IRS efficacy, including recent data showing that > 90% of people are not protected by IRS between 7 and 10 p.m. This is due to the fact that they remain outdoors and that most people are not protected from mosquito bites after 10 p.m. because they either sleep outdoors without IRS protection or indoors without an ITN. Moreover, people have large amounts of clothing hanging on walls where mosquitoes can rest instead of IRS-treated walls. Finally, other components are important to consider in implementing IRS among which: (i) Vector resistance management strategies are sometimes poorly understood; this is actually different from the need to replace one insecticide with another after the emergence of resistance; (ii) African countries should prepare to finance IRS themselves. CONCLUSION: To curtail residual malaria transmission, additional interventions able to target vectors escaping IRS should be prioritized.

Quebec's Multi-Party Observatory on Zoonoses and Adaptation to Climate Change.

Climate change has been linked with the establishment and geographical expansion of zoonotic diseases, an example of which is the well-documented increase in human cases of Lyme disease in Quebec, Canada. As temperatures continue to increase in Quebec, it is anticipated that several zoonotic diseases will be affected. In response to the growing zoonotic issues facing public health authorities, Quebec's Multi-Party Observatory on Zoonoses and Adaptation to Climate Change (Observatoire multipartite québécois sur les zoonoses et l'adaptation aux changements climatiques) (the Observatory) was founded in 2015 as part of the Quebec government's Climate Change Action Plan (Plan d'action 2013-2020 sur les changements climatiques). The Observatory was designed to bring together agencies involved in formulating public policy and experts from the disciplines of human health, animal health and environmental sciences, in a manner similar to the innovative "One World, One Health" approach. The Observatory provides a platform for knowledge sharing and consensus building among representatives of public policy decision makers and scientists. Its main objectives are to anticipate and prioritize potential issues associated with zoonotic diseases in Quebec, in order to support applicable risk management and climate change adaptation. This article describes what the Observatory is, what it does and outlines its plans for the future.

Dengue-induced autophagy, virus replication and protection from cell death require ER stress (PERK) pathway activation.

A virus that reproduces in a host without killing cells can easily establish a successful infection. Previously, we showed that dengue-2, a virus that threatens 40% of the world, induces autophagy, enabling dengue to reproduce in cells without triggering cell death. Autophagy further protects the virus-laden cells from further insults. In this study, we evaluate how it does so; we show that dengue upregulates host pathways that increase autophagy, namely endoplasmic reticulum (ER) stress and ataxia telangiectasia mutated (ATM) signaling followed by production of reactive oxygen species (ROS). Inhibition of ER stress or ATM signaling abrogates the dengue-conferred protection against other cell stressors. Direct inhibition of ER stress response in infected cells decreases autophagosome turnover, reduces ROS production and limits reproduction of dengue virus. Blocking ATM activation, which is an early response to infection, decreases transcription of ER stress response proteins, but ATM has limited impact on production of ROS and virus titers. Production of ROS determines only late-onset autophagy in infected cells and is not necessary for dengue-induced protection from stressors. Collectively, these results demonstrate that among the multiple autophagy-inducing pathways during infection, ER stress signaling is more important to viral replication and protection of cells than either ATM or ROS-mediated signaling. To limit virus production and survival of dengue-infected cells, one must address the earliest phase of autophagy, induced by ER stress.

Prasugrel vs. clopidogrel in acute coronary syndrome patients treated with prasugrel.

WHAT IS KNOWN AND OBJECTIVE: Current guidelines recommend a combination of clopidogrel and aspirin for management of patients who have experienced an acute coronary syndrome (ACS). Additional antiplatelet agents have been recently approved. Few comparative effectiveness studies are available for these new agents. Accordingly, we evaluated effect on time to hospital admission and resource utilization (number of hospitalizations, ER visits and outpatient visits) of prasugrel vs. clopidogrel in prasugrel-treated patients as assessed in a matched cohort. METHODS: Based on the Truven Health Analytics MarketScan database from 01 January 2009 through 31 July 2012, a retrospective prasugrel-clopidogrel matched cohort was created. Inferences for average treatment effect over 1 and 12 months on time to hospitalization and resource utilization were performed by (i) frequentist Kaplan-Meier estimation with a Cox proportional hazard model and Lin's cost history method for censored resource utilization outcomes and (ii) Bayesian discrete-time hazard and negative binomial models. RESULTS AND DISCUSSION: The 10,963 matched pairs were well balanced on baseline characteristics. Frequentist analyses of time to hospital admission over 365 days and mean all-cause resource utilization over 30 and 365 days showed no statistical differences between prasugrel and clopidogrel (P-values > 0·05). Based on Bayesian analysis of time to admission over 12 months, there was positive evidence of equivalence (0·987 probability of equivalence at a 10% equivalence margin and a Bayes factor of 0·611). Although the frequentist analyses for number of all-cause hospitalizations showed a lack of a significant difference at Months 1 and 12, the Bayesian data analysis showed positive evidence of superiority of clopidogrel at Month 1 (Bayes factor: 5·369); however, at Month 12, there was little evidence of superiority of one treatment over the other (Bayes factor: 0·422). WHAT IS NEW AND CONCLUSION: Using frequentist and Bayesian data analyses, in prasugrel-treated patients, clopidogrel was equivalent to prasugrel for time to hospital admission over 12 months and there was positive evidence that it was superior to prasugrel for number of hospitalizations over the first month of treatment.

Multilaboratory study of epidemiological cutoff values for detection of resistance in eight Candida species to fluconazole, posaconazole, and voriconazole.

Although epidemiological cutoff values (ECVs) have been established for Candida spp. and the triazoles, they are based on MIC data from a single laboratory. We have established ECVs for eight Candida species and fluconazole, posaconazole, and voriconazole based on wild-type (WT) MIC distributions for isolates of C. albicans (n=11,241 isolates), C. glabrata (7,538), C. parapsilosis (6,023), C. tropicalis (3,748), C. krusei (1,073), C. lusitaniae (574), C. guilliermondii (373), and C. dubliniensis (162). The 24-h CLSI broth microdilution MICs were collated from multiple laboratories (in Canada, Brazil, Europe, Mexico, Peru, and the United States). The ECVs for distributions originating from ≥6 laboratories, which included ≥95% of the modeled WT population, for fluconazole, posaconazole, and voriconazole were, respectively, 0.5, 0.06 and 0.03 µg/ml for C. albicans, 0.5, 0.25, and 0.03 µg/ml for C. dubliniensis, 8, 1, and 0.25 µg/ml for C. glabrata, 8, 0.5, and 0.12 µg/ml for C. guilliermondii, 32, 0.5, and 0.25 µg/ml for C. krusei, 1, 0.06, and 0.06 µg/ml for C. lusitaniae, 1, 0.25, and 0.03 µg/ml for C. parapsilosis, and 1, 0.12, and 0.06 µg/ml for C. tropicalis. The low number of MICs (<100) for other less prevalent species (C. famata, C. kefyr, C. orthopsilosis, C. rugosa) precluded ECV definition, but their MIC distributions are documented. Evaluation of our ECVs for some species/agent combinations using published individual MICs for 136 isolates (harboring mutations in or upregulation of ERG11, MDR1, CDR1, or CDR2) and 64 WT isolates indicated that our ECVs may be useful in distinguishing WT from non-WT isolates.

Multicenter study of anidulafungin and micafungin MIC distributions and epidemiological cutoff values for eight Candida species and the CLSI M27-A3 broth microdilution method.

Since epidemiological cutoff values (ECVs) using CLSI MICs from multiple laboratories are not available for Candida spp. and the echinocandins, we established ECVs for anidulafungin and micafungin on the basis of wild-type (WT) MIC distributions (for organisms in a species-drug combination with no detectable acquired resistance mechanisms) for 8,210 Candida albicans, 3,102 C. glabrata, 3,976 C. parapsilosis, 2,042 C. tropicalis, 617 C. krusei, 258 C. lusitaniae, 234 C. guilliermondii, and 131 C. dubliniensis isolates. CLSI broth microdilution MIC data gathered from 15 different laboratories in Canada, Europe, Mexico, Peru, and the United States were aggregated to statistically define ECVs. ECVs encompassing 97.5% of the statistically modeled population for anidulafungin and micafungin were, respectively, 0.12 and 0.03 µg/ml for C. albicans, 0.12 and 0.03 µg/ml for C. glabrata, 8 and 4 µg/ml for C. parapsilosis, 0.12 and 0.06 µg/ml for C. tropicalis, 0.25 and 0.25 µg/ml for C. krusei, 1 and 0.5 µg/ml for C. lusitaniae, 8 and 2 µg/ml for C. guilliermondii, and 0.12 and 0.12 µg/ml for C. dubliniensis. Previously reported single and multicenter ECVs defined in the present study were quite similar or within 1 2-fold dilution of each other. For a collection of 230 WT isolates (no fks mutations) and 51 isolates with fks mutations, the species-specific ECVs for anidulafungin and micafungin correctly classified 47 (92.2%) and 51 (100%) of the fks mutants, respectively, as non-WT strains. These ECVs may aid in detecting non-WT isolates with reduced susceptibility to anidulafungin and micafungin due to fks mutations.

Interlaboratory variability of Caspofungin MICs for Candida spp. Using CLSI and EUCAST methods: should the clinical laboratory be testing this agent?

Although Clinical and Laboratory Standards Institute (CLSI) clinical breakpoints (CBPs) are available for interpreting echinocandin MICs for Candida spp., epidemiologic cutoff values (ECVs) based on collective MIC data from multiple laboratories have not been defined. While collating CLSI caspofungin MICs for 145 to 11,550 Candida isolates from 17 laboratories (Brazil, Canada, Europe, Mexico, Peru, and the United States), we observed an extraordinary amount of modal variability (wide ranges) among laboratories as well as truncated and bimodal MIC distributions. The species-specific modes across different laboratories ranged from 0.016 to 0.5 µg/ml for C. albicans and C. tropicalis, 0.031 to 0.5 µg/ml for C. glabrata, and 0.063 to 1 µg/ml for C. krusei. Variability was also similar among MIC distributions for C. dubliniensis and C. lusitaniae. The exceptions were C. parapsilosis and C. guilliermondii MIC distributions, where most modes were within one 2-fold dilution of each other. These findings were consistent with available data from the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (403 to 2,556 MICs) for C. albicans, C. glabrata, C. krusei, and C. tropicalis. Although many factors (caspofungin powder source, stock solution solvent, powder storage time length and temperature, and MIC determination testing parameters) were examined as a potential cause of such unprecedented variability, a single specific cause was not identified. Therefore, it seems highly likely that the use of the CLSI species-specific caspofungin CBPs could lead to reporting an excessive number of wild-type (WT) isolates (e.g., C. glabrata and C. krusei) as either non-WT or resistant isolates. Until this problem is resolved, routine testing or reporting of CLSI caspofungin MICs for Candida is not recommended; micafungin or anidulafungin data could be used instead.

Endemic human blastomycosis in Quebec, Canada, 1988-2011.

Blastomycosis is a systemic fungal infection found in various parts of the world. A review of literature for Quebec, Canada revealed only few case reports with the most recent one dating back to 1993. However, whether Quebec represents an important endemic region for blastomycosis in North America is unknown. In this work we reviewed 158 cases of human blastomycosis documented in Quebec during 1988-2011 using microbiological records available from the provincial public health laboratory. The estimated annual incidence of blastomycosis in the province is was ~0·133 cases per 100 000 individuals with the highest rates of 0·79 and 0·46 cases per 100 000 recorded in South-eastern and South-western Quebec. Moreover, the annual incidence rate significantly increased over the past 20 years. This study for the first time establishes Quebec as an important endemic region for Blastomyces dermatitidis.

Pre- and post-partum mild underfeeding influences gene expression in the reproductive tract of cyclic dairy cows.

Undernutrition before and after calving has a detrimental effect on the fertility of dairy cows. The effect of nutritional stress was previously reported to influence gene expression in key tissues for metabolic health and reproduction such as the liver and the genital tract early after calving, but not at breeding, that is, between 70 and 90 days post-partum. This study investigated the effects of pre- and post-partum mild underfeeding on global gene expression in the oviduct, endometrium and corpus luteum of eight multiparous Holstein cows during the early and middle phases of an induced cycle 80 days post-partum. Four control cows received 100% of energy and protein requirements during the dry period and after calving, while four underfed received 80% of control diet. Oestrous synchronization treatment was used to induce ovulation on D80 post-partum. Oviducts, ovaries and the anterior part of each uterine horn were recovered surgically 4, 8, 12 and 15 days after ovulation. Corpora lutea were dissected from the ovaries, and the endometrium was separated from the stroma and myometrium in each uterine horn. The oviduct segments were comprised of ampulla and isthmus. RNAs from ipsi- and contralateral samples were pooled on an equal weight basis. In each tissue, gene expression was assessed on a custom bovine 10K array. No differentially expressed gene (DEG) in the corpus luteum was identified between underfed and control, conversely to 293 DEGs in the oviduct vs 1 in the endometrium under a false discovery rate (FDR) < 0.10 and 1370 DEGs vs 3, respectively, under FDR < 0.15. Additionally, we used dedicated statistics (regularized canonical correlation analysis) to correlate the post-partum patterns of six plasma metabolites and hormones related to energy metabolism measured weekly between calving and D80 with gene expression. High correlations were observed between post-partum patterns of IGF-1, insulin, ß-hydroxybutyrate and the expression in the oviduct of genes related to reproductive system disease, connective tissue disorders and metabolic disease. Moreover, we found special interest in the literature to retinoic acid-related genes (e.g. FABP5/CRABP2) that might indicate abnormalities in post-partum tissue repair mechanisms. In conclusion, this experiment highlights relationships between underfeeding and gene expression in the oviduct and endometrium after ovulation in cyclic Holstein cows. This might help to explain the effect of mild undernutrition on fertilization failure and early embryonic mortality in post-partum dairy cows.

Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole.

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Nonlinearity testing in the case of non Gaussian surrogates, applied to improving analysis of synchronicity in uterine contraction.

Surrogates are commonly used to test a particular hypothesis on time series. The parameter commonly used in the literature to test these hypotheses is the z score. The z score assumes that the distribution of the statistics obtained on the surrogates is Gaussian. In this paper, we propose the use of a more general parameter than the z score that will also work in the case of non-Gaussian distribution of the statistics. We also derive a statistical test, based on the fitting of the distribution of the surrogate measure profile, in order to test the initial hypothesis. We validate the proposed approach on both synthetic signals and real uterine EMG signals by using the nonlinear correlation coefficient as initial statistic. We further show that this corrected nonlinear correlation coefficient can discriminate between pregnancy contractions and labor in a monkey, but the uncorrected nonlinear correlation coefficient cannot. This makes the corrected nonlinear correlation coefficient a promising candidate in a future application for preterm labor prediction in humans.

Developmental regulation of prostacyclin synthase and prostacyclin receptors in the ovine uterus and conceptus during the peri-implantation period.

This study documents the expression of prostacyclin (PGI2) synthase (PTGIS) and PGI2 receptors in the trophoblast and uterus of the ewe at the time of maternal recognition of pregnancy (i.e. days 7, 9, 12, 14 and 17). The membrane receptor for PGI2 (PTGIR) and the nuclear receptors, i.e. peroxisome proliferator-activated receptors (PPAR) and their heterodimer partners the retinoid X receptors (RXR), were analysed. In the endometrium, PTGIS transcript and protein were expressed at day 9 of pregnancy and levels declined from days 12 to 17. Immunohistochemistry and in situ hybridization indicated that PTGIS was mainly located in the luminal epithelium of the endometrium. Endometrial PTGIR, PPARA, PPARG and RXRG expression was regulated during the peri-implantation period whereas PPARD, RXRA and RXRB were consistently expressed. In the trophoblast, PTGIS transcript levels rose as development progressed and peaked at day 17. PTGIR and PPARA transcripts peaked before day 12 and then declined and became nearly undetectable by day 17, whereas PPARD and PPARG transcript levels rose steadily from days 12 to 17. Because the PPARs and the RXRs display different expression profiles, we suggest that different heterodimers may form and support distinct functions as development proceeds. Our results also underline the importance of PTGIS and PPARD in the trophoblast and PTGIR in the uterus, suggesting that PGI2 is of both uterine and trophoblastic origin and is involved in a complex signalling pathway at around the time of implantation in the ewe.

Reconsideration of the proposed luteotropic and luteoprotective actions of ovine placental lactogen in sheep: in vivo and in vitro studies.

Ovine placental lactogen (oPL) is produced by the conceptus trophectoderm and is secreted into both the maternal and fetal circulations. The present study was designed to examine in vivo the luteotropic effect of recombinant oPL (roPL), as determined by monitoring progesterone concentration and cycle length (experiment 1), and the antioxidative and antiapoptotic effects of roPL, as determined respectively by monitoring antioxidant enzymatic activity and apoptosis in the corpus luteum (CL) of cyclic ewes (experiment 2). We also studied whether roPL is capable of stimulating progesterone secretion in vitro by cultured luteal tissue of functionally active CL obtained from day-10 cyclic ewes (experiment 3) and day-60 pregnant ewes (experiment 4). Circulating concentrations of progesterone and cycle length were not affected by treatment of ewes with 80 microg/kg body weight per day of roPL (n = 4 ewes) for 10 days beginning on day 11 post-estrus, as compared with saline-treated ewes (n = 4 ewes). Luteolysis occurred between days 15 and 16 post-estrus in the four saline-treated ewes and in 3/4 roPL-treated ewes. The activities of the key antioxidant enzymes copper-zinc superoxide dismutase (Cu,Zn-SOD), manganese SOD (Mn-SOD), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) were unaffected by treatment of ewes with 80 microg/kg per day of roPL (n = 4 ewes) for 3 days, between days 11 and 14 post-estrus, as compared with saline-treated ewes (n = 4 ewes). In situ TUNEL method revealed that the number of apoptotic cells was not different between the two groups of ewes. There was no significant change in progesterone secretion by explants from day-10 estrous cycle (n = 3 ewes) or day-60 pregnancy (n = 3 ewes) CL cultured with different concentrations (10, 100 and 1000 ng/ml) of roPL, whereas treatment with oLH at the concentration of 100 or 1000 ng/ml caused a significant increase in progesterone secretion by explants from day-10 estrous cycle CL (P < 0.05) and by explants from day-60 pregnancy CL (P < 0.01). In conclusion, our results demonstrate that oPL has no luteotropic and/or luteoprotective actions in sheep, either in vivo or in vitro.

Identification of differentially regulated genes during elongation and early implantation in the ovine trophoblast using complementary DNA array screening.

Following hatching, pre-elongated conceptuses undergo elongation by intense proliferation, until implantation. We investigated the changes in gene expression associated with these physiological events using human cDNA arrays containing 2370 known genes. Comparison of pre-elongated, elongated, and implanting trophoblasts allowed the determination of 313 expressed genes, 63 of which were differentially regulated. These were classified into four functional families. Pre-elongated trophoblasts were characterized by preferential expression of genes involved in protein trafficking, whereas only latter developmental stages expressed cell signaling genes and receptors. Among the 63 developmentally regulated genes, four exhibited the highest levels of expression (TMSB10, CTNNA1, NMP1, and CX3CL1). Each of these also represents a functional family and display a specific expression pattern. One of them, CX3CL1 (CX3C chemokine, also known as fractalkine), is a chemokine that seems to have potential importance in trophoblast development, and which deserves further clarification of its role in implantation.