Unified Total Synthesis of Tetracyclic Diquinane Lycopodium Alkaloids (+)-Paniculatine, (-)-Magellanine, and (+)-Magellaninone.

A unified route for the total synthesis of three tetracyclic diquinane Lycopodium alkaloids (+)-paniculatine, (-)-magellanine, and (+)-magellaninone has been accomplished in 13-14 overall steps based on late-stage diverse transformations from an advanced tetracyclic common intermediate. In the established synthesis, quick formation of the two five-membered rings was efficiently achieved by an intramolecular reductive coupling of ketone-carbonyl and ester-carbonyl and an organocatalytic intramolecular Michael addition of aldehyde-derived enamine to an internal enone functionality with satisfactory redox and step economies and excellent stereoselectivities, providing the requisite tricyclic carbo-framework possessing multiple dense stereogenic centers, and an intramolecular reductive amination finally furnished the essential piperidine ring.

The impacts of laser zona thinning on hatching and implantation of vitrified-warmed mouse embryos.

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.

Progesterone, the maternal immune system and the onset of parturition in the mouse.

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.

Cloning, identification and function analysis of a Chibby homolog from Litopenaeus vannamei.

Chibby, a vital inhibitor molecule of Wnt/ß-catenin signaling pathway, participates in development and stem cell differentiation through the regulation of ß-catenin. Our previous studies have demonstrated that Litopenaeus vannamei ß-catenin (Lv-ß-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-ß-catenin. Moreover, the relationship of Lv-ß-catenin, Lv-Chibby and WSSV069 was investigated. It was shown that Lv-Chibby enhanced the interaction between Lv-ß-catenin and WSSV069. Interestingly, WSSV069 promoted the interaction between Lv-ß-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-ß-catenin. In sum, these results implied that Wnt/ß-catenin signal pathway plays an important role in the defense against virus, and Chibby could be modulated by WSSV to regulate the signal pathway.

Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells.

Cyclic AMP (cAMP) is the archetypal smooth muscle relaxant, mediating the effects of many hormones and drugs. However, recently PGI(2) , acting via cAMP/PKA, was found to increase contraction-associated protein expression in myometrial cells and to promote oxytocin-driven myometrial contractility. Cyclo-oxygenase-2 (COX-2) is the rate-limiting enzyme in prostaglandin synthesis, which is critical to the onset and progression of human labour. We have investigated the impact of cAMP on myometrial COX-2 expression, synthesis and activity. Three cAMP agonists (8-bromo-cAMP, forskolin and rolipram) increased COX-2 mRNA expression and further studies confirmed that this was associated with COX-2 protein synthesis and activity (increased PGE(2) and PGI(2) in culture supernatant) in primary cultures of human myometrial cells. These effects were neither reproduced by specific agonists nor inhibited by specific inhibitors of known cAMP-effectors (PKA, EPAC and AMPK). We then used shRNA to knockdown the same effectors and another recently described cAMP-effector PDZ-GEF(1-2) , without changing the response to cAMP. We found that MAPK activation mediated the cAMP effects on COX-2 expression and that PGE(2) acts through EP-2 to activate MAPK and increase COX-2. These data provide further evidence in support of a dual role for cAMP in the regulation of myometrial function.

Enantioselective Total Synthesis of (+)-Sieboldine A and Analogues Thereof.

An 11-step enantioselective total synthesis of (+)-sieboldine A (1) has been accomplished from (5R)-methylcyclohex-2-en-1-one (16), in which an intramolecular ketone/ester reductive coupling followed by one-pot acidic treatment to quickly construct the unique oxa-spiroacetal and a TsOH-catalyzed displacement to directly form the characteristic N-hydroxyazacyclononane ring successfully served as the key methodologies. Moreover, several full-skeleton analogues of 1 were also synthesized on the basis of the advanced intermediates, and their inhibitory effects on electric eel acetylcholinesterase were examined.

Modification of the Calculation Method for Dynamic Reserves in Tight Sandstone Gas Reservoirs.

The determination of dynamic reserves is important for tight sandstone gas reservoirs in production. Based on the geological and gas data of the Yan'an gas field, the influence of pressure on the properties of natural gas is studied by mathematical methods. At the same time, the modified flowing material balance equation is established considering the changes in gas viscosity and compressibility. The result shows that (1) the viscosity of natural gas increases rapidly with pressure; (2) the deviation factor decreases with pressure (P < 15 MPa) and then increases (P > 15 MPa) with temperature; (3) the compressibility decreases rapidly with pressure and increases with temperature; (4) compared with the results of the material balance method, the average error of the flowing material balance method is 33.95%, and the accuracy of the modified flowing material balance method is higher with an average error of 1.25%; and (5) a large change in the production will affect the accuracy of the modified flowing material balance method, especially a shut-in for a long time before the pressure drop production is calculated at a certain time, so data points that are relatively consistent should be selected as far as possible to calculate the dynamic reserves. The findings of this study can help in the accurate evaluation of dynamic reserves of the tight gas reservoir in the Yan'an gas field and are an important guide for the formulation of a rational plan for the gas reservoir and its economic and efficient development.

PKA and AKIP1 interact to mediate cAMP-driven COX-2 expression: A potentially pivotal interaction in preterm and term labour.

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-1ß-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-1ß-induced COX-2 mRNA expression and reduced the IL-1ß-induced increase in nuclear levels of p65 and c-jun. Forskolin alone and with IL-1ß 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-1ß-stimulated OTR mRNA expression. AKIP1 was up-regulated by IL-1ß, 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.

Labour classified by cervical dilatation & fetal membrane rupture demonstrates differential impact on RNA-seq data for human myometrium tissues.

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.

Molecular cloning and characterization of a class II ADP ribosylation factor from the shrimp Marsupenaeus japonicus.

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.

Changes in cAMP effector predominance are associated with increased oxytocin receptor expression in twin but not infection-associated or idiopathic preterm labour.

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.

The interaction between protein kinase A and progesterone on basal and inflammation-induced myometrial oxytocin receptor expression.

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-1ß 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-1ß-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1ß-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-1ß-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-1ß 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.

Genomic organization of the translationally controlled tumor protein (TCTP) gene from shrimp Marsupenaeus japonicus.

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.

Mild Hypothermia Prevents NO-Induced Cytotoxicity in Human Neuroblastoma Cells Via Induction of COX-2.

The cold-inducible protein RBM3 mediates hypothermic neuroprotection against nitric oxide (NO)-induced cell death. Meanwhile, it is well-known that cyclooxygenase-2 (COX-2) is upregulated by RBM3 in several types of cells; however, it is still unclear whether COX-2 contributes to the neuroprotective effects of mild hypothermia/RBM3 against NO-induced cell death. Using human SH-SY5Y neuroblastoma cells, it was revealed that NO remarkably downregulates the expression of COX-2 at both mRNA and protein levels. When COX-2 was silenced using siRNA technique, cells became more sensitive to NO-induced cell death. Conversely, the overexpression of COX-2 significantly prevented NO-induced cell death in SH-SY5Y cells, indicating a pro-survival role of COX-2. Upon mild hypothermia pre-treatment, COX-2 was notably induced at both mRNA and protein levels; however, COX-2 silencing abrogated hypothermia-related neuroprotection against NO-induced cell death. Furthermore, it was revealed that either silencing or overexpression of RBM3 had no effects on the expression of COX-2 in SH-SY5Y cells. These findings suggest that mild hypothermia could protect neuroblastoma cells against NO-induced cell death by inducing COX-2 in a RBM3-independent manner.

Myometrial Transcriptional Signatures of Human Parturition.

The process of parturition involves the transformation of the quiescent myometrium (uterine smooth muscle) to the highly contractile laboring state. This is thought to be driven by changes in gene expression in myometrial cells. Despite the existence of multiple myometrial gene expression studies, the transcriptional programs that initiate labor are not known. Here, we integrated three transcriptome datasets, one novel (NCBI Gene Expression Ominibus: GSE80172) and two existing, to characterize the gene expression changes in myometrium associated with the onset of labor at term. Computational analyses including classification, singular value decomposition, pathway enrichment, and network inference were applied to individual and combined datasets. Outcomes across studies were integrated with multiple protein and pathway databases to build a myometrial parturition signaling network. A high-confidence (significant across all studies) set of 126 labor genes were identified and machine learning models exhibited high reproducibility between studies. Labor signatures included both known (interleukins, cytokines) and unknown (apoptosis, MYC, cell proliferation/differentiation) pathways while cyclic AMP signaling and muscle relaxation were associated with non-labor. These signatures accurately classified and characterized the stages of labor. The data-derived parturition signaling networks provide new genes/signaling interactions to understand phenotype-specific processes and aid in future studies of parturition.

Corrigendum: Myometrial Transcriptional Signatures of Human Parturition.

[This corrects the article DOI: 10.3389/fgene.2019.00185.].

Difference between hemocyanin subunits from shrimp Penaeus japonicus in anti-WSSV defense.

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.

Genomic organization, promoter characterization and expression profiles of an antiviral gene PmAV from the shrimp Penaeus monodon.

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.

Effect of crystallization of settled aluminum hydroxide precipitate on "dissolved Al".

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.

Cyclic AMP Effectors Regulate Myometrial Oxytocin Receptor Expression.

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-1ß 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-1ß 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.