Mouse preimplantation embryo responses to culture medium osmolarity include increased expression of CCM2 and p38 MAPK activation

BACKGROUND: Mechanisms that confer an ability to respond positively to environmental osmolarity are fundamental to ensuring embryo survival during the preimplantation period. Activation of p38 mitogen-activated protein kinase (MAPK) occurs following exposure to hyperosmotic treatment. Recently, a novel scaffolding protein called Osmosensing Scaffold for MEKK3 (OSM) was linked to p38 MAPK activation in response to sorbitol-induced hypertonicity. The human ortholog of OSM is cerebral cavernous malformation 2 (CCM2). The present study was conducted to investigate whether CCM2 is expressed during mouse preimplantation development and to determine whether this scaffolding protein is associated with p38 MAPK activation following exposure of preimplantation embryos to hyperosmotic environments. RESULTS: Our results indicate that Ccm2 along with upstream p38 MAPK pathway constituents (Map3k3, Map2k3, Map2k6, and Map2k4) are expressed throughout mouse preimplantation development. CCM2, MAP3K3 and the phosphorylated forms of MAP2K3/MAP2K6 and MAP2K4 were also detected throughout preimplantation development. Embryo culture in hyperosmotic media increased p38 MAPK activity in conjunction with elevated CCM2 levels. CONCLUSION: These results define the expression of upstream activators of p38 MAPK during preimplantation development and indicate that embryo responses to hyperosmotic environments include elevation of CCM2 and activation of p38 MAPK

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten\u27s medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways

PP2Cdelta (Ppm1d, WIP1), an endogenous inhibitor of p38 MAPK, is regulated along with Trp53 and Cdkn2a following p38 MAPK inhibition during mouse preimplantation development.

Preimplantation embryos utilize mitogen-activated protein kinase signaling (MAPK) pathways to relay signals from the external environment to prepare appropriate responses and adaptations to a changing milieu. It is therefore important to investigate how MAPK pathways are regulated during preimplantation development. This study was conducted to investigate whether PP2Cdelta (Ppm1d, WIP1) is expressed during mouse preimplantation development and to determine the influences of p38 MAPK inhibition on expression of Trp53 (p53), Ppm1d, (WIP1), and Cdkn2a (p16) during mouse preimplantation development. Our results indicate that Trp53, Ppm1d, and Cdkn2a mRNAs and TRP53 and PP2Cdelta proteins are expressed throughout mouse preimplantation development. Treatment of 2-cell embryos with SB220025 (potent inhibitor of p38 MAPK alpha/beta/MAPK 14/11) significantly increased Trp53, Ppm1d and Cdkn2a and Mapk14 mRNA levels at 12 and 24 hr. Treatment of 8-cell embryos with SB220025 for 12 hr increased Trp53, Ppm1d, and Cdkn2a mRNA levels, but not Mapk14 mRNA levels. Treatment of 8-cell embryos for 24 hr increased Trp53, and Ppm1d mRNA levels, but decreased Cdkn2a and Mapk14 mRNA levels. Therefore, blockade of p38 MAPK activity is associated with embryo stage specific influences on Trp53, Ppm1d, Cdkn2a, and Mapk14 expression during mouse preimplantation development. These results define downstream targets of p38 MAPK during preimplantation development and indicate that the p38 MAPK pathway regulates Trp53, Ppm1d, and Cdkn2a expression. This study increases our understanding of the mechanisms controlling preimplantation development and of the interactions between preimplantation embryos and their culture environments

The ecotypic differentiation of populations: Altitudinal distribution of variation in Festuca

The investigation of the chromosome numbers of the fescue populations on Ben Cruachon shows that there are several distinct chromosome races. The setaceous-leaved sexual fescues are almost entirely diploid but a tetraploid also occurs; the broad-leaved sexual fescues include hexaploid and octoploid races as well as some heptaploids: the viviparous rescues are without exception tetraploid. 2. The viviparous fescues may be found throughout the area and occur all along each of the three gradients. In the sotaceous-leaved sexual fescue the tetraploid occurs at sea-level but the diploid race extends as high as 2,600 feet on Ban Oruaohon. The hexaploid race of broad-leaved sexual feseue has a range coincident with that of the sexual diploid although its occurrence on the hill is rather sporadic and the plants are rather difficult to identify. The octopiold and heptaploid races of the broad-leaved sexual fescues occur only at sea-level. 3. Chromosome counts of setaceous4eaved sexual fescues from various parts of Scotland, the Scottish islands, England and Wales indicate that the two chromosome races have a definite pattern of geographical distribution. The diploids occur in the North and islands whilst tetraploids are found in the South of Scotland, England and Wales but further investigations are necessary before any definite conclusions can be drawn. 4. The setaceous-leaved sexual fescues have been found to be capable of free interbreeding but are sexually distinct from the broad-leaved sexual rescues. 5. The hereditary variation which is present in each population makes it difficult to fit the plants into taxonomic groups but for reference purposes the setaceous-leaved sexual rescues are called Festuca ovine, the broad-leaved sexual rescues, Festueca. supra, and the viviparoue feeoues, yestuca yivipore. 6. A key for the identification of British viviperous fescues adapted by Wilmott from that proposed by Turesson is given in full and its application to the viviparous rescues from Ben Cruachan discussed. 7. The distribution of hereditary variation in F. ovina, F. rubre. and F. va on the three gradlente is discussed in the light of the statistical results obtained from the measurement data and an attempt is made to deduce reasonable, explanations through correlation with environmental conditions. 8. The problem of the origin of hereditary variation in the viviparous rescues is discussed: mention is made of the occurrence of partial and complete sexual flowers on hunger inflorescences late in the season and also of the production of completely sexual plants from the bulbils of a viviparous plant. 9. The taxonomic units of species, subspecies and variety are discussed with special reference to the possibility and advisability of equating them with the units of experimental taxonomy. 10. The numerous difficulties involved in taking into account the hereditary variation in populations can only be adequately resolved by applying the concept of the character gradient with reference to the related environment influence 11. The areal ecotype is a useful but limited concept since the information which it supplies is little more than that the population in question is tolerant of the ecological conditions. The acoclinel, however, takes into consideration the trends of hereditary variation which occur along an environmental gradient, end ecpclInpl"esatue4mav be defined for reference purpose. It is understandable that where a number of creel populations are investigated they way be found to occur on an environments gradient end they may in that ease be called ecoclineal subspecies such a classification permits of further definition within each ecoclinal subspecies of internal trends of variation. 12. The distribution of the hereditary variation within the fescue populations (F. ovina and V. vivioare) of Den Oruachan in respect of habit of growth may he defined as following a climatic gradient. In this case, three ecoclinal ecotypes have been named for reference purposes, erecta, ascrindent, end decumbent'. The climatic ecocline in respect of habit of growth is quite plain in spite of some irregularity at the lower levels, an irregularity which in part, at any rate, is probably due to the small else end comparative isolation of the populations. Climatic ecoclines in respect of several other attributes may also be traced. 103. 13. The chromosome races within F. rubra which occur at sea-level may be found to have distinct habitat preferences but no definite statement can be made until further research which is now being carried out, has been completed. 14. A review of the situation makes it clear that the units of orthodox taxonomy and those of experimental taxonomy must remain distinct. The classification of orthodox taxonomy has proved its usefulness and will continue to do so but the knowledge gained through the genecological approach will supplement it in a most valuable manner, 15. The study of trends of variation is not only of very great academic interest but it can also provide important data which are vital for the work of the plant breeder and for the economic success of agricultural crops

Mitogen-activated protein kinase (MAPK) pathways mediate embryonic responses to culture medium osmolarity by regulating Aquaporin 3 and 9 expression and localization, as well as embryonic apoptosis.

BACKGROUND: In order to advance the development of culture conditions and increase the potential for supporting normal preimplantation embryo development in vitro, it is critical to define the mechanisms that early embryos utilize to survive in culture. We investigated the mechanisms that embryos employ in response to culture medium osmolarity. We hypothesized that mitogen-activated protein kinase (MAPK) pathways mediate responses to hyperosmotic stress by regulating Aquaporin (AQP) 3 and 9 expression as well as embryonic apoptosis. METHODS: Real-time reverse transcription and polymerase chain reaction and whole-mount immunofluorescence were used to determine the relative mRNA levels and protein localization patterns of AQP 3 and 9 after hyperosmotic medium treatment. RESULTS: At 6 and 24 h, a significant increase in Aqp 3 and 9 mRNA was observed in the sucrose hyperosmotic treatment compared with standard medium and glycerol controls. Blockade of MAPK14/11 negated the increase in Aqp 3 and 9 mRNA levels, whereas culture in a MAPK8 blocker did not. Hyperosmotic sucrose treatment significantly increased embryonic apoptosis which was negated in the presence of MAPK8 blocker, but not MAPK14/11 blocker. CONCLUSIONS: MAPK14/11 activation is a component of the rapid adaptive stress response mechanism that includes the effects of AQP mRNA expression and protein localization, whereas the MAPK8 pathway is a regulator of apoptosis

The dynamics of HCV-specific antibody responses in HIV/HCV patients on long-term antiretroviral therapy

Antibody responses have not been fully characterised in chronically HIV/HCV patients receiving antiretroviral therapy (ART). Seventeen HIV/HCV patients receiving ART were followed for a median (range) interval of 597 (186–766) weeks. Prior to ART, HIV/HCV patients had lower levels of antibodies reactive with HCV core and JFH-1, and lower genotype cross-reactive neutralising antibodies (nAb) titres, than HCV patients. Levels of JFH-1 reactive antibody increased on ART, irrespective of CD4+ T-cell counts or changes in serum ALT levels. The appearance of nAb coincided with control of HCV viral replication in five HIV/HCV patients. In other patients, HCV viral loads remained elevated despite nAb responses. Sustained virological responses following HCV therapy were associated with reduced antibody responses to JFH-1 and core but elevated responses to non-structural proteins. We conclude that nAb responses alone may fail to clear HCV, but contribute to control of viral replication in some HIV/HCV patients responding to ART

Near infra-red luminescent osmium labelled gold nanoparticles for cellular imaging and singlet oxygen generation

Osmium(ii) complexes have attractive properties for potential theranostic agents given their anticancer activitiy, their redox potentials favourable for biological transformations within cancer cells and their luminescence in the near infrared (NIR) region. To achieve localised detection and delivery, gold nanoparticles (AuNP) provide an attractive scaffold to attach multiple luminescent agents on a single particle and provide a multimodal platform for detection and loaclaised delivery. We have developed 13 nm and 25 nm AuNP decorated with an osmium complex based on 1,10-phenantholine and surface active bipyridine ligands, OsPhenSS for live cell imaging and singlet oxygen generation, notated as OsPhenSS·AuNP13 and OsPhenSS·AuNP25. The AuNP designs not only allow versatile modalities for localisation of the probe but also water solubility for the osmium metal complex. The osmium decorated nanoparticles OsPhenSS·AuNP13 and OsPhenSS·AuNP25 display characteristic NIR luminescence from the osmium(ii) 3MLCT at 785 nm in aqueous solutions with visible excitation. Upon incubation of the nanoparticles in lung cancer and breast carcinoma the luminescence signature of osmium and the gold reflectance reveal localisation in the cytoplasmic and perinuclear compartments. Excitation of the nanoparticles at 552 nm in the presence of a ROS indicator revealed a marked increase in the green fluorescence from the indicator, consistent with photo-induced ROS generation. The detection of singlet oxygen by time-resolved luminescence studies of the osmium and the nanoparticle probes further demonstrates the dual activity of the osmium-based nanoprobes for imaging and therapy. The introduction of gold nanoparticles for carrying osmium imaging probes allows a novel versatile strategy combining detection and localised therapies at the nanoscale

Point-of-Care Tests to Strengthen Health Systems and Save Newborn Lives: The Case of Syphilis

Rosanna Peeling and colleagues describe their experience of introducing point-of-care testing to screen for syphilis in pregnant women living in low- and middle-income countries

The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure

Acetaminophen (APAP) is the main cause of acute liver failure in the West. Specific efficacious therapies for acute liver failure (ALF) are limited and time-dependent. The mechanisms that drive irreversible acute liver failure remain poorly characterized. Here we report that the recently discovered platelet receptor CLEC-2 (C-type lectin-like receptor) perpetuates and worsens liver damage after toxic liver injury. Our data demonstrate that blocking platelet CLEC-2 signalling enhances liver recovery from acute toxic liver injuries (APAP and carbon tetrachloride) by increasing tumour necrosis factor-α (TNF-α) production which then enhances reparative hepatic neutrophil recruitment. We provide data from humans and mice demonstrating that platelet CLEC-2 influences the hepatic sterile inflammatory response and that this can be manipulated for therapeutic benefit in acute liver injury. Since CLEC-2 mediated platelet activation is independent of major haemostatic pathways, blocking this pathway represents a coagulopathy-sparing, specific and novel therapy in acute liver failure

Dissemination of Metarhizium anisopliae of low and high virulence by mating behavior in Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>Dengue is a viral disease transmitted by <it>Aedes </it>mosquitoes. It is a threat for public health worldwide and its primary vector <it>Aedes aegypti </it>is becoming resistant to chemical insecticides. These factors have encouraged studies to evaluate entomopathogenic fungi against the vector. Here we evaluated mortality, infection, insemination and fecundity rates in <it>A. aegypti </it>females after infection by autodissemination with two Mexican strains of <it>Metarhizium anisopliae</it>.</p> <p>Methods</p> <p>Two <it>M. anisopliae </it>strains were tested: The Ma-CBG-1 least virulent (lv), and the Ma-CBG-2 highly virulent (hv) strain. The lv was tested as non mosquito-passed (NMP), and mosquito-passed (MP), while the hv was examined only as MP version, therefore including the control four treatments were used. In the first bioassay virulence of fungal strains towards female mosquitoes was determined by indirect exposure for 48 hours to conidia-impregnated paper. In the second bioassay autodissemination of fungal conidia from fungus-contaminated males to females was evaluated. Daily mortality allowed computation of survival curves and calculation of the LT₅₀by the Kaplan-Meier model. All combinations of fungal sporulation and mating insemination across the four treatments were analyzed by χ². The mean fecundity was analyzed by ANOVA and means contrasted with the Ryan test.</p> <p>Results</p> <p>Indirect exposure to conidia allowed a faster rate of mortality, but exposure to a fungal-contaminated male was also an effective method of infecting female mosquitoes. All females confined with the hv strain-contaminated male died in fifteen days with a LT₅₀of 7.57 (± 0.45) where the control was 24.82 (± 0.92). For the lv strain, it was possible to increase fungal virulence by passing the strain through mosquitoes. 85% of females exposed to hv-contaminated males became infected and of them just 10% were inseminated; control insemination was 46%. The hv strain reduced fecundity by up to 99%, and the lv strain caused a 40% reduction in fecundity.</p> <p>Conclusions</p> <p>The hv isolate infringed a high mortality, allowed a low rate of insemination, and reduced fecundity to nearly zero in females confined with a fungus-contaminated male. This pathogenic impact exerted through sexual transmission makes the hv strain of <it>M. anisopliae </it>worthy of further research.</p