Development of a droplet digital PCR method for detection of Streptococcus agalactiae.

BACKGROUND: Streptococcus agalactiae (GBS) is the causative pathogen of puerperal sepsis in pregnant women and pneumonia, sepsis and meningitis in infants. Infection of GBS is responsible for the increased morbidity in pregnant women and the elderly, and bring challenges to clinical diagnosis and treatment. However, culture-based approaches to detect S.agalactiae is time-consuming with limited sensitivity. Besides, real-time quantitative PCR demands expensive instruments with tedious steps. Thus, we aim to establish a new detection method for more accurate and rapid detection of S.agalactiae. RESULTS: The ddPCR primer targeted the CpsE gene showed better amplified efficiency in the reaction. The limit of detection for GBS DNA with ddPCR was able to reach 5 pg/µL. Moreover, no positive amplified signals could be detected in the reactions which served 11 non-GBS strains DNA as templates. Furthermore, the coefficient of variation of this method was 4.5%, indicating excellent repeatability of ddPCR assay. CONCLUSIONS: In our study, ddPCR was performed as a rapid detection of S.agalactiae with high sensitivity and specificity. This technique can promote the accuracy of the diagnosis of GBS infection and provide a scientific basis for clinical treatment.

The Dual Role of the NFATc2/galectin-9 Axis in Modulating Tumor-Initiating Cell Phenotypes and Immune Suppression in Lung Adenocarcinoma.

Tumor-initiating cells (TICs) resilience and an immunosuppressive microenvironment are aggressive oncogenic phenotypes that contribute to unsatisfactory long-term outcomes in lung adenocarcinoma (LUAD) patients. The molecular mechanisms mediating the interaction between TICs and immune tolerance have not been elucidated. The role of Galectin-9 in oncogenesis and immunosuppressive microenvironment is still unknown. This study explored the potential role of galectin-9 in TIC regulation and immune modulation in LUAD. The results show that galectin-9 supports TIC properties in LUAD. Co-culture of patient-derived organoids and matched peripheral blood mononuclear cells showed that tumor-secreted galectin-9 suppressed T cell cytotoxicity and induced regulatory T cells (Tregs). Clinically, galectin-9 is upregulated in human LUAD. High expression of galectin-9 predicted poor recurrence-free survival and correlated with high levels of Treg infiltration. LGALS9, the gene encoding galectin-9, is found to be transcriptionally regulated by the nuclear factor of activated T cells 2 (NFATc2), a previously reported TIC regulator, via in silico prediction and luciferase reporter assays. Overall, the results suggest that the NFATc2/galectin-9 axis plays a dual role in TIC regulation and immune suppression.

Targeting GSTP1 as Therapeutic Strategy against Lung Adenocarcinoma Stemness and Resistance to Tyrosine Kinase Inhibitors.

Glutathione S-transferase pi (GSTP1), a phase II detoxification enzyme, is known to be overexpressed and mediates chemotherapeutic resistance in lung cancer. However, whether GSTP1 supports cancer stem cells (CSCs) and the underlying mechanisms in lung adenocarcinoma (LUAD) remain largely unknown. This study unveiled that GSTP1 is upregulated in lung CSCs and supports tumor self-renewal, metastasis, and resistance to targeted tyrosine kinase inhibitors of LUAD both in vitro and in vivo. Mechanistically, CaMK2A (calcium/calmodulin-dependent protein kinase 2 isoform A)/NRF2 (nuclear factor erythroid 2-related factor 2)/GSTP1 is uncovered as a regulatory axis under hypoxia. CaMK2A increased GSTP1 expression through phosphorylating the Sersine558 residue of NRF2 and promoting its nuclear translocation, a novel mechanism for NRF2 activation apart from conventional oxidization-dependent activation. Upregulation of GSTP1 in turn suppressed reactive oxygen species levels and supported CSC phenotypes. Clinically, GSTP1 analyzed by immunohistochemistry is upregulated in a proportion of LUAD and serves as a prognostic marker for survival. Using patient-derived organoids from an ALK-translocated LUAD, the therapeutic potential of a specific GSTP1 inhibitor ezatiostat in combination treatment with the ALK inhibitor crizotinib is demonstrated. This study demonstrates GSTP1 to be a promising therapeutic target for long-term control of LUAD through targeting CSCs.

PDE5 inhibitors, sildenafil and vardenafil, reverse multidrug resistance by inhibiting the efflux function of multidrug resistance protein 7 (ATP-binding Cassette C10) transporter.

Phosphodiesterase type 5 (PDE5) inhibitors are widely used in the treatment of male erectile dysfunction and pulmonary hypertension. Recently, several groups have evaluated the ability of PDE5 inhibitors for their anticancer activities. Previously, we had shown that sildenafil, vardenafil and tadalafil could reverse P-glycoprotein (ATP-binding cassette B1)-mediated MDR. In the present study, we determined whether these PDE5 inhibitors have the potential to reverse multidrug resistance protein 7 (MRP7; ATP-binding cassette C10)-mediated MDR. We found that sildenafil and vardenafil dose-dependently enhanced the sensitivity of MRP7-transfected HEK293 cells to paclitaxel, docetaxel and vinblastine, while tadalafil had only a minimal effect. Accumulation and efflux experiments demonstrated that sildenafil and vardenafil increased the intracellular accumulation of [(3)H]-paclitaxel by inhibiting the efflux of [(3 H]-paclitaxel in HEK/MRP7 cells. In addition, immunoblot and immunofluorescence analyses indicated that no significant alterations of MRP7 protein expression and localization in plasma membranes were found after treatment with sildenafil, vardenafil or tadalafil. These results demonstrate that sildenafil and vardenafil reverse MRP7-mediated a MDR through inhibition of the drug efflux function of MRP7. Our findings indicate a potentially novel use of PDE5 inhibitors as an adjuvant chemotherapeutic agent in clinical practice.

Enhanced chemosensitization in multidrug-resistant human breast cancer cells by inhibition of IL-6 and IL-8 production.

Drug resistance remains a major hurdle to successful cancer treatment. Many mechanisms such as overexpression of multidrug-resistance related proteins, increased drug metabolism, decreased apoptosis, and impairment of signal transduction pathway can contribute multidrug resistance (MDR). Recent studies strongly suggest a close link between cytokines and drug resistance. To identify new targets involved in drug resistance, we established a multidrug-resistant human breast cancer cell line MCF-7/R and examined the cytokine profile using cytokine antibody array technology. Among 120 cytokines/chemokines screened, IL-6, IL-8, and 13 other proteins were found to be markedly increased in drug-resistant MCF-7/R cell line as compared to sensitive MCF-7/S cell line, while 7 proteins were specifically reduced in drug-resistant MCF-7/R cells. Neutralizing antibodies against IL-6 and IL-8 partially reversed the drug resistance of MCF-7/R to paclitaxel and doxorubicin, while a neutralizing antibody against MCP-1 had no significant effect. Inhibition of endogenous IL-6 or IL-8 by siRNA technology significantly enhanced drug sensitivity of MCF-7/R cells. Furthermore, overexpression of IL-6 or IL-8 expression by transfection increased the ADM resistance in MCF-7/S cells. Our data suggest that increased expression levels of IL-6 and IL-8 may contribute to MDR in human breast cancer cells.

BBA, a derivative of 23-hydroxybetulinic acid, potently reverses ABCB1-mediated drug resistance in vitro and in vivo.

23-O-(1,4'-Bipiperidine-1-carbonyl)betulinic acid (BBA), a synthetic derivative of 23-hydroxybetulinic acid (23-HBA), shows a reversal effect on multidrug resistance (MDR) in our preliminary screening. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 has been reported in recent studies to be a major factor contributing to MDR. Our study results showed that BBA enhanced the cytotoxicity of ABCB1 substrates and increased the accumulation of doxorubicin or rhodamine123 in ABCB1 overexpressing cells, but had no effect on non ABCB1 substrate, such as cisplatin; what's more, BBA slightly reversed ABCG2-mediated resistance to SN-38, but did not affect the ABCC1-mediated MDR. Further studies on the mechanism indicated that BBA did not alter the expression of ABCB1 at mRNA or protein levels, but affected the ABCB1 ATPase activity by stimulating the basal activity at lower concentrations and inhibiting the activity at higher concentrations. In addition, BBA inhibited the verapamil-stimulated ABCB1 ATPase activity and the photolabeling of ABCB1 with [(125)I] iodoarylazidoprazosin in a concentration-dependent manner, indicating that BBA directly interacts with ABCB1. The docking study confirmed this notion that BBA could bind to the drug binding site(s) on ABCB1, but its binding position was only partially overlapping with that of verapamil or iodoarylazidoprazosin. Importantly, BBA increased the inhibitory effect of paclitaxel in ABCB1 overexpressing KB-C2 cell xenografts in nude mice. Taken together, our findings suggest that BBA can reverse ABCB1-mediated MDR by inhibiting its efflux function of ABCB1, which supports the development of BBA as a novel potential MDR reversal agent used in the clinic.

Enhancing chemosensitivity in ABCB1- and ABCG2-overexpressing cells and cancer stem-like cells by an Aurora kinase inhibitor CCT129202.

Imidazopyridine CCT129202 is an inhibitor of Aurora kinase activity and displays a favorable antineoplastic effect in preclinical studies. Here, we investigated the enhanced effect of CCT129202 on the cytotoxicity of chemotherapeutic drugs in multidrug resistant (MDR) cells with overexpression of ATP-binding cassette (ABC) transporters and cancer stem-like cells. CCT129202 of more than 90% cell survival concentration significantly enhanced the cytotoxicity of substrate drugs and increased the intracellular accumulations of doxorubicin and rhodamine 123 in ABCB1 and ABCG2 overexpressing cells, while no effect was found on parental sensitive cells. Interestingly, CCT129202 also potentiated the sensitivity of cancer stem-like cells to doxorubicin. Importantly, CCT129202 increased the inhibitory effect of vincristine and paclitaxel on ABCB1 overexpressing KBv200 cell xenografts in nude mice and human esophageal cancer tissue overexpressing ABCB1 ex vivo, respectively. Furthermore, the ATPase activity of ABCB1 was inhibited by CCT129202. Homology modeling predicted the binding conformation of CCT129202 within the large hydrophobic cavity of ABCB1. On the other hand, CCT129202 neither apparently altered the expression levels of ABCB1 and ABCG2 nor inhibited the activity of Aurora kinases in MDR cells under the concentration of reversal MDR. In conclusion, CCT129202 significantly reversed ABCB1- and ABCG2-mediated MDR in vitro, in vivo and ex vivo by inhibiting the function of their transporters and enhanced the eradication of cancer stem-like cells by chemotherapeutic agents. CCT129202 may be a candidate as MDR reversal agent for antineoplastic combination therapy and merits further clinical investigation.

Angiotensin(1-7) activates MAS-1 and upregulates CFTR to promote insulin secretion in pancreatic ß-cells: the association with type 2 diabetes.

Objective: The beneficial effect of angiotensin(1-7) (Ang(1-7)), via the activation of its receptor, MAS-1, has been noted in diabetes treatment; however, how Ang(1-7) or MAS-1 affects insulin secretion remains elusive and whether the endogenous level of Ang(1-7) or MAS-1 is altered in diabetic individuals remains unexplored. We recently identified an important role of cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated Cl- channel, in the regulation of insulin secretion. Here, we tested the possible involvement of CFTR in mediating Ang(1-7)'s effect on insulin secretion and measured the level of Ang(1-7), MAS-1 as well as CFTR in the blood of individuals with or without type 2 diabetes. Methods: Ang(1-7)/MAS-1/CFTR pathway was determined by specific inhibitors, gene manipulation, Western blotting as well as insulin ELISA in a pancreatic ß-cell line, RINm5F. Human blood samples were collected from 333 individuals with (n = 197) and without (n = 136) type 2 diabetes. Ang(1-7), MAS-1 and CFTR levels in the human blood were determined by ELISA. Results: In RINm5F cells, Ang(1-7) induced intracellular cAMP increase, cAMP-response element binding protein (CREB) activation, enhanced CFTR expression and potentiated glucose-stimulated insulin secretion, which were abolished by a selective CFTR inhibitor, RNAi-knockdown of CFTR, or inhibition of MAS-1. In human subjects, the blood levels of MAS-1 and CFTR, but not Ang(1-7), were significantly higher in individuals with type 2 diabetes as compared to those in non-diabetic healthy subjects. In addition, blood levels of MAS-1 and CFTR were in significant positive correlation in type-2 diabetic but not non-diabetic subjects. Conclusion: These results suggested that MAS-1 and CFTR as key players in mediating Ang(1-7)-promoted insulin secretion in pancreatic ß-cells; MAS-1 and CFTR are positively correlated and both upregulated in type 2 diabetes.

Potassium channel-related genes are a novel prognostic signature for the tumor microenvironment of renal clear cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) accounts for 80% of renal cell carcinomas (RCCs), and its morbidity and prognosis are unfavorable. Surgical resection is the first-line treatment for ccRCC, but the oncogenesis of ccRCC is very complex. With the development of high-throughput sequencing technology, it is necessary to analyze the transcriptome to determine more effective treatment methods. The tumor microenvironment (TME) is composed of tumor cells, various immune-infiltrating cells, fibroblasts, many cytokines, and catalysts. It is a complex system with a dynamic balance that plays an essential role in tumor growth, invasion, and metastasis. Previous studies have confirmed that potassium channels can affect the immune system, especially T lymphocytes that require potassium channel activation. However, the effect of potassium channels on the TME of ccRCC remains to be studied. Therefore, this study aims to construct a prognostic signature for ccRCC patients based on potassium ion channel-related genes (PCRGs), assess patient risk scores, and divide patients into high- and low-risk groups based on the cutoff value. In addition, we investigated whether there were differences in immune cell infiltration, immune activator expression, somatic mutations, and chemotherapeutic responses between the high- and low-risk groups. Our results demonstrate that the PCRG signature can accurately assess patient prognosis and the tumor microenvironment and predict chemotherapeutic responses. In summary, the PCRG signature could serve as an auxiliary tool for the precision treatment of ccRCC.

Photonic Nanojet-Mediated Optogenetics.

Optogenetics has become a widely used technique in neuroscience research, capable of controlling neuronal activity with high spatiotemporal precision and cell-type specificity. Expressing exogenous opsins in the selected cells can induce neuronal activation upon light irradiation, and the activation depends on the power of incident light. However, high optical power can also lead to off-target neuronal activation or even cell damage. Limiting the incident power, but enhancing power distribution to the targeted neurons, can improve optogenetic efficiency and reduce off-target effects. Here, the use of optical lenses made of polystyrene microspheres is demonstrated to achieve effective focusing of the incident light of relatively low power to neighboring neurons via photonic jets. The presence of microspheres significantly localizes and enhances the power density to the target neurons both in vitro and ex vivo, resulting in increased inward current and evoked action potentials. In vivo results show optogenetic stimulation with microspheres that can evoke significantly more motor behavior and neuronal activation at lowered power density. In all, a proof-of-concept of a strategy is demonstrated to increase the efficacy of optogenetic neuromodulation using pulses of reduced optical power.

[Wastewater Treatment Effects of Ferric-carbon Micro-electrolysis and Zeolite in Constructed Wetlands].

Ferric-carbon micro-electrolysis fillers and zeolite have been increasingly used as substrates in constructed wetlands due to their good wastewater pollution-removal efficiencies. To explore the effects of different fillers on wastewater treatment in constructed wetlands, four constructed wetlands were examined with vertical subsurface flow areas filled with ferric-carbon micro-electrolysis filler+gravel (CW-A), ferric-carbon micro-electrolysis filler+zeolite (CW-B), zeolite (CW-C), and gravel (CW-D). In addition, intermittent aeration was used to improve the dissolved oxygen (DO) environment. The results showed that, compared with CW-D, the ferric-carbon micro-electrolysis filler significantly increased the dissolved oxygen (DO, P<0.05) and pH (P<0.05) of the effluent from the wetlands. The mean removal efficiency of chemical oxygen demand (COD) in the four constructed wetlands were more than 95% (P>0.05). For TN, the mean removal efficiency of CW-A,-B, and-C was 7.94% (P<0.05), 9.29% (P<0.05), and 3.63% (P<0.05) higher than that of CW-D, respectively. The contribution of ferric-carbon micro-electrolysis filler and zeolite to improving the TN removal efficiency of the constructed wetlands was 73.55% and 26.45%, respectively. The mean removal efficiency of NH4+ in the four wetlands ranged from 67.93% to 76.90%, and compared with CW-D, the other treatments significantly improved the removal efficiency of NH4+ (P<0.05). The ferric-carbon micro-electrolysis filler had an excellent removal effect on NO3-, with a removal efficiency of more than 99%, which was significantly higher than the constructed wetlands without ferric-carbon micro-electrolysis (P<0.05). Considering the treatment effect of the organic pollutants and the nitrogen-containing pollutants, CW-B achieved the best removal efficiency in constructed wetlands with intermittent aeration.

Involvement of kisspeptin in androgen-induced hypothalamic endoplasmic reticulum stress and its rescuing effect in PCOS rats.

Endoplasmic reticulum (ER) stress, with adaptive unfolded protein response (UPR), is a key link between obesity, insulin resistance and type 2 diabetes, all of which are often present in the most common endocrine-metabolic disorder in women of reproductive age, polycystic ovary syndrome (PCOS), which is characterized with hyperandrogenism. However, the link between excess androgen and endoplasmic reticulum (ER) stress/insulin resistance in patients with polycystic ovary syndrome (PCOS) is unknown. An unexpected role of kisspeptin was reported in the regulation of UPR pathways and its involvement in the androgen-induced ER stress in hypothalamic neuronal cells. To evaluate the relationship of kisspeptin and ER stress, we detected kisspeptin and other factors in blood plasm of PCOS patients, rat models and hypothalamic neuronal cells. We detected higher testosterone and lower kisspeptin levels in the plasma of PCOS than that in non-PCOS women. We established a PCOS rat model by dihydrotestosterone (DHT) chronic exposure, and observed significantly downregulated kisspeptin expression and activated UPR pathways in PCOS rat hypothalamus compared to that in controls. Inhibition or knockdown of kisspeptin completely mimicked the enhancing effect of DHT on UPR pathways in a hypothalamic neuronal cell line, GT1-7. Kp10, the most potent peptide of kisspeptin, effectively reversed or suppressed the activated UPR pathways induced by DHT or thapsigargin, an ER stress activator, in GT1-7 cells, as well as in the hypothalamus in PCOS rats. Similarly, kisspeptin attenuated thapsigargin-induced Ca2+ response and the DHT- induced insulin resistance in GT1-7 cells. Collectively, the present study has revealed an unexpected protective role of kisspeptin against ER stress and insulin resistance in the hypothalamus and has provided a new treatment strategy targeting hypothalamic ER stress and insulin resistance with kisspeptin as a potential therapeutic agent.

[Effect of Plastic Film Mulching on Methane and Nitrous Oxide Emissions from the Ridges and Furrows of a Vegetable Field].

Investigate the effects of plastic film mulching on CH4 and N2O emissions from a vegetable field, a one-year in situ field observation was conducted using a static opaque chamber in a pepper-radish cropping system at the Key Field Station for Monitoring of Eco-Environment of Purple Soil of the Ministry of Agriculture of China at Southwest University, Chongqing. Two treatments (conventional and film mulching) were used to study the influence of film mulching on CH4 and N2O emissions. The results showed that mulching significantly increased the annual average soil pH (P<0.01), annual surface and subsurface (5 cm) temperature (P<0.05), and soil moisture content during the radish-growing season (P<0.05). The mulching also significantly reduced CH4 emissions in the field ridges (P<0.05); the average CH4 flux from ridges during the pepper-growing season was 0.110 mg·(m2·h)-1 and 0.028 mg·(m2·h)-1, and 0.011 mg·(m2·h)-1 and -0.019 mg·(m2·h)-1 during the radish-growing season, under the conventional and film mulching treatments, respectively. However, across the entire experiment, CH4 flux from field furrows was not significantly different between the two mulching treatments (P>0.05), with mean flux values during the pepper-growing season of 0.058 mg·(m2·h)-1 and 0.057 mg·(m2·h)-1, and 0.083 mg·(m2·h)-1 and 0.092 mg·(m2·h)-1 during the radish-growing season, for conventional and plastic film mulching, respectively. Except for the conventional treatment during the pepper-growing season, CH4 emissions from ridges were significantly higher than from furrows, but for other treatments, including conventional and film mulching treatments during radish-growing season and film mulching treatment during the pepper-growing season, the CH4 emissions from furrows were all significantly higher than those from ridges. This was related to the stable anoxic environment created in furrows under high rainfall conditions in Southwest China. The N2O emission flux from the ridges during the pepper-growing season was 65.41 µg·(m2·h)-1 and 68.39 µg·(m2·h)-1 under the conventional and film mulching treatments, respectively, and the N2O emission flux during the radish-growing season was 78.43 µg·(m2·h)-1 and 66.19 µg·(m2·h)-1, respectively. The N2O flux between conventional treatment and film mulching treatment in ridges or furrows were not significantly different (P>0.05), while the N2O emissions from the ridges were significantly higher than that from the furrows. CH4 emission flux was significantly positively correlated with surface and subsurface temperature, while N2O emission flux was only significantly positively correlated with alkaline nitrogen and ammonium nitrogen content.

LncRNA AC105942.1 Downregulates hnRNPA2/B1 to Attenuate Vascular Smooth Muscle Cells Proliferation.

The abnormal proliferation of vascular smooth muscle cells (VSMCs) is crucial in the atherosclerosis. Although long noncoding RNAs (lncRNAs) are implicated in a variety of diseases, their roles in activation of VSMCs proliferation and vascular disorder diseases are not well understood. In addition, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) was reported to participate in lncRNAs-mediated function. Herein, we propose to investigate the role of lncRNA AC105942.1 and hnRNPA2/B1 in pathological VSMCs proliferation and the possible mechanisms in vitro. We have identified that lncRNA AC105942.1 was downregulated and hnRNPA2/B1 was upregulated in atherosclerotic plaques compared with normal artery tissues. Enhanced lncRNA AC105942.1 could noticeably inhibit Ang II-induced VSMCs proliferation. Further investigation suggested that lncRNA AC105942.1 could downregulate the expression of hnRNPA2/B1 and then regulate the level of CDK4 and p27. Taken together, our study indicated that lncRNA AC105942.1 downregulated hnRNPA2B1 to protect against the atherosclerosis by suppressing VSMCs proliferation. LncRNA AC105942.1 and hnRNPA2/B1 could represent potential therapeutic and diagnostic targets to atherosclerosis-related diseases.

[Characteristics of Aerosol Optical Depth in the Urban Area of Beibei and Its Correlation with Particle Concentration].

To understand the atmospheric quality of the Beibei District of Chongqing, using the simultaneous observation data of aerosol optical depth and particulate matter concentration in 2014, we analyzed the characteristics of aerosol optical depth (AOD) in the urban area of Beibei and its correlation with particle concentration. The results showed that the annual average of AOD500nm in Beibei District is 1.46±0.69, which varies significantly by month. The highest value in November was 2.90±1.85, and the lowest in September was 0.54±0.05. There is particulate matter pollution in Beibei District. The annual average values of PM2.5 and PM10 are (62±40)µg·m-3 and (94±51)µg·m-3, respectively, which exceed the secondary standard of GB 3095-2012 Ambient Air Quality Standard. The limit values, the daily average over-standard rates of PM2.5 and PM10, are 26% and 15%, respectively. There was significant correlation between fine particle PM2.5 and PM10 concentration of respirable particulate matter. The annual coefficient of determination R2 could reach 0.95 (P<0.01). The correlation between AOD and PM2.5 and PM10 was positive throughout the year. The coefficient of determination R2 was 0.48 and 0.46, respectively, and the coefficient of determination and correlation function were different in different seasons, among which the correlation in winter was the best and the correlation in summer was the worst. AOD and air quality index showed positive correlation characteristics throughout the year, and the coefficient of determination R2 was 0.15 (P<0.05). The AOD value was affected by the comprehensive effects of weather elements. The temperature, humidity, water vapor, and other factor data should also be collected synchronously during the observation period.

PM2.5 exposure aggravates left heart failure induced pulmonary hypertension.

Aim: Particulate matter 2.5 (PM2.5) exposure is high risk to cardiovascular diseases. We investigated the influence of PM2.5 exposure on pulmonary arterial hypertension (PAH) murine model induced by left ventricular (LV) failure. Methods: Thirty 10 weeks old C57BL/6 mice were randomised to four groups: sham group, sham + PM2.5 group, TAC group, and TAC + PM2.5 group. Eight weeks post TAC surgery, right ventricular (RV) and lung remodelling (Sirius Red staining and WGA Staining), heart and lung function (EF and RVSBP), and fibrotic genes (TGF-ti mRNA expression and collagen III protein level in lung tissue were measured. Results: Exposure to PM2.5 augments TAC induced PAH as evidenced by decreased EF value and increased RVSBP, RV cardiomyocytes size, RV and lung fibrosis, and upregulated expression of collagen III and TGF-a in comparison to TAC group in lung tissues. Even the LV EF value was deceased from 79.3 ± 3.4% to 63.4 ± 2.1% when sham group exposed to PM2.5, PM2.5 exposure had no effect on RVSBP, RV cardiomyocytes' size, RV weight/tibia length, RV and lung fibrosis, and expression of collagen III and TGF-a in sham surgery mice. Conclusions: Exposure to PM2.5 aggravates deterioration of LV failure induced PAH.

Elevation of antimüllerian hormone in women with polycystic ovary syndrome undergoing assisted reproduction: effect of insulin.

OBJECTIVE: To measure blood and follicular antimüllerian hormone (AMH) levels in women with polycystic ovary syndrome (PCOS) undergoing assisted reproductive technologies (ART) and to examine the direct action of insulin on AMH expression in human granulosa cells. DESIGN: Prospective clinical and experimental study. SETTING: University Hospital-based laboratory. PATIENT(S): Women with (n = 86) and without (n = 172) PCOS in ART. INTERVENTION(S): Blood, follicular fluid, and luteinized granulosa cells were collected from PCOS and non-PCOS women in ART. MAIN OUTCOME MEASURE(S): Hormone levels in blood and fluid, and gene expression in granulosa cells. RESULT(S): Serum levels of AMH were elevated and inversely correlated with embryo cleavage rate in PCOS women in ART. Significant higher levels of AMH were also found in small and large follicles collected from PCOS women compared with non-PCOS women. Luteinized granulosa cells from PCOS women showed higher expression of AMH and its receptor AMHR2. Direct effect of insulin in increasing the expression of AMH in the isolated luteinized granulosa cells was observed, with the PCOS granulosa cells responding to a high dose of insulin. Cotreatment with AMH attenuated insulin-induced aromatase expression in the luteinized granulosa cells. CONCLUSION(S): These results suggest that insulin may contribute to AMH elevation in PCOS and that AMH counteracts insulin-promoted aromatase expression in granulosa cells.

MRP4 sustains Wnt/ß-catenin signaling for pregnancy, endometriosis and endometrial cancer.

Rationale: Abnormal Wnt/ß-catenin signaling in the endometrium can lead to both embryo implantation failure and severe pathogenic changes of the endometrium such as endometrial cancer and endometriosis. However, how Wnt/ß-catenin signaling is regulated in the endometrium remains elusive. We explored possible regulation of Wnt/ß-catenin signaling by multi-drug resistance protein 4 (MRP4), a potential target in cancer chemotherapy, and investigated the mechanism. Methods: Knockdown of MRP4 was performed in human endometrial cells in vitro or in a mouse embryo-implantation model in vivo. Immunoprecipitation, immunoblotting and immunofluorescence were used to assess protein interaction and stability. Wnt/ß-catenin signaling was assessed by TOPflash reporter assay and quantitative PCR array. Normal and endometriotic human endometrial tissues were examined. Data from human microarray or RNAseq databases of more than 100 participants with endometriosis, endometrial cancer or IVF were analyzed. In vitro and in vivo tumorigenesis was performed. Results: MRP4-knockdown, but not its transporter-function-inhibition, accelerates ß-catenin degradation in human endometrial cells. MRP4 and ß-catenin are co-localized and co-immunoprecipitated in mouse and human endometrium. MRP4-knockdown in mouse uterus reduces ß-catenin levels, downregulates a series of Wnt/ß-catenin target genes and impairs embryo implantation, which are all reversed by blocking ß-catenin degradation. Analysis of human endometrial biopsy samples and available databases reveals significant and positive correlations of MRP4 with ß-catenin and Wnt/ß-catenin target genes in the receptive endometrium in IVF, ectopic endometriotic lesions and endometrial cancers. Knockdown of MRP4 also inhibits in vitro and in vivo endometrial tumorigenesis. Conclusion: A previously undefined role of MRP4 in stabilizing ß-catenin to sustain Wnt/ß-catenin signaling in endometrial cells is revealed for both embryo implantation and endometrial disorders, suggesting MRP4 as a theranostic target for endometrial diseases associated with Wnt/ß-catenin signaling abnormality.

Activation of the epithelial sodium channel (ENaC) leads to cytokine profile shift to pro-inflammatory in labor.

The shift of cytokine profile from anti- to pro-inflammatory is the most recognizable sign of labor, although the underlying mechanism remains elusive. Here, we report that the epithelial sodium channel (ENaC) is upregulated and activated in the uterus at labor in mice. Mechanical activation of ENaC results in phosphorylation of CREB and upregulation of pro-inflammatory cytokines as well as COX-2/PGE2 in uterine epithelial cells. ENaC expression is also upregulated in mice with RU486-induced preterm labor as well as in women with preterm labor. Interference with ENaC attenuates mechanically stimulated uterine contractions and significantly delays the RU486-induced preterm labor in mice. Analysis of a human transcriptome database for maternal-fetus tissue/blood collected at onset of human term and preterm births reveals significant and positive correlation of ENaC with labor-associated pro-inflammatory factors in labored birth groups (both term and preterm), but not in non-labored birth groups. Taken together, the present finding reveals a pro-inflammatory role of ENaC in labor at term and preterm, suggesting it as a potential target for the prevention and treatment of preterm labor.