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Purpose Phosphorus (P) is a limiting nutrient in many managed forests. To further understand the risks and benefits of biochars as sustainable P source in forest management, an improved mechanistic understanding of its interactions in root systems is required. Methods A rhizobox experiment was conducted to observe root response of P. sylvestris (Scots pine) seedlings to different biochars in comparison to triple superphosphate (TSP) fertiliser as a P source. Three types of wood-derived biochar were compared: biochar from mixed softwood pellets (“Reference biochar”); from the vascular cambium zone of Picea sitchensis (“VCZ biochar”) and from mixed softwood pellets infused with TSP (“Processed biochar”). These biochars presented a range of available P from low to high. Seedling root development was spatially analysed using GIS software. Results The total length of P. sylvestris roots did not significantly differ between treatments. However, seedling roots showed strong preference for soil proximal to VCZ biochar and strong avoidance to TSP fertiliser. There was a milder avoidance effect for Processed biochar. Differences in root responses could be explained by available P: roots favored a moderate, sustained P source and avoided high available P sources. The avoidance effect can be attributed partially to lower soil pH around TSP fertiliser. Conclusion The extent concentration and duration of P availability affects the root response of P. sylverstris to P sources. Under P-deficient conditions, P. sylvestris root growth was markedly improved by introducing biochar with a certain P concentration, and VCZ biochar has potential as an effective source of P in forest establishment.

Optimized slurry management targeted to increase nitrogen use efficiency (NUE) can be fundamental for limiting fertilizer N losses from agricultural grasslands causing eutrophication of ground- and surface water as well as air pollution. However, a holistic assessment of both agronomic and environmental impacts including key soil functions is still missing. This study aims to provide such information by assessing the impacts of traditional vs. modern slurry application techniques on NUE, hydrological and gaseous N losses, productivity and fodder quality, soil organic nitrogen formation and total N balances. In a plot-scale grassland experiment 15N enriched slurry was applied after the first cut in early summer. The application treatments included: (1) traditional slurry broadcast spreading under dry weather; (2) application like (1) followed by a heavy rainfall event to increase slurry infiltration into soil; (3) broadcast spreading of slurry diluted with water; (4) injection of slurry into soil via shallow slits; and (5) injection of slurry into soil via deep slits. Variant (4) and (5) represent modern trailing shoe injections requiring extensive machinery. Fates of fertilizer N such as plant uptake, immobilization in soil and microbial biomass as well as NO3 leaching were investigated by 15N tracing approaches in order to create full N balances. For this, biomass harvest and soil sampling were conducted after a growth period of 2 months. Here, we will present first results of our work that are expected to provide highly relevant decision support for grassland management.

Local environments such as soils and waterways, but also landscape-scale environments such as agricultural areas, are often classified as oxic based on the dominant conditions in such systems. Still, anoxic conditions do occur within these “oxic” landscapes, their upland and lowland soils, sediments and creeks at a wide range of spatiotemporal scales, and can represent hot spots and hot moments for greenhouse gas emissions but also carbon storage due to their strongly deviating biogeochemical character.Within the project “Bavarian landscapes under climate change”, located in the crystalline Bavarian Forest, Germany, we characterize and quantify the role of anoxic spots for greenhouse gas emissions and carbon storage at the scale of agricultural landscapes, including managed grassland and cropland soils, riparian soils and creeks including their special features such as beaver dams. Our focus is on mesoscale anoxic events, at the 1-10 m2 scale, that are the result of temporary conditions, such as flooding or fine sediment deposition. We place these mesoscale events into the context of the whole agricultural landscape and watershed, and characterize the carbon species, gaseous emissions, carbon stocks, and organic carbon degradation dynamics in the different oxic and anoxic parts of our larger system, both aquatic and terrestrial. First results indicate that the enhanced greenhouse gas emissions, but also carbon storage, can be large enough to be relevant offsets of the larger scale (whole stream, riparian zone) carbon dynamics. Ignoring these spatially or temporally relatively small events or locations in upscaling or modeling can thus lead to important underestimations of carbon storage, as well as to offsets of the greenhouse gas balance of an area.

The impacts of land use on organic carbon (OC) and nitrogen (N) in different ecosystems have been widely studied. However, less attention has been paid to its influence on the change of total phosphorus (Pt) and its two fractions, inorganic P (Pi) and organic P (Po), along slopes. In addition, since N and P are the main factors causing the eutrophication of aquatic ecosystems, it is essential to link the terrestrial and aquatic ecosystems.Two slope-floodplain-creek catenas were chosen in the low mountain region of the eastern Bavarian Forest. The slopes of the two catenas have been extensively managed for grassland and cropland use, respectively. The floodplain was under grassland use. They were fertilized with sludge, occasionally with farmyard manure, under the same regime. Additional carbonic magnesium lime was applied annually at the cropland. Soil profiles at the top, middle and bottom slope, as well as the floodplain area, were sampled according to their horizons and replicated with three soil cores in each catena. Liquid-nitrogen freeze-cores of the aquatic sediments were sampled to extend each catena into the below-slope creek.We found the grassland slope had higher OC and N content in the topsoil (0-10 cm) and higher Pt and Po content in both the topsoil (0-40 cm) and subsoil (40-60 cm). The P content in the topsoil (0-40cm) of the grassland slope was dominated by Po, while the cropland slope was Pi -dominated. The OC and N stocks on the grassland slope were significantly higher than at the corresponding position on the cropland slope. Except for the lowest P stock found on the top slope of grassland, the Pt stock in topsoil (0-20 cm) was not land-use affected while the Po stock was significantly higher on the grassland slope. An accumulation of OC, N and Po in topsoil (0-20 cm) from top to bottom of the slope was observed. In the floodplain, upslope cropland use decreased the OC, N, Pt content and stocks, but reversely for Po. The upslope land use effect on OC, N and P content in the creek sediment was limited.In conclusion, this study provides a field-based observation on the change of OC, N and P (Pt and Po) under different land use upslope from terrestrial to aquatic systems through two slope-floodplain-creek catenas.

In order to study the stress sensitivity of tight sand reservoir and evaluate their impact on the productivity, physical and mathematical simulation is carried out under the scanning electron microscopy (SEM), the casting thin sections and the stress sensitivity test technology. The results show that porosity and permeability decreases with pressure, the damage mode of porosity and the permeability can be described as “Falling quickly then falling slowly.” The relative loss rate of porosity is from 4.88% to 8.46%, meanwhile, permeability has stronger stress sensitivity and the relative loss rate of permeability is from 63.86% to 86.14%. The porosity stress sensitivity is connected with the microscopic pore structure, pore size and rock particles compressive capacity, and permeability is mainly affected by the pore distribution and pore size distribution. A productivity equation of tight reservoir in which the stress sensitivity of the porosity and permeability is considered shows that as the reservoir properties become worse, the stronger the rock sensitivity, the higher the degree of impact on the production. This study provides reference for reducing sensitivity damage in tight sandstone reservoirs and improving gas recovery.

The determination of dynamic reserves is an important basis for rational production allocation. Based on the Yan’an Gas Field, the modified flowing material balance equation is established considering the changes of gas viscosity and compressibility. At the same time, a reasonable production allocation method for gas wells is developed. The result shows: ① μ gCg decreases with pressure and increases with temperature. ② The error of the modified flowing material balance method is smaller compared with the flow material balance method, and the error is 1.25 %. ③ The great change of the production will affect the accuracy of the modified flow material balance method, especially the shut-in for a long time before the pressure drop production is calculated at a certain time, so the points with relatively constant should be selected as far as possible. ④ When the dynamic reserves are used for allocation, the initial decline rate decreases by 74.65 % on average, and the stable production period increases by 21.28 % and the stable production period increases by 1.79 % compared with the allocation method of open flow rate. ⑤ When there is a large error of dynamic reserves, it will lead to production deviation from the reasonable value. The findings of this study can help for the accurate evaluation of dynamic reserves and reasonable production allocation of tight gas wells in the Yan’an gas field, and is an important guide for the formulation of a rational plan for the gas reservoir and its economic and efficient development.

An 11-step enantioselective total synthesis of (+)-sieboldine A (

The determination of dynamic reserves of gas well is an important basis for rational production allocation and development of a single well. The commonly used flow material balance method (FMB method) uses the slope of the curve of wellhead pressure and cumulative production after stable production of gas well to replace the slope of the curve of average formation pressure and cumulative production to calculate the controlled reserves of single well. However, based on the theoretical calculation, the FMB method ignores the change of natural gas compression coefficient, viscosity and deviation coefficient in the production process. After considering these changes, the slope of the curve of the relationship between bottom hole pressure and cumulative production and the slope of the curve of the relationship between average formation pressure and cumulative production are not equal. In order to solve this problem, the influence of pressure on each parameter is considered, and the equation of modified flowing material balance method is derived. The application of Yan'an gas field in Ordos Basin shows that: compared with the results of the material balance method, the result of the flow material balance method is smaller, and the maximum error is 58.816%. The consequence of the modified mobile material balance method is more accurate, and the average error is 2.114%, which has good applicability. This study provides technical support for an accurate evaluation of dynamic reserves of tight gas wells in Yan'an gas field, and has important guiding significance for economic and efficient development of gas reservoir.

The formation of sandstone-hosted uranium deposits has been linked to upward migration of hydrocarbons in many sedimentary basins. However, hydrocarbon-induced diagenetic alterations associated with uranium mineralization and preservation are rarely discussed. Here, we report the bleaching of red sandstone to green in the Middle Jurassic Zhiluo Formation from the uranium deposits in the northern Ordos Basin, China. The original gray sandstone underwent oxidation and was reddened by the downward flow of meteoric water from the northern margin of the basin during the Late Cretaceous–Cenozoic uplift. This process resulted in the leaching of preore-stage uranium concentration and oxidation of preore pyrite in the organic matter-rich sandstone. Uranyl and sulfate ions were carried downward through the host sandstone by oxygenated groundwater. Meanwhile, hydrocarbons migrated upward and reacted with the downward flowing oxidizing fluids, which induced uranium mineralization at the redox interface between the red and gray sandstone where iron was reduced to Fe2+ and incorporated into pyrite cement. The highly negative 34S values (−58.0‰ to −33.4‰ V-CDT) of the pyrite indicate that it originated from bacterial sulfate reduction during hydrocarbon biodegradation. Abundant organic acids and CO2 were generated from hydrocarbon oxidation, resulting in the dissolution of feldspars and the precipitation of kaolinite in the mineralized sandstone. With the formation of the Cenozoic Hetao graben to the north, the gravity-driven flow of meteoric water was limited, resulting in progressive halting of epigenetic uranium mineralization. Then, the upward hydrocarbons dominated the fluid system throughout the northern slope of the basin and bleached the previous red sandstone updip, which set up a strong reducing environment and protected the early formed uranium deposits at the paleo-redox front from oxidation and remobilization. Due to the oxygen and sulfur supply absence, the iron released from hematite during bleaching was immobilized and incorporated into Fe-rich chlorite that imparts the green color, rather than into pyrite or reprecipitation as hematite. This study provides insights for the processes of hydrocarbon-water-rock interactions associated with uranium mineralization and preservation, which can serve as exploration guides for sandstone-hosted uranium deposits.

Previously, we showed that cAMP increased COX-2 expression in myometrial cells via MAPK. Here, we have extended these observations, using primary myometrial cell cultures to show that the cAMP agonist, forskolin, enhances IL-1β-driven COX-2 expression. We then explored the role of A-kinase interacting protein (AKIP1), which modulates the effect of PKA on p65 activation. AKIP1 knockdown reversed the effect of forskolin, such that its addition inhibited IL-1β-induced COX-2 mRNA expression and reduced the IL-1β-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1β increased IκBα mRNA expression suggesting that in the context of inflammation and in the presence of AKIP1, cAMP enhances p65 activation. AKIP1 knockdown reversed these changes. Interestingly, AKIP1 knockdown had minimal effect on the ability of forskolin to repress either basal OTR expression or IL-1β-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1β, but not stretch and was repressed by cAMP. The mRNA expression of AKIP1 increased in early labour in tandem with an increase in COX-2 mRNA and protein. AKIP1 protein levels were also increased with inflammation and stretch-induced preterm labour. Our results identify a second important cAMP effector-switch occurring at term in human myometrium and suggest that a hitherto unrecognized interaction may exist between AKIP1, NFκB and AP-1. These data add to the proposition that cAMP acts as a key regulator of human myometrial contractility.

We previously reported that at term pregnancy, a decline in myometrial protein kinase A (PKA) activity leads to an exchange protein activated by cyclic AMP (Epac1)-dependent increase in oxytocin receptor (OTR) expression, promoting the onset of labour. Here, we studied the changes in the cyclic adenosine monophosphate (cAMP) effector system present in different phenotypes of preterm labour (PTL). Myometrial biopsies obtained from women with phenotypically distinct forms of PTL and the levels of PKA and OTR were examined. Although we found similar changes in the cAMP effector pathway in all forms of PTL, only in the case of twin PTL (T-PTL) was myometrial OTR levels increased in association with these results. Although there were several changes in the mRNA levels of components of the cAMP synthetic pathway, the total myometrial cAMP levels did not change with the onset of any subtype of PTL. With regards to the expression of cAMP-responsive genes, we found that the mRNA levels of 4 of the 5 cAMP-down-regulated genes were increased in T-PTL, similar to our findings in term labour. These data signify that although changes in the cAMP effector system were common to all forms of PTL, only in T-PTL were OTR levels increased. Similarly, the mRNA levels of cAMP-repressed genes were only increased in T-PTL supporting the concept that the decline in PKA levels influences myometrial function driving the onset of T-PTL.

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1β to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1β-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1β-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1β-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1β alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.

High throughput sequencing has previously identified differentially expressed genes (DEGs) and enriched signalling networks in human myometrium for term (≥37 weeks) gestation labour, when defined as a singular state of activity at comparison to the non-labouring state. However, transcriptome changes that occur during transition from early to established labour (defined as ≤3 and >3 cm cervical dilatation, respectively) and potentially altered by fetal membrane rupture (ROM), when adapting from onset to completion of childbirth, remained to be defined. In the present study, we assessed whether differences for these two clinically observable factors of labour are associated with different myometrial transcriptome profiles. Analysis of our tissue (‘bulk’) RNA-seq data (NCBI Gene Expression Omnibus: GSE80172) with classification of labour into four groups, each compared to the same non-labour group, identified more DEGs for early than established labour; ROM was the strongest up-regulator of DEGs. We propose that lower DEGs frequency for early labour and/or ROM negative myometrium was attributed to bulk RNA-seq limitations associated with tissue heterogeneity, as well as the possibility that processes other than gene transcription are of more importance at labour onset. Integrative analysis with future data from additional samples, which have at least equivalent refined clinical classification for labour status, and alternative omics approaches will help to explain what truly contributes to transcriptomic changes that are critical for labour onset. Lastly, we identified five DEGs common to all labour groupings; two of which (AREG and PER3) were validated by qPCR and not differentially expressed in placenta and choriodecidua.

Tabular–type uranium ore deposits (the Hangjinqi and Daying deposits) have recently been found in the Middle Jurassic Zhiluo Formation, north of the Ordos Basin, China. Petrographic observations, the chemical composition of U minerals determined by EMPA and fs–LA–ICP–MS, whole rock geochemistry and the microthermometric study of fluid inclusions have been integrated to characterize the genetic conditions of the U mineralization in the Hangjinqi sandstone–hosted deposit. Two different groups of U minerals have been identified. One group includes coffinite(I) associated with vanadium–rich micas. Coffinite(I) is enriched in vanadium (V) and devoid of iron (Fe) and yttrium (Y) and has a LREE–enriched chondrite–normalized REE pattern. The U minerals of this group are similar to meteoric fluid infiltration related deposits. The second group has coeval coffinite(II) and coarsely crystalline calcite cement. Coffinite(II) is enriched in Y and Fe and depleted in V and is marked by a flat chondrite–normalized REE pattern, which is compatible with typical hydrothermal genetic deposits with high–salinity mineralizing fluids. The temperature and salinity of the primary aqueous inclusions in the ore–stage calcite are 120–180 °C and 8.00–16.34% (eq. wt% NaCl), respectively. These mineral assemblages, temperatures and salinities indicate that the Hangjinqi deposit was affected by two distinct types of ore–bearing fluids: low–salinity meteoric waters and high–salinity hydrothermal fluids. The meteoric fluids event began at 97 ± 5 Ma with the titling of the northern Ordos Basin and the uplift of the Hetao region to the north. Hydrothermal U mineralization occurred since 39 ± 2 Ma with the rifting of the Hetao graben. Thus, the previous biogenic model for the U mineralization should be modified in the uraniferous region of the north Ordos Basin.

The factors that initiate human labor are poorly understood. We have tested the hypothesis that a decline in cAMP/protein kinase A (PKA) function leads to the onset of labor. Initially, we identified myometrial cAMP/PKA-responsive genes (six up-regulated and five down-regulated genes) and assessed their expression in myometrial samples taken from different stages of pregnancy and labor. We found that the oxytocin receptor (OTR) was one of the cAMP-repressed genes, and, given the importance of OTR in the labor process, we studied the mechanisms involved in greater detail using small interfering RNA, chemical agonists, and antagonists of the cAMP effectors. We found that cAMP-repressed genes, including OTR, increased with the onset of labor. Our in vitro studies showed that cAMP acting via PKA reduced OTR expression but that in the absence of PKA, cAMP acts via exchange protein activated by cAMP (EPAC) to increase OTR expression. In early labor myometrial samples, PKA levels and activity declined and Epac1 levels increased, perhaps accounting for the increase in myometrial OTR mRNA and protein levels at this time. In vitro exposure of myometrial cells to stretch and IL-1β increased OTR levels and reduced basal and forskolin-stimulated cAMP and PKA activity, as judged by phospho-cAMP response element-binding protein levels, but neither stretch nor IL-1β had any effect on PKA or EPAC1 levels. In summary, there is a reduction in the activity of the cAMP/PKA pathway with the onset of human labor potentially playing a critical role in regulating OTR expression and the transition from myometrial quiescence to activation.

Embryo vitrification has advantages in assisted reproduction yet it also induces zona hardening. Laser zona thinning (LZT) is considered as a solution yet its efficacy and security have not been well studied. In this study, we used vitrified-warmed morulae from 2-month-old and 10-month-old ICR female mice as model to investigate the impacts that LZT treatment brings to the in vitro hatching process and implantation by analyzing hatching rate, implantation rate, and blastocyst quality. The results showed that the fully hatched rate was significantly higher after LZT treatment for both young (25.7% vs. 16.2%, P 0.05) and aged (36.6% vs. 13.2%, P 0.01) mice. For zona-thinned morulae in young mice, its onset of hatching occurred earlier (28.6% vs. 8.8%, P 0.01) at D4 and with a greater percentage of U-shaped hatching at D5 (48.3% vs. 33.0%, P 0.05). LZT treatment did not induce expression change of apoptosis-related genes in all groups (P 0.05), but for young mice, the total cell number of day 5 blastocyst in zona-thinned group was significantly less than that of the control group (40.6 ± 5.1 vs. 59.9 ± 14.5, P 0.01). At last, there was an increasing implantation rate in zona-thinned compared to the control group for young (63.8% vs. 52.5%, P 0.05) and aged (55.6% vs. 47.2%; P 0.05) mice after embryos were bilaterally transferred in the same recipient. In conclusion, the significant increase of fully hatched rate after LZT treatment is related to the advanced onset of hatching as well as the enhancement of superior hatching structure, and LZT also lead to a better implantation after embryo transfer.

When aluminum salts are added to water at around neutral pH, a precipitate of Al hydroxide is formed very rapidly. Initially the precipitate is in the form of nano-scale primary particles, which then aggregate to form flocs. The nature of the flocs depends greatly on the solution composition, for instance on the presence of humic acid (HA), which not only increases the size of the primary nanoparticles, but also decreases the connection points between them. The nanoparticles become smaller with aging, both with and without HA, as a result of crystallization. The aggregated amorphous nanoparticles (settled flocs) undergo a room temperature structural modification best characterized as a disorder-to-order transition, following elimination of water. During this process, the apparent Al concentration in the supernatant of water increases with age. The "dissolved Al" concentration in the supernatant becomes higher with increasing pH and, to some extent, in the presence of HA. However, it can be shown that the "dissolved Al" in the supernatant exists in the form of crystalline nano-particles or larger clusters, which are detached from the settled flocs. TEM results confirmed that HA only adsorbed on the surface of nano-particles during the coagulation process, which shows precipitate nanoparticles formed firstly during sweep coagulation before the adsorption of HA or complexed Al3+-HA. However, the adsorbed outer layer of HA does not change the crystallization process for the inner part of nano-particles. This laboratory study may have implications for the release of Al from sediments into lake water, following addition of coagulants to lower phosphorus concentrations.

Chibby, a vital inhibitor molecule of Wnt/β-catenin signaling pathway, participates in development and stem cell differentiation through the regulation of β-catenin. Our previous studies have demonstrated that Litopenaeus vannamei β-catenin (Lv-β-catenin) was involved in WSSV infection and could inhibit virus replication by modulating the host immune system. In the study, a Chibby homolog from L. vannamei (designed as Lv-Chibby) was isolated and its role in WSSV infection was investigated. Sequence analysis suggested that Lv-Chibby was a novel homolog of Chibby family. It could transcript in all examined tissues, including hemocyte, gill, intestine, hepatopancreas, muscle and heart. Real-time quantitative PCR demonstrated that Lv-Chibby could take part in WSSV infection and be down-regulated by WSSV. Further studies confirmed that Lv-Chibby was able to interact with Lv-β-catenin. Moreover, the relationship of Lv-β-catenin, Lv-Chibby and WSSV069 was investigated. It was shown that Lv-Chibby enhanced the interaction between Lv-β-catenin and WSSV069. Interestingly, WSSV069 promoted the interaction between Lv-β-catenin and Lv-Chibby under high concentration, while low concentration of WSSV069 inhibited their interaction. A subsequent immunofluorescence assay revealed that WSSV069 appeared to reduce the nuclear entry of Lv-β-catenin. In sum, these results implied that Wnt/β-catenin signal pathway plays an important role in the defense against virus, and Chibby could be modulated by WSSV to regulate the signal pathway.

The role of progesterone (P4) in the regulation of the local (uterine) and systemic innate immune system, myometrial expression of connexin 43 (Cx-43) and cyclooxygenase 2 (COX-2), and the onset of parturition was examined in (i) naïve mice delivering at term; (ii) E16 mice treated with RU486 (P4-antagonist) to induce preterm parturition; and (iii) in mice treated with P4 to prevent term parturition. In naïve mice, myometrial neutrophil and monocyte numbers peaked at E18 and declined with the onset of parturition. In contrast, circulating monocytes did not change and although neutrophils were increased with pregnancy, they did not change across gestation. The myometrial mRNA and protein levels of most chemokines/cytokines, Cx-43, and COX-2 increased with, but not before, parturition. With RU486-induced parturition, myometrial and systemic neutrophil numbers increased before and myometrial monocyte numbers increased with parturition only. Myometrial chemokine/cytokine mRNA abundance increased with parturition, but protein levels peaked earlier at between 4.5 and 9 h post-RU486. Cx-43, but not COX-2, mRNA expression and protein levels increased prior to the onset of parturition. In mice treated with P4, the gestation-linked increase in myometrial monocyte, but not neutrophil, numbers was prevented, and expression of Cx-43 and COX-2 was reduced. On E20 of P4 supplementation, myometrial chemokine/cytokine and leukocyte numbers, but not Cx-43 and COX-2 expression, increased. These data show that during pregnancy P4 controls myometrial monocyte infiltration, cytokine and prolabor factor synthesis via mRNA-dependent and independent mechanisms and, with prolonged P4 supplementation, P4 action is repressed resulting in increased myometrial inflammation.

Cyclic AMP (cAMP) has been shown to promote progesterone and glucocorticoid action in a variety of cellular settings. In this study, we have used human myometrial cells to investigate whether cAMP potentiates the ability of progesterone to repress IL-1β-driven COX-2 expression. We found that forskolin enhanced progesterone-repression of IL-1β-driven COX-2 expression in association with delayed IL-1β-induced nuclear phospho-p65 entry and reduced NF-κB binding to the COX-2 promoter. Further, forskolin enhanced the progesterone-induced expression of FKBP5 and 11βHSD1, progesterone-driven activity of a progesterone response element (PRE) and progesterone receptor (PR)-B binding to a transfected PRE. In addition, forskolin treatment increased PR-B levels and reduced the PR-A:PR-B ratio while acutely decreasing the association between PR and nuclear receptor co-repressor (NCoR) and reducing NCoR levels after 6h. These findings are of importance in situations where enhancing progesterone activity is desirable, for example in the management of endometrial cancer, the promotion of endometrial receptivity or the maintenance of myometrial quiescence during pregnancy.

Myometrial explants represent a superior model compared with cell culture models for the study of human myometrial progesterone (P4) signalling in parturition.Gene expression analysis showed myometrial explants closely resemble the in vivo condition and the anti-inflammatory action of P4 is not lost with labour onset.Circulating P4 levels decline before the onset of parturition in most animals, but not in humans. This has led to the suggestion that there is a functional withdrawal of P4 action at the myometrial level prior to labour onset. However, to date, no evidence of a loss of P4 function has been provided, with studies hampered by a lack of a physiologically relevant model.Myometrial biopsies obtained at Caesarean section were dissected into explants after a portion was immediately snap frozen (t = 0). Microarray analysis was used to compare gene expression of t = 0 with paired (i) explants, (ii) passage 4 myometrial cell cultures or (iii) the hTERT myometrial cell line. Western blotting and chemokine/cytokine assays were used to study P4 signalling in myometrial explants.Gene expression comparison of t = 0 to the three models demonstrated that explants more closely resemble the in vivo status. At the protein level, explants maintain both P4 receptor (PR) and glucocorticoid receptor (GR) levels versus t = 0 whereas cells only maintain GR levels. Additionally, treatment with 1 µM P4 led to a reduction in interleukin-1 (IL-1) β-driven cyclooxygenase-2 in explants but not in cells. P4 signalling in explants was PR-mediated and associated with a repression of p65 and c-Jun phosphorylation. Furthermore, the anti-inflammatory action of P4 was maintained after labour onset.There is evidence of basal inflammation in the myometrial explant model.Myometrial explants constitute a novel model to study P4 signalling in the myometrium and can be used to further elucidate the mechanisms of P4 action in human labour.Data deposited at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=gvmpggkurbgxfqfacc=GSE77830.This work was supported by grants from the Joint Research Committee of the Westminster Medical School Research Trust, Borne (No. 1067412-7; a sub-charity of the Chelsea and Westminster Health Charity) and the Imperial NIHR Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NHS or the Department of Health. The authors have no conflict of interest.

Progesterone administration reduces the risk of preterm labor in high-risk women with singleton pregnancies but has no effect in women with a multiple pregnancy.We investigated whether progesterone is able to inhibit stretch-induced gene expression and/or whether stretch in turn inhibits progesterone action, perhaps providing an explanation for the functional progesterone withdrawal associated with human labor.In a series of in vitro studies using primary cultures of human myometrial cells, we found that preincubation with progesterone did not block stretch-induced ERK1/2 activation and cyclooxygenase-2 mRNA expression. Furthermore, we found that stretch did not alter the ability of progesterone to: 1) modulate progesterone-responsive gene expression; 2) activate a luciferase-linked progesterone response element; or 3) repress IL-1β-driven cyclooxygenase-2 mRNA expression. We did find that stretch reduced the expression of progesterone receptor mRNA via nuclear factor κB activation but that this did not alter myometrial progesterone response.These data show that progesterone does not inhibit stretch-induced MAPK activation or gene expression, possibly explaining why progesterone is ineffective in the prevention of preterm labor in multiple pregnancy. Although stretch did reduce progesterone receptor expression in a nuclear factor κB-dependent manner, this was not sufficient to inhibit progesterone action, suggesting that it is not responsible for the functional progesterone withdrawal observed with the onset of human labor.

The shrimp translationally controlled tumor protein (TCTP) gene was previously found up-regulated in response to viral infection. In order to further understand the transcriptional regulation of TCTP gene expression under viral challenge, the 5'-flanking promoter region of the TCTP gene in shrimp Marsupenaeus japonicus (Mj-TCTP) was cloned by using genomic walking method. The sequence shows that this region contains many important potential binding sites for transcription factors, such as activator protein1 (AP1), cAMP response element binding protein (CREB), Ets like transcription factor-1 (Elk-1), suggesting its expression is highly regulated. The promoter activity of this 5'-flanking region was confirmed by fusing to the enhanced green fluorescence protein (EGFP) gene and testing in Trichoplusia ni High Five cell line. Along with the introns and exons cloned in this study, the Mj-TCTP DNA sequence was reported here as the first TCTP gene with complete genomic organization in crustaceans.

Progesterone (P4) maintains uterine quiescence during pregnancy and its functional withdrawal is associated with increased prostaglandin synthesis and the onset of labor. In primary human myometrial cells, the glucocorticoid receptor (GR) rather than the P4 receptor mediates P4 antagonism of IL-1β-induced cyclooxygenase-2 (COX-2) expression, the rate-limiting enzyme in prostaglandin synthesis. We now report that P4 also acts via GR to induce MAPK phosphatase (MKP)-1 and knockdown of MKP-1 impairs the ability of P4 to repress IL-1β-dependent COX-2 induction. Microarray analysis revealed that P4 repressed preferentially activator protein-1-responsive genes in response to IL-1β. Consistent with these observations, we found that the ability of P4 to reduce c-Jun activation was lost upon GR as well as MKP-1 knockdown. Interestingly, c-Jun levels in human myometrial cells declined upon GR and MKP-1 knockdown, which suggests the presence of an activator protein-1 feedback loop. This is supported by our observation that c-Jun levels declined after an initial rise in primary myometrial cells treated with phorbol 12-myrisatate 13-acetate, a potent activator of c-Jun N-terminal kinase. Finally, we show that MKP-1 is an intermediate in P4-mediated repression of some but not all IL-1β-responsive genes. For example, P4 repression of IL11 and IRAK3 was maintained upon MKP-1 knockdown. Taken together, the data show that P4 acts via GR to drive MKP-1 expression, which in turn inhibits IL-1β-dependent c-Jun activation and COX-2 expression.

Inflammation plays a central role in many human diseases. Human parturition also resembles an inflammatory reaction, where progesterone (P4) and progesterone receptors (PRs) have already been demonstrated to suppress contraction-associated gene expression. In our previous studies, we have found that the progesterone actions, including progesterone-induced gene expression and progesterone's anti-inflammatory effect, are mediated by PR, GR or both. In this study, we used microarrays (GSE68171) to find P4 and IL-1β responsive genes and IL-1β responsive genes which were repressed by P4. These data may provide a broader view of gene networks and cellular functions regulated by P4 and IL-1β in human myometrial cells. These data will also help us understand the role of PR and GR in human parturition.

Progesterone is widely used to prolong gestation in women at risk of preterm labour (PTL), and acts at least in part via the inhibition of inflammatory cytokine-induced prostaglandin synthesis. This study investigates the mechanisms responsible for this inhibition in human myometrial cells. We used reporter constructs to demonstrate that interleukin 1beta (IL-1β) inhibits progesterone driven PRE activation via p65 activation and that IL-1β reduced progesterone driven gene expression (FKBP5). Conversely, we found that the activity of a p65-driven NFκB reporter construct was reduced by overexpression of progesterone receptor B (PRB) alone and that this was enhanced by the addition of MPA and that both MPA and progesterone suppressed IL-1β-driven cyclo-oxygenase-2 (COX-2) expression. We found that over-expressed Halo-tagged PRB, but not PRA, bound to p65 and that in IL-1β-treated cells, with no overexpression of either PR or p65, activated p65 bound to PR. However, we found that the ability of MPA to repress IL-1β-driven COX-2 expression was not enhanced by overexpression of either PRB or PRA and that although the combined PR and GR antagonist Ru486 blocked the effects of progesterone and MPA, the specific PR antagonist, Org31710, did not, suggesting that progesterone and MPA act via GR and not PR. Knockdown using siRNA confirmed that both MPA and progesterone acted via GR and not PR or AR to repress IL-1β-driven COX-2 expression. We conclude that progesterone acts via GR to repress IL-1β-driven COX-2 activation and that although the interaction between p65 and PRB may be involved in the repression of progesterone driven gene expression it does not seem to be responsible for progesterone repression of IL-1β-induced COX-2 expression.

In shrimp, small GTPases in the Ras superfamily can regulate hemolytic phagocytosis against WSSV infection. However, the ADP ribosylation factors (Arfs), also belonging to the regulatory GTP-binding proteins and playing a central role in membrane trafficking, have not been reported yet in shrimp and their relationship with WSSV infection is completely unknown to date. Here, a novel class II Arf (designated as MjArf4) was cloned and characterized from the shrimp Marsupenaeus japonicus. Like other Arfs, MjArf4 contains an N-terminal myristoylated site, a p loop, switch regions, as well as an interswitch region. In High Five cells, when MjArf4 was in its GDP-bound form, it dispersed into the whole cell, whereas in the GTP-bound form it promoted formation of a punctuate Golgi-like structure, indicating that the MjArf4 distribution was dependent on its GDP/GTP binding. After challenge with WSSV, the mRNA level of MjArf4 was up-regulated significantly as WSSV propagated. Thus, a member of the Arf family was characterized for the first time in shrimp and found to be involved in WSSV infection.

Hemocyanin, the respiratory protein of arthropods and mollusks, was found to function against bacterial and fungal invasion. In this study, we show that this protein complex could delay the infection of white spot syndrome virus (WSSV) in vivo and cloned two hemocyanin subunits genes (PjHcL and PjHcY) from the shrimp Penaeus japonicus. Further investigation of their transcriptional regulation during viral invasion showed that in contrast to subunit PjHcY, PjHcL expression could be strongly induced by WSSV infection. These findings not only reveal an important role of hemocyanin in shrimp antiviral defense but also suggest a possible discrepancy between the two subunits in shrimp innate immunity.

Diseases caused by viruses are the greatest challenge to worldwide shrimp aquaculture. PmAV is the first antiviral gene identified from the shrimp and its mRNA level is up-regulated in response to viral infection ( Luo et al., 2003 ). The transcriptional regulation of PmAV is critical since the expression of this gene is regulated in response to viral stimuli. In this study, we show that the Penaeus monodon PmAV gene contains four exons, separated by three introns. The 5′-flanking sequence of PmAV gene consists of a typical TATA box, several potential regulatory elements, and a compound microsatellite repeat as well. Interestingly, a TATA box and several regulatory elements also exist in the first intron (intron 1). Both intron 1 and the microsatellite are involved in transcriptional regulation of the PmAV gene. PmAV promoter activity depends on the existence of intron 1 while the microsatellite in 5′-flanking sequence acts as a negative regulative element. In addition, using Real-time PCR, we found that PmAV was highly expressed in the hepatopancreas and was up-regulated from the second day post-infection with the increase of viral load. These results extend our previous findings and provide insights into the molecular regulation of PmAV gene expression, which will be helpful for shrimp viral disease control.