Colorimetric aptasensor based on temporally controllable light-stimulated oxidase-mimicking fluorescein for the sensitive detection of exosomes in mild conditions.

Analysis of exosomes provides important information for rapid and non-invasive screening of tumors. However, sensitive and convenient detection of exosomes remains technically challenging to date. Herein, a colorimetric aptasensor based on the light-stimulated oxidase-mimicking activity of FITC was constructed for detecting ovarian cancer (OC) exosomes. The aptasensor contained an EpCAM aptamer to capture OC exosomes. Cholesterol and fluorescein (FITC) were used to modify either end of the DNA (DNA anchor). The DNA anchor could combine with exosomes through a hydrophobic reaction between cholesterol and the lipid membrane. FITC oxidized 3,3',5,5'-tetramethylbenzidine (TMB) under a 365 nm LED light source in a temporally controllable manner under mild conditions, causing the solution to change from colorless to blue, and the corresponding UV-vis absorbance increased. Based on this principle, the exosomes were qualitatively analyzed by observing the color change with the naked eye. In parallel, the exosome concentration was also detected using UV-vis spectrophotometry. The linear range was from 2 × 105 to 100 × 105 particles per mL with a limit of detection of 1.77 × 105 particles per mL. The developed aptasensor also exhibited favorable selectivity and could discriminate the exosomes from OC cells and normal cells. Besides, the receiver operating characteristic (ROC) curve demonstrates that it is possible to distinguish between patients with OC and healthy donors (HDs) using exosomes as the biomarker. Our technology may expand the applications of DNA-based detection method-enabled OC diagnostic tools.

Bioinformatics identification and validation of biomarkers and infiltrating immune cells in endometriosis.

Background: Endometriosis (EM) is a common gynecological disorder that often leads to irregular menstruation and infertility. The pathogenesis of EM remains unclear and delays in diagnosis are common. Thus, it is urgent to explore potential biomarkers and underlying molecular mechanisms for EM diagnosis and therapies. Methods: Three EM-related datasets (GSE11691, GSE25628, and GSE86534) were downloaded from the Gene Expression Omnibus (GEO) which were integrated into a combined dataset after removing batch effect. Differentially expressed immune cell-related genes were obtained by CIBERSORT, WGCNA, and the identification of differentially expressed genes. Random forest model (RF), support vector machine model (SVM), and generalized linear model (GLM) were then constructed and the biomarkers for EM were determined. A nomogram evaluating the risk of disease was constructed and the validity was assessed by the calibration curve, DCA curve, and clinical impact curve. Single-gene Gene Set Enrichment Analysis (GSEA)was performed to explore the molecular mechanisms of biomarkers. The ceRNA regulatory network of biomarkers was created by Cytoscape and potential target drugs were obtained in the DGIdb database (Drug-Gene Interaction database).The expression levels of biomarkers from clinical samples was quantified by RT-qPCR. Results: The ratio of eight immune cells was significantly different between the eutopic and ectopic endometrium samples. A total of eight differentially expressed immune cell-related genes were investigated. The SVM model was a relatively suitable model for the prediction of EM and five genes (CXCL12, PDGFRL, AGTR1, PTGER3, and S1PR1) were selected from the model as biomarkers. The calibration curve, DCA curve, and clinical impact curve indicated that the nomogram based on the five biomarkers had a robust ability to predict disease. Single gene GSEA result suggested that all five biomarkers were involved in labyrinthine layer morphogenesis and transmembrane transport-related biological processes in EM. A ceRNA regulatory network containing 184 nodes and 251 edges was constructed. Seven drugs targeting CXCL12, 49 drugs targeting AGTR1, 16 drugs targeting PTGER3, and 21 drugs targeting S1PR1 were extracted as potential drugs for EM therapy. Finally, the expression of PDGFRL and S1PR1 in clinical samples was validated by RT-qPCR, which was consistent with the result of public database. Conclusions: In summary, we identified five biomarkers (CXCL12, PDGFRL, AGTR1, PTGER3, and S1PR1) and constructed diagnostic model, furthermore predicted the potential therapeutic drugs for EM. Collectively, these findings provide new insights into EM diagnosis and treatment.

Expression Level of Keratin 7 in Epithelial Ovarian Cancer and Malignant Metastasis of Benign Epithelial Ovarian Tumors.

It was to investigate the diagnostic value of keratin 7 (KRT7) in malignant metastasis of epithelial ovarian cancer and benign epithelial ovarian tumors. From January 2018 to January 2019, 30 fresh tissues of benign epithelial ovarian tumors, 30 fresh tissues of borderline tumors, 30 fresh tissues of metastatic ovarian were collected in The First Affiliated Hospital of Fujian Medical University, and 30 fresh tissues of normal ovarian tissues were collected as the control group. Federation of gynecology and obstetrics (FIGO) staging criteria: 25 cases of stage I, 26 cases of stage II, 16 cases of stage III, and 23 cases of stage IV. The relative expression of KRT7 was detected by real-time fluorescence quantitative PCR, and the relationship between KRT7 expression and epithelial ovarian cancer grading was analyzed. The results showed that the positive expression rate of KRT7 was 12.1% in normal ovarian tissues, 28.4% in benign epithelial ovarian tumors, 53.5% in borderline tumors, and 24.2% in metastatic ovarian cancer. With the increase of tumor stage malignancy, the relative expression of KRT7 decreased significantly, but there was no significant difference between stage I and stage II, stage III and stage IV (P > 0.05). The difference between stage I and stage III, and stage IV was significant (P < 0.05). Patients with epithelial ovarian cancer had a significant difference compared with the control group (P < 0.05). In summary, compared with the control group, the expression of KRT7 in patients with benign epithelial ovarian tumors and borderline tumors was significantly decreased. The expression level of KRT7 in benign epithelial ovarian tumors was lower than that in borderline tumors. The expression of KRT7 was related to the occurrence, development, and deterioration of ovarian cancer, which provided a basis for targeted therapy of tumors.

A Deep Learning Quantification Algorithm for HER2 Scoring of Gastric Cancer.

Gastric cancer is the third most common cause of cancer-related death in the world. Human epidermal growth factor receptor 2 (HER2) positive is an important subtype of gastric cancer, which can provide significant diagnostic information for gastric cancer pathologists. However, pathologists usually use a semi-quantitative assessment method to assign HER2 scores for gastric cancer by repeatedly comparing hematoxylin and eosin (H&E) whole slide images (WSIs) with their HER2 immunohistochemical WSIs one by one under the microscope. It is a repetitive, tedious, and highly subjective process. Additionally, WSIs have billions of pixels in an image, which poses computational challenges to Computer-Aided Diagnosis (CAD) systems. This study proposed a deep learning algorithm for HER2 quantification evaluation of gastric cancer. Different from other studies that use convolutional neural networks for extracting feature maps or pre-processing on WSIs, we proposed a novel automatic HER2 scoring framework in this study. In order to accelerate the computational process, we proposed to use the re-parameterization scheme to separate the training model from the deployment model, which significantly speedup the inference process. To the best of our knowledge, this is the first study to provide a deep learning quantification algorithm for HER2 scoring of gastric cancer to assist the pathologist's diagnosis. Experiment results have demonstrated the effectiveness of our proposed method with an accuracy of 0.94 for the HER2 scoring prediction.

O-Net: A Novel Framework With Deep Fusion of CNN and Transformer for Simultaneous Segmentation and Classification.

The application of deep learning in the medical field has continuously made huge breakthroughs in recent years. Based on convolutional neural network (CNN), the U-Net framework has become the benchmark of the medical image segmentation task. However, this framework cannot fully learn global information and remote semantic information. The transformer structure has been demonstrated to capture global information relatively better than the U-Net, but the ability to learn local information is not as good as CNN. Therefore, we propose a novel network referred to as the O-Net, which combines the advantages of CNN and transformer to fully use both the global and the local information for improving medical image segmentation and classification. In the encoder part of our proposed O-Net framework, we combine the CNN and the Swin Transformer to acquire both global and local contextual features. In the decoder part, the results of the Swin Transformer and the CNN blocks are fused to get the final results. We have evaluated the proposed network on the synapse multi-organ CT dataset and the ISIC 2017 challenge dataset for the segmentation task. The classification network is simultaneously trained by using the encoder weights of the segmentation network. The experimental results show that our proposed O-Net achieves superior segmentation performance than state-of-the-art approaches, and the segmentation results are beneficial for improving the accuracy of the classification task. The codes and models of this study are available at https://github.com/ortonwang/O-Net.

DLC1 Is a Prognosis-Related Biomarker Correlated With Tumor Microenvironment Remodeling in Endometrial Carcinoma.

BACKGROUND AND AIM: Deleted in liver cancer 1 (DLC1) is confirmed as a metastasis suppressor gene in endometrial carcinoma (EC). However, its functional mechanisms remain unclear. This study aimed to explore the relationship between DLC1 expression and EC. METHODS: The Cancer Genome Atlas database was used for evaluating the expression of DLC1 in pan-cancer. CIBERSORT was used to assess the relationship between DLC1 and tumor immune infiltration. We applied real-time quantitative polymerase chain reaction to determine the expression of DLC1 in EC and adjacent normal tissue samples. The targeting endogenous protein levels were assessed using the dataset from the cBioPortal database. RESULTS: DLC1 expression negatively correlated with the clinical characteristics (clinical stage, histologic grade) and positively correlated with the survival of patients with uterine corpus EC (UCEC). The gene set enrichment analysis displayed that the low-expression DLC1 group was enriched in metabolic pathways. Concomitantly, the high-expression DLC1 group was enriched in tumor immune-related activities. The CIBERSORT analysis showed that the number of resting memory CD4 T cells and resting mast cells positively correlated with DLC1 expression, while the number of macrophages M2 had a negative correlation, indicating that DLC1 played a key role in mediating immune cell infiltration. The target gene validation confirmed that DLC1 expression was downregulated in tumor samples. The target protein level was consistently downregulated in tumor samples. CONCLUSIONS: DLC1 levels might be useful in predicting the prognosis of patients with UCEC and especially governing the status of tumor microenvironment transition from immune-dominant to metabolic-dominant. The findings shed a different light on the immune therapeutics of UCEC.

A ratiometric electrochemical biosensor via alkaline phosphatase mediated dissolution of nano-MnO2 and Ru(III) redox recycling for the determination of dimethoate.

A sensitive and ratiometric electrochemical biosensor was developed for the determination of dimethoate via alkaline phosphatase (ALP) mediated dissolution of nano-MnO2 and [Ru(NH3)6]3+(Ru(III)) redox recycling. The electroactive probe Ru(III) was adsorbed on the nano-MnO2 with the high specific surface area through electrostatic interaction to form the MnO2-Ru(III) nanocomposite, which was then fixed on the surface of the glassy carbon electrode. When the dimethoate inhibited the catalytic activity of ALP in a homogeneous system, the hydrolysate L-ascorbic acid (AA) produced by ALP hydrolysis of L-ascorbic acid-trisodium 2-phosphate (AAP) decreased. The solution was then incubated with a glassy carbon electrode modified by MnO2-Ru(III). At this time, only a small amount of MnO2-Ru(III) was decomposed and Ru(III) was rapidly electroreduced to Ru(II) on the surface of the electrode. The in-situ produced Ru(II) was chemically oxidized back to Ru(III) by Fe(III). The redox recycling of Ru(III) was completed and the Ru(III) reduction current signal was amplified. The process consumed part of Fe(III) to reduce the reduction current signal of Fe(III), and the ratio of the two reduction currents (IRu(III)/IFe(III)) increased significantly. The IRu(III)/IFe(III) value increased with the increase of dimethoate concentration in the linear range of 0.01-300 ng mL-1, and the detection limit was 6.3 pg mL-1. It has been successfully applied to the determination of dimethoate in oilseed rape and lettuce with a satisfactory result.

XPO1-Mediated EIF1AX Cytoplasmic Relocation Promotes Tumor Migration and Invasion in Endometrial Carcinoma.

Dysregulation of eukaryotic translation initiation factor 1A, X-linked (EIF1AX), has been implicated in the pathogenesis of some cancers. However, the role of EIF1AX in endometrial carcinoma (EC) remains unknown. We investigated the EIF1AX expression in EC patients and assessed its tumorigenesis-associated function and nucleocytoplasmic transport mechanism in vitro and in vivo. The results indicated that the cytoplasmic EIF1AX expression showed a gradual increase when going from endometrium normal tissue, simple endometrial hyperplasia, complex endometrial hyperplasia, and endometrial atypical hyperplasia to EC, while vice versa for the nuclear EIF1AX expression. In addition, the cytoplasmic EIF1AX expression was positively correlated with histologic type, high International Federation of Gynecology and Obstetrics (FIGO) grade, advanced FIGO stage, deeper infiltration, high Ki67 index, and shorter recurrence-free survival in EC patients. In vitro, short hairpin RNA-mediated EIF1AX depletion or SV40NLS-mediated EIF1AX import into the nucleus in multiple human EC cells potently suppressed cell migration and invasion, epithelial-mesenchymal transition, and lung metastasis. Moreover, exportin 1 induced the transport of EIF1AX from the nucleus to the cytoplasm that could be inhibited by leptomycin B treatment or the mutation in the EIF1AX location sequence. These results demonstrate that cytoplasmic EIF1AX may play a key role in the incidence and promotion of EC, and thus, targeting EIF1AX or its nucleocytoplasmic transport process may offer an effective new therapeutic approach to EC.

Genetic interactions among Brca1, Brca2, Palb2, and Trp53 in mammary tumor development.

Inherited mutations in BRCA1, BRCA2, and PALB2 cause a high risk of breast cancer. Here, we conducted parallel conditional knockout (CKO) of Brca1, Palb2, and Brca2, individually and in combination, along with one copy of Trp53, in the mammary gland of nulliparous female mice. We observed a functional equivalence of the three genes in their basic tumor-suppressive activity, a linear epistasis of Palb2 and Brca2, but complementary roles of Brca1 and Palb2 in mammary tumor suppression, as combined ablation of either Palb2 or Brca2 with Brca1 led to delayed tumor formation. Whole-exome sequencing (WES) revealed both similarities and differences between Brca1 and Palb2 or Brca2 null tumors. Analyses of mouse mammary glands and cultured human cells showed that combined loss of BRCA1 and PALB2 led to high levels of reactive oxygen species (ROS) and increased apoptosis, implicating oxidative stress in the delayed tumor development in Brca1;Palb2 double CKO mice. The functional complementarity between BRCA1 and PALB2/BRCA2 and the role of ROS in tumorigenesis require further investigation.

Dysbiosis of the endometrial microbiota and its association with inflammatory cytokines in endometrial cancer.

The underlying molecular mechanisms involved in the pathogenesis of endometrial cancer (EC) are still not well understood. Our goal was to investigate the composition of the endometrial microbiota and the association with inflammatory cytokines in EC. Endometrial microbiota profiles of women with EC (n = 25) and benign uterine lesions (BUL, n = 25) were assessed by 16S ribosomal RNA gene amplicon sequencing. The expression levels of interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-17 (IL-17) mRNA and protein in the endometrial tissues of the two groups were determined by real-time quantitative polymerase chain reaction and Western blot, respectively. There were significant differences in alpha diversity based on the observed operational taxonomic units (P = .002), Pielou evenness (P = .001), and Shannon index (P < .001) between EC and BUL groups. Significant differences were also found in Bray-Curtis (P = .001) and unweighted UniFrac (P = .001) beta diversity measures between the two groups. At the genus level, Micrococcus was more abundant in the EC group. Pseudoramibacter_Eubacterium, Rhodobacter, Vogesella, Bilophila, Rheinheimera, and Megamonas were enriched in the BUL group. There were no differences in IL-8 and IL-17 protein levels between the two groups, except IL-6 protein levels. However, the mRNA expression levels of IL-6, IL-8, and IL-17 were significantly different. Moreover, the relative abundances of Micrococcus was positively correlated with IL-6, and IL-17 mRNA levels. In conclusion, our results suggested that dysbiosis of endometrial microbiota and the inflammatory cytokines were associated with Micrococcus in EC patients, which might be useful for exploration of the mechanism between the endometrial microbiota and inflammatory responses in future studies.

Development and Characterization of A New Dimethicone Nanoemulsion and its Application for Electronic Gastroscopy Examination.

PURPOSE: Although the effective and safe medical defoamers, dimethicone (DM) and simethicone (SM) are widely used in electronic gastroscope examination (EGE), their preparations are presented in the form of suspensions or emulsions, these are untransparent or milk-like in appearance and can easily cause misdiagnosis as a result of an unclear field of vision if the doctor does not master the amount of defoamer or operates incorrectly. At the same time, it is also difficult to wash out the camera and pipeline, due to the large oil droplets of preparations. The purpose of this study was to develop a new clear and transparent oil in water (O/W) DM nanoemulsions (DMNs) and observe the effect of application in EGE. METHODS: The oil phase was chosen for its antifoaming activity and viscosity. The emulsifier and co-emulsifier were selected according to the solubility of the oil phase in them. The water titration method was used to make the pseudoternary phase diagrams of nanoemulsions and optimize the prescription composition. DM-in-water nanoemulsion was prepared by the low energy method and evaluated for appearance, antifoaming ability, droplet size, and stability. The effect of DMNs utilized in EGEs was also observed. RESULTS: The optimal formulation of DMNs contained CRH-40 as an emulsifier, PEG-400 as a co-emulsifier, DM as oil phase with the viscosity of 10 mPa.s, and their proportion was 4.5:4.5:1, respectively. DMNs obtained the average particle size of 67.98 nm with the polydispersity index (PDI) of 0.332, and 57.14% defoaming rate. The result of using an EGE showed that DMNs were superior in comparison to the emulsions with regard to the defoaming effect, visual clarity, and easy cleanup. CONCLUSION: DMNs were found to provide excellent visual clarity to its other preparations. The novel DMNs is a promising substitute for DM emulsions or suspensions in EGEs.

Activation of NF-κB signaling pathway during HCG-induced VEGF expression in luteal cells.

Vascular endothelial growth factor (VEGF) plays an essential role in luteal angiogenesis, the present study therefore utilized luteal cells cultured in vitro to further investigate the activation and contribution of nuclear factor (NF)-κB to VEGF expression induced by human chorionic gonadotrophin (HCG). The present results showed HCG induced VEGF expression as well as hypoxia-inducible factor (HIF)-1α mRNA and protein expressions, which was blocked by NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Further analysis found that these increases of VEGF and HIF-1α mRNA induced by HCG were also blocked by NF-κB siRNA transfection, which was consistent with PDTC treatment. However, HIF-1α siRNA treatment significantly decreased HCG induced-VEGF expression with no effect on NF-κB mRNA expression. Furthermore, combination of HIF-1α siRNA and PDTC treatment did not further decrease VEGF mRNA expression, and the result of chromatin immunoprecipitation indicated NF-κB may regulate HIF-1α transcription through binding with its promoter. Taken together, the present results clearly demonstrated that NF-κB was activated to regulate VEGF expression by increasing HIF-1α transcription in luteal cells treated with HCG. Therefore, the present study provided a new and important mechanism of luteal angiogenesis during the formation of corpus luteum in mammals.

miR-221 regulates proliferation and apoptosis of ovarian cancer cells by targeting BMF.

To observe the expression of microRNA-221 (miR-221) in ovarian cancer tissues and its effect and associated mechanism on proliferation and apoptosis in the ovarian cancer SKOV3 cell line. The expression of miR-221 and B-cell lymphoma 2 modifying factor (BMF) mRNA in ovarian cancer and para-carcinoma tissues was detected by reverse transcription-quantitative polymerase chain reaction, the expression of BMF was detected by western blot. MicroRNA.org online predicted that BMF was the possible target gene of miR-221, and the regulatory association was validated by a dual-luciferase reporter gene assay. SKOV3 cells were divided into 8 transfection groups: Anti-miR-negative control (NC); anti-miR-221; phosphorylated internal ribosome entry site 2 (pIRES2)-blank, pIRES2-BMF, small interfering (si)-NC, si-BMF, anti-miR-221+si-BMF and anti-miR-221+pIRES2-BMF groups. Cell proliferation was detected by EdU staining flow cytometry. The effect of transfection on cell apoptosis was detected by Annexin V/PI double staining, and the activity of caspase-3 was detected by spectrophotometry. The effect of anti-miR-221 or pIRES2-BMF transfection on SKOV3 cell proliferation was detected by MTT assay and flow cytometry, and the effect on apoptosis was detected by the Annexin V/PI double staining. Compared with para-cancer tissues, the miR-221 expression was significantly upregulated (P<0.001), the BMF mRNA expression was significantly downregulated (P<0.001), and the expression of BMF proteins was significantly downregulated in the ovarian cancer tissues. Dual-luciferase reporter gene assay confirmed a targeted regulatory association between miR-221 and BMF. The anti-miR-221 or pIRES2-BMF transfection significantly upregulated BMF expression in SKOV3 cells, significantly decreased cell proliferation and significantly increased cell apoptosis. The overexpression of BMF may enhance the proapoptotic and proliferation-inhibition effect of anti-miR-221 on SKOV3 cells. The transfection of si-BMF significantly promoted cell proliferation, reduced cell apoptosis and attenuated the proapoptotic and proliferation-inhibition effect of anti-miR-221 on cells. The expression of miR-221 was significantly upregulated and the expression of BMF was significantly down-regulated in ovarian cancer tissues. The overexpression of miR-221 antagonized the apoptosis of ovarian cancer SKOV3 cell and promoted the cell proliferation by targeted inhibition of the expression of BMF, which may serve a role in the pathogenesis of ovarian cancer.

Treatment of Adenomyosis with Subcutaneous Etonogestrel Implants: A Clinical Observational Study in 17 Patients.

BACKGROUND Adenomyosis is a cause of chronic pelvic pain in women of reproductive age. The aim of this study was to investigate the effects of subcutaneous etonogestrel implantation on adenomyosis. MATERIAL AND METHODS A clinical observational study included 17 women with adenomyosis who were treated with subcutaneous etonogestrel implants and followed-up for 12 months. Imaging and clinical observations were undertaken in the 17 patients at baseline (time 0), and at 3 months, 6 months, and 12 months following subcutaneous etonogestrel implantation. The following imaging and clinical findings were compared between baseline (time 0) and 12-month follow-up: menstrual bleeding pattern, dysmenorrhea, visual analog scale (VAS) pain score, uterine volume, serum cancer antigen 125 (CA125) levels, hemoglobin, follicle-stimulating hormone (FSH) levels, luteinizing hormone levels, serum estradiol levels, and any treatment side effects. RESULTS All 17 patients treated with etonogestrel implants completed the 12-month follow-up, at which time, the mean hemoglobin level (127.08±2.56 g/L) was significantly higher compared with that at baseline (94.54±5.47 g/L; P<0.01); uterine volume, serum CA125, and VAS score for dysmenorrhea at 12 months (118.03±12.83 cm³, 34.58±9.66 U/mL, and 1.45±0.35, respectively) were significantly lower when compared with baseline (198.53±39.47 cm³, 100.41±49.89 U/mL, and 7.62±0.74, respectively) (P<0.01, for all). However, changes in bleeding pattern and amenorrhoea occurred after treatment in some women. CONCLUSIONS Subcutaneous etonogestrel was effective in reducing some symptoms and signs of adenomyosis, including dysmenorrhea, anemia, serum CA125, and uterine volume.

Upregulated expression of HOXC8 is associated with poor prognosis of cervical cancer.

Homeobox C8 (HOXC8) is a transcription factor that has been reported to regulate numerous genes associated with tumor progression. However, its function in cervical cancer (CC) remains to be elucidated. In the present study, the expression level of HOXC8 was examined in CC tissues and cell lines using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Additionally, CC cell lines were transfected with small interfering RNAs (siRNAs) to downregulate the expression of HOX8 and assess cell proliferation using Cell Counting Kit-8. The results demonstrated a significantly increased expression of HOXC8 in CC tissues and cell lines compared with non-tumor tissues, and a normal cervical cell line, respectively. Additionally, the downregulation of HOXC8, which was achieved by siRNA transfection, significantly inhibited the proliferation rate of CC cell lines. Kaplan-Meier curves demonstrated that the increased expression of HOXC8 was associated with poor prognosis of patients with CC. Additionally, univariate and multivariate analysis revealed that HOXC8 was a significant and independent predictor for overall survival of patients with CC. In conclusion, the results of the present study suggest that HOXC8 may be involved in the progression of CC and may serve as a therapeutic target for CC.

Accumulated autophagosomes and excessive apoptosis during the luteal development of pregnant rats.

Autophagy plays an important role in the regression of pseudopregnant corpus luteum, whereas the involvement of autophagy in the pregnant luteolysis still remains unknown. Therefore, the present study was designed to investigate the levels and effects of accumulated autophagosomes on excessive apoptosis during the luteal development of pregnant rats. Ovaries were obtained from the female rats at the early, middle or late phase of the pregnancy, which correspondingly had three groups; including the early (ELP), middle (MLP) and late luteal phase (LLP). The results of autophagy-associated protein LC-3 clearly showed that autophagy expressed during the pregnant CL and significantly increased at LLP, while the expression changes of apoptosis related proteins cleaved caspase-3 and Bax were similar with LC-3 expression changes, indicating autophagy may be involved in the initiation of pregnant luteolysis through the induction of cellular apoptosis at LLP of pregnant ovaries. The present study was further examined the expressions of other two autophagy-associated proteins p62 and LAMP-2, since the degradation failure of autophagosomes contributed to cellular apoptosis. The results demonstrated p62 protein was accumulated at LLP while its mRNA was maintained during the whole luteal development of pregnant rats. Interestingly, the expressions of LAMP-2 mRNA and premature protein were significantly increased at MLP and LLP, while the expression of mature LAMP-2 increased at MLP and then decreased at LLP, implying autophagosomes were accumulated at LLP. Together, to our knowledge, the present study firstly demonstrated that the insufficient of lysosomal functions contributed to the impaired degradation of autophagosomes and then activated caspase-3 dependent apoptotic pathway during the pregnant luteolysis of rat ovaries, which will provide a new insight into the important mechanism regulating the luteolysis of the pregnant ovaries in mammals.

The effects of poloxamer 188 on the autophagy induced by traumatic brain injury.

Poloxamer 188 (P188) has been reported to reseal plasma membranes and attenuate TBI-induced neuronal death by suppressing apoptosis. Recent studies also confirm increased autophagy after traumatic brain injury (TBI). The present study aimed to investigate the effects of plasmalemmal resealing by P188 on neuronal autophagy in TBI. Scratch test was performed in rat cell line PC-12 in vitro, followed by immunofluorescence analysis of LC3 24h after PC-12 cell stretch-injury in vitro. CD1 mice were randomized into saline and P188-treatment groups (both undergoing intravenous injection of 4mg/ml, 100µl via the caudal vein 30min after TBI) as well as sham group. To analyze the effect of P188 on autophagy, the LC3 protein levels were assessed by western blotting 1h, 6h, 12h, 24h, and 48h after TBI. The autophagy-associated protein levels of Beclin-1, Bcl-2, and p62 were likewise determined. In vitro, the scratch test showed that the wound healing rate was significantly improved at 12h and 24h in P188 groups, and LC3 immunofluorescence analysis indicated that P188 induced extensive formation of LC3 puncta in PC-12 cells. In vivo, western blotting analyses revealed elevations of the LC3-II/LC3-I and Beclin-1/bcl-2 ratios as well as downregulation of p62 in the saline group, in contrast with the more significant increases of LC3-II/LC3-I and Beclin-1/bcl-2 ratios and the further downregulation of p62 in P188-treated group. These results revealed that plasma membranes were resealed after TBI, in which P188 aggravated autophagy in vivo.

Health expenditures spent for prevention, economic performance, and social welfare.

BACKGROUND: Countries with limited resources in economic downturns often reduce government expenditures, of which spending on preventive healthcare with no apparent immediate health impact might be cut down first. This research aims to find the optimum share of preventive health expenditure to gross domestic product (GDP) and investigate the implications of preventive health services on economic performance and the population's wellbeing. METHODS: We develop the economic growth model to undertake health-economic analyses and parameterize for Taiwan setting. Based on the US experiences over the period from 1975 to 2013, this research further examines the model's predictions on the relationship between preventive health expenditure and economic performance. RESULTS: Theoretical analysis and numerical simulations show that an inverse U-shaped relationship exists between the proportion of GDP spent on prevention and social welfare, as well as between the proportion spent on prevention and economic growth. Empirical analysis shows an under-investment in prevention in Taiwan. The spending of preventive healthcare in Taiwan government was 0.0027 GDP in 2014, while the optimization levels for economic development and social welfare would be 0 · 0119 and 0 · 0203, respectively. There is a statistically significant nonlinear relationship between health expenditure on prevention and the estimated real impact of economic performance from US experiences. The welfare-maximizing proportion of preventive expenditure is usually greater than the proportion maximizing economic growth, indicating a conflict between economic growth and welfare after a marginal share. CONCLUSION: Our findings indicate that it is worthwhile increasing investment on prevention up until an optimization level for economic development and social welfare. Such levels could also be estimated in other economies.

The neuroprotective effect of apelin-13 in a mouse model of intracerebral hemorrhage.

Adipocytokine apelin-13 is a peptide which could reportedly protect the brain against ischemic reperfusion injury and traumatic brain injury (TBI). Whether apelin-13 has any roles to play in intracerebral hemorrhage (ICH) has not been clarified. We aimed to investigate the roles of apelin-13 in ICH and effects on ICH-induced apoptosis. Firstly, CD-1 mice were subjected to infusion of Type IV collagenase (to induce ICH) or saline (for shams) into the left striatum. ICH animals received intracerebroventricular administration of vehicle, apelin-13 (50µg dissolved in 5µl saline) immediately after ICH. The motor function and the cerebral water content (CWC) as well as blood brain barrier (BBB) disruption were measured, coupled with determination of ICH-induced neural cell death by Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL). The apoptosis-associated proteins caspase-3 and Bcl-2 as well as the brain edema-associated proteins aquaporin-4 (AQP4) and MMP-9 were all assessed with western blotting. The results showed that apelin-13 decreased CWC and reduced Evans blue leakage into injured hemispheres, with the motor function significantly improved. Additionally, apelin-13 also acutely decreased the number of ICH-induced TUNEL-positive (TUNEL(+)) cells at 48h after ICH. The expressions of AQP4, MMP-9, caspse-3 and Bcl-2 were all downregulated by apelin-13 at 24h and 48h after ICH. All these results revealed that apelin-13 attenuated brain edema and reduced cellular death by suppressing apoptosis after ICH in mice.