Rayleigh-Instability-Induced Bismuth Nanorod@Nitrogen-Doped Carbon Nanotubes as A Long Cycling and High Rate Anode for Sodium-Ion Batteries.

Sodium-ion battery (SIB) as one of the most promising large-scale energy storage devices has drawn great attention in recent years. However, the development of SIBs is limited by the lacking of proper anodes with long cycling lifespans and large reversible capacities. Here we present rational synthesis of Rayleigh-instability-induced bismuth nanorods encapsulated in N-doped carbon nanotubes (Bi@N-C) using Bi2S3 nanobelts as the template for high-performance SIB. The Bi@N-C electrode delivers superior sodium storage performance in half cells, including a high specific capacity (410 mA h g-1 at 50 mA g-1), long cycling lifespan (1000 cycles), and superior rate capability (368 mA h g-1 at 2 A g-1). When coupled with homemade Na3V2(PO4)3/C in full cells, this electrode also exhibits excellent performances with high power density of 1190 W kg-1 and energy density of 119 Wh kg-1total. The exceptional performance of Bi@N-C is ascribed to the unique nanorod@nanotube structure, which can accommodate volume expansion of Bi during cycling and stabilize the solid electrolyte interphase layer and improve the electronic conductivity.

Functional characterization of three novel dense granule proteins in Neospora caninum using the CRISPR-Cas9 system.

Neospora caninum is an obligate intracellular parasite that infects a wide range of mammalian species, and particularly causes abortions in cattle and nervous system dysfunction in dogs. Dense granule proteins (GRAs) are thought to play an important role in the mediation of host-parasite interactions and facilitating parasitism. However, a large number of potential GRAs remain uncharacterized, and the functions of most of the identified GRAs have not been elucidated. Previously, we screened a large number GRAs including NcGRA27 and NcGRA61 using the proximity-dependent biotin identification (BioID) technique. Here, we identified a novel GRA protein NcGRA85 and used C-terminal endogenous gene tagging to determine its localization at the parasitophorous vacuole (PV) in the tachyzoite. We successfully disrupted three gra genes (NcGRA27, NcGRA61 and NcGRA85) of N. caninum NC1 strain using CRISPR-Cas9-mediated homologous recombination and phenotyped the single knockout strain. The NcGRA61 and NcGRA85 genes were not essential for parasite replication and growth in vitro and for virulence during infection of mice, as observed by replication assays, plaque assays and in vitro virulence assays in mice. Deletion of the NcGRA27 gene in the NC1 strain reduced the in vitro replication and growth of the parasite, as well as the pathogenicity of the NC1 strain in mice. In summary, our findings provide a basis for in-depth studies of N. caninum pathogenesis and demonstrate the importance of NcGRA27 in parasite growth and virulence, most likely a new virulence factor of N. caninum.

Prevalence and molecular characterization of Trichomonas gallinae from pigeons in Anhui, China.

Trichomonas gallinae, a protozoan parasite causing avian trichomonosis, exhibits a widespread global prevalence. It primarily affects the upper digestive tract of birds and has resulted in significant ecological problems worldwide. This study aimed to investigate the prevalence and genotypes of T. gallinae in Anhui Province, China. A total of 1612 oropharyngeal swab samples were collected from pigeon farms in Anhui Province to determine the prevalence of T. gallinae infection. The results revealed 565 (35.1%) positive samples of T. gallinae. Significant differences in infection rates were observed among different regions and age groups. Furthermore, the ITS1/5.8 S/ITS2 region was amplified, sequenced, and subjected to phylogenetic analysis. Genotypes A and B of T. gallinae were identified, and genotype B was the dominant genotype in Anhui Province. This is the first report on the prevalence and molecular characterization of T. gallinae in Anhui Province, China. Additionally, we integrated reports on the prevalence and genotype of T. gallinae in relevant provinces in China.

GRA-GCN: Dense Granule Protein Prediction in Apicomplexa Protozoa Through Graph Convolutional Network.

Dense granule proteins (GRAs) are secreted by Apicomplexa protozoa, which are closely related to an extensive variety of farm animal diseases. Predicting GRAs is an integral part in prevention and treatment of parasitic diseases. Considering that biological experiment approach is time-consuming and labor-intensive, computational method is a superior choice. Hence, developing an effective computational method for GRAs prediction is of urgency. In this paper, we present a novel computational method named GRA-GCN through graph convolutional network. In terms of the graph theory, the GRAs prediction can be regarded as a node classification task. GRA-GCN leverages k-nearest neighbor algorithm to construct the feature graph for aggregating more informative representation. To our knowledge, this is the first attempt to utilize computational approach for GRAs prediction. Evaluated by 5-fold cross-validations, the GRA-GCN method achieves satisfactory performance, and is superior to four classic machine learning-based methods and three state-of-the-art models. The analysis of the comprehensive experiment results and a case study could offer valuable information for understanding complex mechanisms, and would contribute to accurate prediction of GRAs. Moreover, we also implement a web server at http://dgpd.tlds.cc/GRAGCN/index/, for facilitating the process of using our model.

Epidemiological survey of tick-borne pathogens in dogs in Anhui Province, China.

Tick-borne diseases have continued to increase worldwide in both developing and many developed countries due to the widespread of different tick species and tick's adaptability to different climatic weather. In order to investigate the prevalence of the tick-borne pathogens, EDTA-anticoagulated whole blood samples were aseptically collected from 765 pet dogs in twenty veterinary clinics located in sixteen prefecture-level cities in Anhui Province, China, and the samples were examined and analyzed for tick-borne pathogens using both microscopy and PCR. Our result analysis revealed 17(2.22%) positive samples to Babesia spp and 4(0.52%) positive samples to Hepatozoon spp, of which case of co-infection was recorded in Lu'An and Chuzhou. The BLAST analysis results of the 18S rRNA gene revealed that the dogs were infected with Babesia gibsoni and Hepatozoon canis. All samples were negative for Anaplasma spp., Ehrlichia spp., and Rickettsia spp. This is the first molecular report of B. gibsoni and H. canis in dogs in Anhui, China.

Development and validation of a machine learning algorithm prediction for dense granule proteins in Apicomplexa.

BACKGROUND: Apicomplexa consist of numerous pathogenic parasitic protistan genera that invade host cells and reside and replicate within the parasitophorous vacuole (PV). Through this interface, the parasite exchanges nutrients and affects transport and immune modulation. During the intracellular life-cycle, the specialized secretory organelles of the parasite secrete an array of proteins, among which dense granule proteins (GRAs) play a major role in the modification of the PV. Despite this important role of GRAs, a large number of potential GRAs remain unidentified in Apicomplexa. METHODS: A multi-view attention graph convolutional network (MVA-GCN) prediction model with multiple features was constructed using a combination of machine learning and genomic datasets, and the prediction was performed on selected Neospora caninum protein data. The candidate GRAs were verified by a CRISPR/Cas9 gene editing system, and the complete NcGRA64(a,b) gene knockout strain was constructed and the phenotypes of the mutant were analyzed. RESULTS: The MVA-GCN prediction model was used to screen N. caninum candidate GRAs, and two novel GRAs (NcGRA64a and NcGRA64b) were verified by gene endogenous tagging. Knockout of complete genes of NcGRA64(a,b) in N. caninum did not affect the parasite's growth and replication in vitro and virulence in vivo. CONCLUSIONS: Our study showcases the utility of the MVA-GCN deep learning model for mining Apicomplexa GRAs in genomic datasets, and the prediction model also has certain potential in mining other functional proteins of apicomplexan parasites.

DGPD: a knowledge database of dense granule proteins of the Apicomplexa.

Apicomplexan parasites cause severe diseases in human and livestock. Dense granule proteins (GRAs), specific to the Apicomplexa, participate in the maintenance of intracellular parasitism of host cells. GRAs have better immunogenicity and they can be emerged as important players in vaccine development. Although studies on GRAs have increased gradually in recent years, due to incompleteness and complexity of data collection, biologists have difficulty in the comprehensive utilization of information. Thus, there is a desperate need of user-friendly resource to integrate with existing GRAs. In this paper, we developed the Dense Granule Protein Database (DGPD), the first knowledge database dedicated to the integration and analysis of typical GRAs properties. The current version of DGPD includes annotated GRAs metadata of 245 samples derived from multiple web repositories and literature mining, involving five species that cause common diseases (Plasmodium falciparum, Toxoplasma gondii, Hammondia hammondi, Neospora caninum and Cystoisospora suis). We explored the baseline characteristics of GRAs and found that the number of introns and transmembrane domains in GRAs are markedly different from those of non-GRAs. Furthermore, we utilized the data in DGPD to explore the prediction algorithms for GRAs. We hope DGPD will be a good database for researchers to study GRAs. Database URL: http://dgpd.tlds.cc/DGPD/index/.

Development and application of an indirect ELISA to detect antibodies to Neospora caninum in cattle based on a chimeric protein rSRS2-SAG1-GRA7.

Neospora caninum is an important apicomplexan parasite causing neosporosis in cattle. The disease is recognized as one of the most important cause of reproductive problems and abortion in cattle worldwide. In this context, we developed an indirect enzyme-linked immunosorbent assays (ELISA) with chimeric protein rSRS2-SAG1-GRA7 to diagnose antibodies to Neospora-infection. This indirect ELISA was compared to indirect fluorescent antibody test (IFAT) and western blotting (WB), and the sensitivity and specificity results of ELISA were calculated to be 86.7 and 96.1%, respectively. The overall coincidence rate was 92.6% using IFAT and WB. Additionally, 329 aborting dairy cattle serum samples were tested using this ELISA to evaluate the prevalence of N. caninum in Ningxia, China. The positive rate of N. caninum in these farms was from 19.05 to 57.89%, and the mean rate was 41.64% (±11.01%), indicating that infection with N. caninum may be one of the important causes of cattle abortion in this region. This established rSRS2-SAG1-GRA7 indirect ELISA is capable for detecting the antibodies against N. caninum, and it could be a useful screening tool for monitoring the epidemiology of neosporosis in cattle.

Scaffold-Free Endometrial Organoids Respond to Excess Androgens Associated With Polycystic Ovarian Syndrome.

CONTEXT: Polycystic ovary syndrome (PCOS) is a prevalent disorder in reproductive aged women associated with a number of endocrine and metabolic complications, including increased risk of endometrial cancer. OBJECTIVE: To study the effect of the characteristic increased androgen levels in PCOS on the endometrium, a novel scaffold-free multicellular endometrial organoid was established. DESIGN: Human endometrial organoids were constructed using primary endometrial epithelial and stromal cells from endometrial tissues. Organoids were treated for 14 days with physiologic levels of estradiol and testosterone to mimic a normal follicular phase or PCOS hormone profiles. Organoids were harvested for immunostaining and ribonucleic acid sequencing. SETTING: Academic institution. PATIENTS: Endometrial tissues from 10 premenopausal women undergoing hysterectomy for benign pathologies were obtained following written consent. MAIN OUTCOME MEASURES: Organoid architecture, cell specific markers, functional markers, proliferation, and gene expression were measured. RESULTS: A method to generate scaffold-free endometrial organoids containing epithelial and stromal cells was established. These organoids exhibited distinct organization with epithelial cells lining the outer surface and stromal cells in the center of the organoids. Epithelial cells were polarized, organoids expressed cell type specific and functional markers, as well as androgen, estrogen, and progesterone receptors. Treatment with PCOS hormones increased cell proliferation and dysregulated genes in endometrial organoids. CONCLUSIONS: A new multicellular, scaffold-free endometrial organoid system was established that resembled physiology of the native endometrium. Excess androgens in PCOS promoted cell proliferation in endometrial organoids, revealing new mechanisms of PCOS-associated with risk of endometrial neoplasia.

Generation of Multicellular Human Primary Endometrial Organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

Low Thermal Conductivity and Optimized Thermoelectric Properties of p-Type Te-Sb2Se3: Synergistic Effect of Doping and Defect Engineering.

Elemental tellurium exhibits a promising thermoelectric performance, largely due to the optimization of the carrier concentration stemming from effective chemical doping. In this study, we demonstrate a novel approach to realize the collaborative manipulation of the electrical and thermal transport properties in the Te system via introduction of Sb2Se3. A series of p-type Te1-x(Sb2Se3)x (0 ≤ x ≤ 0.2) samples were fabricated through the melting method followed by spark plasma sintering. Electrically, antimony as a successful dopant enables a remarkable improvement of the carrier concentration, from ∼1018 to ∼1019 cm-3, thus resulting in a desired power factor across the entire temperature range. Thermally, utilization of defects engineering containing point defects, grain boundaries, dislocations, and secondary phase precipitates effectively reduces the lattice thermal conductivity. The coexistence of multi-frequency phonon scattering centers derived from the addition of Sb2Se3 leads to a minimum lattice thermal conductivity of 0.5 W m-1 K-1, approaching the amorphous limit. As a result, Te0.95(Sb2Se3)0.05 shows the highest figure of merit ZT ∼1 at 600 K, comparable to that of the toxic Te(As) thermoelectrics. This work not only points out that synergistic effects of both doping and defect engineering play a vital role in decoupling the thermoelectric parameters in the Te1-x(Sb2Se3)x system but also gives a referential strategy for a higher thermoelectric performance in other Te-based materials.

Transcriptional cross talk between the forkhead transcription factor forkhead box O1A and the progesterone receptor coordinates cell cycle regulation and differentiation in human endometrial stromal cells.

Differentiation of human endometrial stromal cells (HESCs) into decidual cells is associated with induction of the forkhead transcription factor forkhead box O1A (FOXO1). We performed a genomic screen to identify decidua-specific genes under FOXO1 control. Primary HESCs were transfected with small interfering RNA targeting FOXO1 or with nontargeting control small interfering RNA before treatment with a cAMP analogue and the progestin, medroxyprogesterone acetate for 72 h. Total RNA was processed for whole genome analysis using high-density oligonucleotide arrays. We identified 3405 significantly regulated genes upon decidualization of HESCs, 507 (15.3%) of which were aberrantly expressed upon FOXO1 knockdown. Among the most up-regulated FOXO1-dependent transcriptional targets were WNT signaling-related genes (WNT4, WNT16 ), the insulin receptor (INSR), differentiation markers (PRL, IGFBP1, and LEFTY2), and the cyclin-dependent kinase inhibitor p57(Kip2) (CDKN1C). Analysis of FOXO1-dependent down-regulated genes uncovered several factors involved in cell cycle regulation, including CCNB1, CCNB2, MCM5, CDC2 and NEK2. Cell viability assay and cell cycle analysis demonstrated that FOXO1 silencing promotes proliferation of differentiating HESCs. Using a glutathione-S-transferase pull-down assay, we confirmed that FOXO1 interacts with progesterone receptor, irrespectively of the presence of ligand. In agreement, knockdown of PR disrupted the regulation of FOXO1 target genes involved in differentiation (IGFBP1, PRL, and WNT4) and cell cycle regulation (CDKN1, CCNB2 and CDC2) in HESCs treated with either cAMP plus medroxyprogesterone acetate or with cAMP alone. Together, the data demonstrate that FOXO1 engages in transcriptional cross talk with progesterone receptor to coordinate cell cycle regulation and differentiation of HESCs.

FOXO1A differentially regulates genes of decidualization.

The forkhead box O1A (FOXO1A) has been identified as one gene that is up-regulated early in the decidualization process. To further investigate the role of FOXO1A during this process, six genes, IGFBP1, PRL, TIMP3, LAMB1, CNR1, and DCN, shown to be up-regulated during decidualization, were chosen as potential targets of FOXO1A action. Treatment of human endometrial stromal cells with hormones (estradiol and medroxyprogesterone acetate) plus dibutyryl cAMP (H+dbcAMP) for 48 h increased expression of IGFBP1, PRL, TIMP3, CNR1, and DCN but not LAMB1, as measured by real-time PCR. Silencing of FOXO1A using small interfering RNA oligonucleotides decreased IGFBP1 and DCN levels and increased CNR1, TIMP3, and PRL levels. LAMB1 was not affected. When FOXO1A was overexpressed in human endometrial stromal cells, expression of IGFBP1, DCN, and PRL increased, whereas levels of TIMP3 and CNR1 decreased. Addition of H+dbcAMP caused an increased expression of IGFBP1, PRL, and DCN beyond that of FOXO1A alone. TIMP3 and CNR1 levels decreased even further in response to H+dbcAMP compared with FOXO1A alone. LAMB1, which was unresponsive to FOXO1A, decreased when H+dbcAMP was added. Overexpressing FOXO1A also caused a change in cell shape, in that the stromal fibroblasts acquired a rounded, epithelioid appearance. Finally, reporter studies showed that cotransfection of FOXO1A significantly increased PRL promoter activity but not TIMP3 promoter activity. Addition of H+dbcAMP resulted in a significant increase in PRL promoter activity and a significant decrease in TIMP3 promoter activity. In summary, this study demonstrates the versatile nature of FOXO1A in the regulation of a number of decidualization-specific genes.

Inactivation of AKT induces cellular senescence in uterine leiomyoma.

Uterine leiomyomas (fibroids) are a major public health problem. Current medical treatments with GnRH analogs do not provide long-term benefit. Thus, permanent shrinkage or inhibition of fibroid growth via medical means remains a challenge. The AKT pathway is a major growth and survival pathway for fibroids. We propose that AKT inhibition results in a transient regulation of specific mechanisms that ultimately drive cells into cellular senescence or cell death. In this study, we investigated specific mechanisms of AKT inhibition that resulted in senescence. We observed that administration of MK-2206, an allosteric AKT inhibitor, increased levels of reactive oxygen species, up-regulated the microRNA miR-182 and several senescence-associated genes (including p16, p53, p21, and ß-galactosidase), and drove leiomyoma cells into stress-induced premature senescence (SIPS). Moreover, induction of SIPS was mediated by HMGA2, which colocalized to senescence-associated heterochromatin foci. This study provides a conceivable molecular mechanism of SIPS by AKT inhibition in fibroids.

Increased AKT or MEK1/2 activity influences progesterone receptor levels and localization in endometriosis.

CONTEXT: Endometriosis is characterized by progesterone resistance and hyperactivity of the AKT and MAPK pathways. Kinases can cause posttranslational modifications of the progesterone receptor (PR) to influence cellular localization and protein stability. OBJECTIVE: The objective of this study was to determine whether the increased AKT or MAPK kinase-1/2 (MEK1/2) activity observed in endometriotic stromal cells (OSIS) from ovarian endometriomas influences levels of PR protein. In turn, the effects of inhibiting AKT or MEK1/2 in the presence of the progestin R5020 on cell viability were investigated. RESULTS: Inhibiting AKT with MK-2206 or MEK1/2 with U0126 for 24 hours in the absence of R5020 increased total and nuclear PRA and PRB protein levels in OSIS but not in eutopic endometrial stromal cells from disease-free patients from disease-free patients. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. Trends toward decreased volumes of sc grafted endometriosis tissues were demonstrated with MK-2206 and progesterone. CONCLUSIONS: Inhibition of AKT or MEK1/2 increased total and nuclear PR protein in OSIS. MK-2206 and R5020 decreased OSIS viability and increased apoptosis. The AKT and MAPK pathways may be potential molecular targets for the treatment of endometriosis.

Increased activation of the PI3K/AKT pathway compromises decidualization of stromal cells from endometriosis.

CONTEXT: Endometriosis affects approximately 10% of women in the United States and causes pain and infertility. Decidualization of endometrial stromal cells from women with endometriosis is aberrant. OBJECTIVE: The objective of this study was to investigate a potential mechanism for the inadequate decidual response in stromal cells from ovarian endometriomas. DESIGN: Stromal cells of the endometrium from women without endometriosis (HSC) or from ovarian endometriomas (OsisSC) were grown in culture and treated with 10 µm LY294002 or 250 nm MK2206, 100 nm medroxyprogesterone acetate (M), and 0.5 mm dibutyryl cAMP (A) or infection with 100 multiplicity of infection adenoviral constructs containing wild-type Forkhead box O1 or triple-mutant FOXO1. Real-time PCR was used to measure the expression of FOXO1, IGF binding protein-1 (IGFBP1), and prolactin (PRL) mRNA, and Western blot and immunohistochemical staining were used to detect the levels of progesterone receptor (PR), FOXO1, AKT, and p(Ser473)-AKT protein in vitro or in vivo. RESULTS: Expression of the decidua-specific genes, IGFBP1 and PRL, were significantly lower in OsisSC compared with normal HSC in response to M+A treatment. Basal expression levels of PRA, PRB, and FOXO1 proteins were dramatically lower in OsisSC. Overexpression of triple-mutant FOXO1 increased mRNA levels of IGFBP1 and PRL in OsisSC in the presence of M+A, whereas the overexpression of wild-type FOXO1 had no effect. AKT was highly phosphorylated in OsisSC compared with HSC and inhibition of phosphatidylinositol 3-kinase, with LY294002, increased levels of FOXO1 protein as well as IGFBP1 mRNA in the presence of M+A. Moreover, inhibition of AKT with MK2206, an allosteric AKT inhibitor, dramatically increased the accumulation of nuclear FOXO1 as well as expression of IGFBP1. Finally, immunohistochemical staining demonstrated higher p(Ser473)-AKT and lower FOXO1 levels in endometriosis tissues, compared with normal endometrial tissues. CONCLUSIONS: In endometriotic stromal cells, overactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway contributes to the reduced expression of the decidua-specific gene, IGFBP1, potentially through reduced levels of nuclear FOXO1.

Inhibition of AKT with the orally active allosteric AKT inhibitor, MK-2206, sensitizes endometrial cancer cells to progestin.

Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.

Progesterone receptor-B induction of BIRC3 protects endometrial cancer cells from AP1-59-mediated apoptosis.

Progesterone is a growth inhibitory hormone in the endometrium. While progestins can be used for the treatment of well-differentiated endometrial cancers, resistance to progestin therapy occurs for reasons that remain unclear. We have previously demonstrated that progesterone receptors (PR) A and B differentially regulate apoptosis in response to overexpression of the forkhead transcription factor, FOXO1. In this study, we further examined the PR-isoform-dependent cellular response to the AKT pathway. Treatment of PRA and PRB-expressing Ishikawa cells (PRA14, PRB23), with an AKT inhibitor API-59CJ-OMe (API-59) promoted apoptosis in the presence and absence of the ligand, R5020 preferentially in PRA14 cells. Upon PR knockdown using small interfering RNA, an increase in apoptosis was observed in PRB23 cells treated with API-59 with or without R5020 while there was no influence in PRA14 cells. Using an apoptosis-focused real-time PCR array, genes regulated by API-59 and R5020 were identified both common and unique to PRA14 and PRB23 cells. BIRC3 was identified as the only gene regulated by R5020 which occurred only in PRB cells. Knockdown of BIRC3 in PRB23 cells promoted a decrease in cell viability in response to API-59 + R5020. Furthermore, the important role of inhibitors of apoptosis (IAPs) in the PRB23 cells to promote cell survival was demonstrated using an antagonist to IAPs, a second mitochondria-derived activator of caspase (Smac also known as DIABLO) mimetic. Treatment of PRB23 cells with Smac mimetic increased apoptosis in response to API-59 + R5020. In summary, our findings indicate a mechanism by which PRB can promote cell survival in the setting of high AKT activity in endometrial cancer cells.

Progestins activate the AKT pathway in leiomyoma cells and promote survival.

CONTEXT: Progesterone has been associated with promoting growth of uterine leiomyomas. The mechanisms involved remain unclear. OBJECTIVE: In this study we investigated the activation of the AKT pathway and its downstream effectors, glycogen synthase kinase-3b and Forkhead box O (FOXO)-1 by progesterone as a mechanism of proliferation and survival of leiomyoma cells. Inhibitors of the AKT pathway were used to demonstrate the role of phosphatidylinositol 3-kinase, AKT, and FOXO1 in contributing to cell proliferation and apoptosis. RESULTS: Treatment of leiomyoma cells with R5020 over a period of 72 h resulted in higher cell numbers compared with untreated cells. When cells were treated with 100 nm R5020 for 1 and 24 h, the levels of phospho(Ser 473)-AKT increased. This increase was inhibited when cells were cotreated with RU486. Treatment of leiomyoma cells with a phosphatidylinositol 3-kinase inhibitor, LY294 dramatically decreased levels of phospho(Ser 473)-AKT, despite R5020 treatment. In addition to increased phospho(Ser 473)-AKT levels, R5020 treatment resulted in an increase in phospho(Ser 256)-FOXO1 and phosphoglycogen synthase kinase-3b. Inhibition of AKT using API-59 decreased proliferation and cell viability even in the presence of R5020. Higher concentrations of API-59-induced apoptosis of leiomyoma cells, even in the presence of R5020. Psammaplysene A increased nuclear FOXO1 levels and did not affect cell proliferation but induced apoptosis of leiomyoma cells. CONCLUSIONS: The progestin, R5020, can rapidly activate the AKT pathway. Inhibition of the AKT pathway inhibits cell proliferation and promotes apoptosis of leiomyoma cells.

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions.

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.