A Chromatographic Method for Separation of Tungsten (W) from Silicate Samples for High-Precision Isotope Analysis Using Negative Thermal Ionization Mass Spectrometry.

A new chromatographic method for isolation of W from large masses of silicate samples (>1 g) for ultrahigh precision isotopic analysis was developed. The purification of W was achieved through two stages of rapid chromatographic separations. In the first step, Ti, Zr, Hf, and W were separated collectively from the sample matrix through an AG1-X8 (100-200 mesh) column with a 10 mL resin volume. Subsequently, W was rapidly separated from Ti and Zr-Hf with high purity by a two-step extraction chromatographic method using 0.6 and 0.3 mL TODGA resin columns (50-100 µm particle size), respectively. The total yield of W, including the anion exchange and the TODGA chromatographic separation steps, is greater than 90%. The procedure was employed to isolate W from rock reference materials GSJ JB-3 and USGS BHVO-2; the separated W was analyzed by TRITON Plus TIMS, yielding a 182W/184W of 0.864898 ± 0.000005 (n = 8, 2 SD) for JB-3 and 182W/184W of 0.864896 ± 0.000006 (n = 5, 2 SD) for BHVO-2, which are in agreement with previously reported values within analytical errors.

Mild changes in the mucosal microbiome during terminal ileum inflammation.

Patients with inflammation in the terminal ileum have high morbidity. In genetically susceptible hosts, chronic intestinal inflammation targeting the resident intestinal microbiota develops, but the microbial signature of the terminal ileum is poorly studied. To improve understanding of the mechanisms underlying the high prevalence of terminal ileum inflammation, we used 16S rRNA sequencing to analyse the mucosa-associated microbiota of the terminal ileum under intestinal homeostasis and inflammation conditions. Mucosal biopsy is the most commonly used sampling technique for assessing microbial communities associated with the intestinal mucosa. Thirty patients (15 with terminal ileum inflammation and 15 controls) underwent colonoscopy and biopsies were taken from the terminal ileum. Diagnosis depended on a combination of endoscopic and histological factors. To determine the composition and diversity of the microbiota, the 16S rRNA was analysed, and a variety of bioinformatics analyses were performed. Among the patients, composition analysis showed that the most abundant phyla identified in the terminal ileum samples were Fusobacteria, Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. At the phylum level, the relative proportion of Bacteroidetes was lower in patients with inflammation than in control patients. In addition, there was an increase in the abundance of the phyla Proteobacteria and Lentisphaerae in patients with inflammation. The abundances of the dominant microbes in the terminal ileum were not significantly different between patients in an inflammatory state and controls. These results confirm that partial dysbiosis of the intestinal mucosa-associated microbiota composition is associated with terminal ileum inflammation.

Sr Isotope Analysis of Picogram-Level Samples by Thermal Ionization Mass Spectrometry Using a Highly Sensitive Silicotungstic Acid Emitter.

Thermal ionization mass spectrometry (TIMS) has shown excellent analytical precision for Sr isotopic ratio analysis, even for small masses of material (0.5-10 ng). However, because of the sensitivity limit of TIMS, it is still not possible to obtain high precision 87Sr/86Sr isotope ratios for picogram-level sample sizes (30-100 pg) due to the lack of a highly sensitive emitter. This study is the first to employ a highly sensitive silicotungstic acid emitter to measure Sr isotopes at the picogram-level using TIMS. This emitter produces a 3-fold enhancement in the ionization efficiency of Sr and not only significantly reduces the required sample size but also has good external precision. Analyses of the NIST 987 standard yield an external reproducibility (2 RSD, n = 8) better than ±0.013% even for 30 pg of Sr. It is possible to yield an internal precision (2 RSE) of ±0.003% for 100 pg of sample using the default 1011 Ohm feedback resistors. This method was verified by using a suite of silicate reference materials. Replicate digestions and analyses ( n = 8) of the basalt standard BCR-2 (87Sr/86Sr = 0.704998 ± 0.000028, 2 SD) at the 326 ± 30 pg level demonstrates that good external reproducibility is reached on ultratrace level silicate samples. This method has a wide variety of potential applications for samples containing ultralow amounts of Sr in geoscience and archeological studies, such as single grains of mica, sphalerite, and pyrite, single mantle melt inclusions, precious extra-terrestrial materials, and human hair to name just a few.

High-precision measurement of (186)Os/(188)Os and (187)Os/(188)Os: isobaric oxide corrections with in-run measured oxygen isotope ratios.

We present a novel method for high precision measurement of (186)Os/(188)Os and (187)Os/(188)Os ratios, applying isobaric oxide interference correction based on in-run measurements of oxygen isotopic ratios. For this purpose, we set up a static data collection routine to measure the main Os(16)O3(-) ion beams with Faraday cups connected to conventional 10(11) amplifiers, and (192)Os(16)O2(17)O(-) and (192)Os(16)O2(18)O(-) ion beams with Faraday cups connected to 10(12) amplifiers. Because of the limited number of Faraday cups, we did not measure (184)Os(16)O3(-) and (189)Os(16)O3(-) simultaneously in-run, but the analytical setup had no significant influence on final (186)Os/(188)Os and (187)Os/(188)Os data. By analyzing UMd, DROsS, an in-house Os solution standard, and several rock reference materials, including WPR-1, WMS-1a, and Gpt-5, the in-run measured oxygen isotopic ratios were proven to present accurate Os isotopic data. However, (186)Os/(188)Os and (187)Os/(188)Os data obtained with in-run O isotopic compositions for the solution standards and rock reference materials show minimal improvement in internal and external precision, compared to the conventional oxygen correction method. We concluded that, the small variations of oxygen isotopes during OsO3(-) analytical sessions are probably not the main source of error for high precision Os isotopic analysis. Nevertheless, use of run-specific O isotopic compositions is still a better choice for Os isotopic data reduction and eliminates the requirement of extra measurements of the oxygen isotopic ratios.

Direct High-Precision Measurements of the (87)Sr/(86)Sr Isotope Ratio in Natural Water without Chemical Separation Using Thermal Ionization Mass Spectrometry Equipped with 10(12) Ω Resistors.

Thermal ionization mass spectrometry (TIMS) allows excellent precision for determining Sr isotope ratios in natural water samples. Traditionally, a chemical separation procedure using cation exchange resin has been employed to obtain a high purity Sr fraction from natural water, which makes sample preparation time-consuming. In this study, we present a rapid and precise method for the direct determination of the Sr isotope ratio of natural water using TIMS equipped with amplifiers with two 10(12) Ω resistors. To eliminate the (87)Rb isobaric interference, Re ribbons are used as filaments, providing a significant advantage over W ribbons in the inhibition of Rb(+) emission, based on systematically examining a series of NIST SRM987 standard doping with various amounts of Rb using Re and W ribbons. To validate the applicability of our method, twenty-two natural water samples, including different water types (rain, snow, river, lake and drinking water), that show a large range in Sr content variations (2.54-922.8 ppb), were collected and analyzed from North and South China. Analytical results show good precision (0.003-0.005%, 2 RSE) and the method was further validated by comparative analysis of the same water with and without chemical separation. The method is simple and rapid, eliminates sample preparation time, and prevents potential contamination during complicated sample-preparation procedures. Therefore, a high sample throughput inherent to the TIMS can be fully utilized.

Defective CFTR-regulated granulosa cell proliferation in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is one of the most frequent causes of female infertility, featured by abnormal hormone profile, chronic oligo/anovulation, and presence of multiple cystic follicles in the ovary. However, the mechanism underlying the abnormal folliculogenesis remains obscure. We have previously demonstrated that CFTR, a cAMP-dependent Cl(-) and HCO3 (-) conducting anion channel, is expressed in the granulosa cells and its expression is downregulated in PCOS rat models and human patients. In this study, we aimed to investigate the possible involvement of downregulation of CFTR in the impaired follicle development in PCOS using two rat PCOS models and primary culture of granulosa cells. Our results indicated that the downregulation of CFTR in the cystic follicles was accompanied by reduced expression of proliferating cell nuclear antigen (PCNA), in rat PCOS models. In addition, knockdown or inhibition of CFTR in granulosa cell culture resulted in reduced cell viability and downregulation of PCNA. We further demonstrated that CFTR regulated both basal and FSH-stimulated granulosa cell proliferation through the HCO3 (-)/sAC/PKA pathway leading to ERK phosphorylation and its downstream target cyclin D2 (Ccnd2) upregulation. Reduced ERK phosphorylation and CCND2 were found in ovaries of rat PCOS model compared with the control. This study suggests that CFTR is required for normal follicle development and that its downregulation in PCOS may inhibit granulosa cell proliferation, resulting in abnormal follicle development in PCOS.

High-precision (143)Nd/(144)Nd ratios from NdO(+) data corrected with in-run measured oxygen isotope ratios.

The NdO(+) technique has been considerably refined in recent years for high-precision measurement of Nd isotope ratios in low-level samples (1-5 ng Nd). As oxygen isotopic compositions may vary significantly with experimental conditions such as filament material, ionization enhancer and the ambient oxygen in the ion source, great "care" should be taken for using correct oxygen isotopic compositions to do the isobaric oxide corrections for the "conventional" NdO(+) method. Our method presented here for NdO(+) data reduction and PrO(+) interference corrections uses the oxygen isotope composition determined in each cycle of the NdO(+) measurements. For that purpose, we measured the small ion signals of (150)Nd(17)O(+) and (150)Nd(18)O(+) with amplifiers equipped with 10(12) Ω feedback resistors, and those of Nd(16)O(+) ion beams with 10(11) Ω amplifiers. Using 10(12) Ω amplifiers facilitates a precise measurement of the very small (150)Nd(17)O(+) and (150)Nd(18)O(+) ion signals and calculation of highly accurate and precise (143)Nd/(144)Nd isotope ratios. The (143)Nd/(144)Nd ratios for JNdi-1 standards and several whole-rock reference materials determined with the method on 4 ng of Nd loads are consistent with previously reported values within analytical error, with internal and external precision (2 RSE and 2 RSD) of better than 20 and 30 ppm, respectively.

1,25-Dihydroxyvitamin D3 reduces early mortality post severe burn injury via alleviating endotoxemia, oxidative stress and inflammation.

Severe burn patients frequently suffer from 1,25-Dihydroxyvitamin D3 (1,25-[OH]2-D3) deficiency. In this study, we investigated the effect of 1,25-[OH]2-D3 on early mortality post severe burn and potential underlying mechanisms. Our results indicate that 1,25-[OH]2-D3 significantly reduced early mortality in mice post severe burn injury. A decrease in serum lipopolysaccharide levels and an increase in serum superoxide dismutase activity were found after administration of 1,25-[OH]2-D3. Furthermore, 1,25-[OH]2-D3 demonstrated protective effects on both intestinal and lung histology and ameliorated lung inflammation. Its anti-inflammatory effect was further confirmed in airway epithelial cells. In conclusion, our study provides evidence that 1,25-[OH]2-D3 has a significant impact on the reduction of early mortality post severe burn injury, possibly through its ability to alleviate endotoxemia, oxidative stress, and inflammation. Our findings highlight the potential of 1,25-[OH]2-D3 to protect the intestinal mucosal barrier in the early stage following major burn injury and opens up new avenues for clinical application of 1,25-[OH]2-D3 in burn patients.

CFTR negatively regulates cyclooxygenase-2-PGE(2) positive feedback loop in inflammation.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent anion channel mostly expressed in epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased cyclooxygenase-2 (COX-2) expression and over-production of prostaglandin E(2) (PGE(2)) in human CF bronchial epithelia cell line (CFBE41o--) with elevated NF-κB activity compared to a wild-type airway epithelial cell line (16HBE14o--). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NF-κB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE(2) involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o-- cells were challenged with LPS as well as PGE(2), indicating possible involvement of CFTR in negative regulation of COX-2/PGE(2). In conclusion, CFTR is a negative regulator of PGE(2)-mediated inflammatory response, defect of which may result in excessive activation of NF-κB, leading to over production of PGE(2) as seen in inflammatory CF tissues.

Simultaneous determination of 143Nd/144Nd and 147Sm/144Nd ratios and Sm-Nd contents from the same filament loaded with purified Sm-Nd aliquot from geological samples by isotope dilution thermal ionization mass spectrometry.

Isotope dilution thermal ionization mass spectrometry (ID-TIMS) is the standard technique used to achieve precise (143)Nd/(144)Nd and (147)Sm/(144)Nd isotope ratios and accurate elemental concentrations of Sm-Nd. However, in previous studies, purified Sm and Nd fractions must be individually loaded onto different filaments for their accurate determination using TIMS because of severe isobaric interferences. Thus, the classical ID-TIMS technique is time consuming and laborious. In this study, a new method is proposed, which is able to acquire both ratios of (143)Nd/(144)Nd and (147)Sm/(144)Nd and concentrations of Sm-Nd simultaneously on the same filament arrangement. The measurement time and filament consumption are reduced by 50% with the current method, and therefore, the operation cost of TIMS is significantly reduced. A mixed (152)Sm-(148)Nd spike was employed to achieve accurate results after spike subtraction and isobaric interference corrections. Results obtained from a series of standard rock samples are in good agreement with recommended values, within ±0.003% for the (143)Nd/(144)Nd ratio and ±1% for the (147)Sm/(144)Nd ratio.

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.

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.

Involvement of CFTR in oviductal HCO3- secretion and its effect on soluble adenylate cyclase-dependent early embryo development.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in electrolyte and fluid transport in epithelial cells, and women with cystic fibrosis (CF), caused by CFTR gene mutations, have a higher incidence of infertility. METHODS: In the present study, we investigated the expression of CFTR in porcine oviduct and its functional role in oviductal HCO(3)(-) secretion and embryo development with RT-PCR, western blot, patch-clamp, short-circuit current (I(sc)), pH measurement and embryo culture. RESULTS: RT-PCR and western blot analysis showed the expression of CFTR mRNA and protein in the oviduct with its localization demonstrated by immunohistochemistry. The whole-cell patch-clamp recording revealed a forskolin (FSK)-activated current with electrophysiological and pharmacological characteristics of CFTR. The I(sc) measurement showed that FSK-stimulated an increase in the I(sc), which could be significantly reduced by CFTR inhibitor or removal of both CO(2) and HCO(3)(-). pH measurement showed a FSK stimulated alkalization at the apical surface, which could be inhibited by CFTR inhibitor, indicating CFTR-mediated HCO(3)(-) secretion. Mouse embryo development from 2-cell to morula or blastocyst stage was significantly inhibited in the absence of HCO(3)(-) or when co-cultured with HCO(3)(-) secretion-deficient CFTR mutant cells as compared with the wild-type. RT-PCR, western blot and immunostaining showed the expression of soluble adenylate cyclase (sAC), the known HCO(3)(-) sensor, in embryos. Treatment with its inhibitors, 2-hydroxyestradiol and KH7, prevented the HCO(3)(-) dependent embryo development. CONCLUSION: The present results suggest that CFTR-mediated oviductal HCO(3)(-) secretion may be vital for sAC-dependent early embryo development, a defect of which may contribute to the reduced fertility seen in women with CF.

[Application of serum protein profiling in diagnosis, prognosis and evaluation of curative effect of pancreatic adenocarcinoma].

OBJECTIVE: To establish decision tree and logistic regression classification models for diagnosing pancreatic adenocarcinoma (PaCa) and for screening serum biomarkers related to evaluation of different stages and curative effects. METHODS: Serum samples obtained from subjects with pancreatic adenocarcinoma (n = 58) and normal pancreas (n = 51) were applied to strong anion exchange chromatography (SAX2) chips for protein profiling by SELDI-TOF-MS to screen multiple serum biomarkers. Biomarker Wizard software and several statistical methods including algorithm of decision tree, logistic regression and ROC curves were used to construct the decision tree or logistic regression classification models. RESULTS: Average of 61 mass peaks were detected at the molecular range of 2000-30,000, ten decision trees with the highest cross validation rate were chosen to construct the classification models, which can differentiate PaCa from normal pancreas with a sensitivity of 83.3% and a specificity of 100%. Logistic regression was used to achieve the AUC (0.976 +/- 0.011, P < 0.001) with a sensitivity of 77.6% - 91.4% and a specificity of 92.2% - 100%. Six mass peaks were combined by logistic regression to achieve the AUC 0.897 +/- 0.054, 0.978 +/- 0.021 and 0.792 +/- 0.107 (P < 0.05) in the three groups (patients at stage I and II, stage II and III, stage III and IV). One mass peak (M/Z 4,016) was screened (P < 0.05) significantly between the preoperative and postoperative PaCa samples and the intensity decreased weeks after operation. CONCLUSION: Decision tree and logistic regression classification models of the mass peaks screened by SELDI-TOF-MS serum profiling can be used to differentiate pancreatic adenocarcinoma from normal pancreas, and is superior to CA 199. The detected mass peaks are helpful for the evaluation of curative effect and prognosis of pancreatic adenocarcinoma.

Regulation of smooth muscle contractility by the epithelium in rat vas deferens: role of ATP-induced release of PGE2.

Recent studies suggest that the epithelium might modulate the contractility of smooth muscle. However, the mechanisms underlying this regulation are unknown. The present study investigated the regulation of smooth muscle contraction by the epithelium in rat vas deferens and the possible factor(s) involved. Exogenously applied ATP inhibited electrical field stimulation (EFS)-evoked smooth muscle contraction in an epithelium-dependent manner. As the effects of ATP on smooth muscle contractility were abrogated by inhibitors of prostaglandin synthesis, but not by those of nitric oxide synthesis, prostaglandins might mediate the effects of ATP. Consistent with this idea, PGE(2) inhibited EFS-evoked smooth muscle contraction independent of the epithelium, while ATP and UTP induced the release of PGE(2) from cultured rat vas deferens epithelial cells, but not smooth muscle cells. The ATP-induced PGE(2) release from vas deferens epithelial cells was abolished by U73122, an inhibitor of phospholipase C (PLC) and BAPTA AM, a Ca(2+) chelator. ATP also transiently increased [Ca(2+)](i) in vas deferens epithelial cells. This effect of ATP on [Ca(2+)](i) was independent of extracellular Ca(2+), but abolished by the P2 receptor antagonist RB2 and U73122. In membrane potential measurements using a voltage-sensitive dye, PGE(2), but not ATP, hyperpolarized vas deferens smooth muscle cells and this effect of PGE(2) was blocked by MDL12330A, an adenylate cyclase inhibitor, and the chromanol 293B, a blocker of cAMP-dependent K(+) channels. Taken together, our results suggest that ATP inhibition of vas deferens smooth muscle contraction is epithelium dependent. The data also suggest that ATP activates P2Y receptor-coupled Ca(2+) mobilization leading to the release of PGE(2) from epithelial cells, which in turn activates cAMP-dependent K(+) channels in smooth muscle cells leading to the hyperpolarization of membrane voltage and the inhibition of vas deferens contraction. Thus, the present findings suggest a novel regulatory mechanism by which the epithelium regulates the contractility of smooth muscle.

Innate immune responses of epididymal epithelial cells to Staphylococcus aureus infection.

The epithelium is an active participant in the host response to infection. We hypothesized that epididymal epithelia play a role in the innate immune responses by sensing the presence of pathogens, expressing and secreting inflammatory cytokines that recruit inflammatory cells in response to invading pathogens. Our results indicated that TNF-alpha and IL-1beta could be secreted by the primary cultured rat epididymal cauda epithelia infected with Staphylococcus aureus. Epididymal epithelial-induced nitric oxide synthase (iNOS) expression was up-regulated after S. aureus infection and nitric oxide (NO) was also found to be produced significantly. NF-kappaB inhibitor BAY11-7082 inhibited TNF-alpha secretion completely and p38 mitogen-activated protein kinases (MAPKs) inhibitor SB203580 decreased TNF-alpha secretion partly, indicating that NF-kappaB and p38 signal pathways were involved in this inflammation response. Toll-like receptor (TLR)-2 and -4 were shown to be expressed in primary cultured rat epididymal epithelia. After infection the level of TLR2 expression was up-regulated rather than TLR4. These results demonstrated that epididymal epithelium have an innate immune response through activation of p38 MAPK and NF-kappaB after TLR2 activation by S. aureus infection.

CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway.

The increase in migratory ability of pancreatic ductal adenocarcinoma cells is a key event in the development of metastasis to the lymph nodes and distant organs. Although the C-C motif chemokine receptor 7 (CCR7) and its ligand, C-C motif chemokine ligand 21 (CCL21), have been revealed to serve an important role in tumor migration, their precise roles and potential underlying mechanisms remain largely unknown. The present study revealed that overexpression of CCR7 significantly promoted BxPC-3 cell migration, accompanied by the induction of anoctamin 6 (ANO6) expression, indicating that ANO6 is a downstream target of CCR7 signaling. Furthermore, the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly increased in CCR7-overexpressing BxPC-3 cells, indicating that ERK may be a potential mediator of CCR7-regulated ANO6 expression in BxPC-3 cells. To characterize the receptor-mediated pathway, a specific ERK inhibitor, U0126, was used, which reduced BxPC-3 cell migration and the expression of ANO6. In summary, the results of the present study demonstrate that CCR7 promoted BxPC-3 cell migration by regulating ANO6 expression perhaps via activation of the ERK signaling pathway.

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.

Glucose-Sensitive CFTR Suppresses Glucagon Secretion by Potentiating KATP Channels in Pancreatic Islet α Cells.

The secretion of glucagon by islet α cells is normally suppressed by high blood glucose, but this suppressibility is impaired in patients with diabetes or cystic fibrosis (CF), a disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a cyclic adenosine monophosphate-activated Cl- channel. However, precisely how glucose regulates glucagon release remains controversial. Here we report that elevated glucagon secretion, together with increased glucose-induced membrane depolarization and Ca2+ response, is found in CFTR mutant (DF508) mice/islets compared with the wild-type. Overexpression of CFTR in AlphaTC1-9 cells results in membrane hyperpolarization and reduced glucagon release, which can be reversed by CFTR inhibition. CFTR is found to potentiate the adenosine triphosphate-sensitive K+ (KATP) channel because membrane depolarization and whole-cell currents sensitive to KATP blockers are significantly greater in wild-type/CFTR-overexpressed α cells compared with that in DF508/non-overexpressed cells. KATP knockdown also reverses the suppressive effect of CFTR overexpression on glucagon secretion. The results reveal that by potentiating KATP channels, CFTR acts as a glucose-sensing negative regulator of glucagon secretion in α cells, a defect of which may contribute to glucose intolerance in CF and other types of diabetes.

Abnormal CFTR Affects Glucagon Production by Islet α Cells in Cystic Fibrosis and Polycystic Ovarian Syndrome.

Glucagon, produced by islet α cells, functions to increase blood glucose. Abnormal glucose levels are often seen in cystic fibrosis (CF), a systematic disease caused by mutations of the CF transmembrane conductance regulator (CFTR), and in polycystic ovarian syndrome (PCOS), an endocrine disorder featured with hyperandrogenism affecting 5-10% women of reproductive age. Here, we explored the role of CFTR in glucagon production in α cells and its possible contribution to glucagon disturbance in CF and PCOS. We found elevated fasting glucagon levels in CFTR mutant (DF508) mice compared to the wildtypes. Glucagon and prohormone convertase 2 (PC2) were also upregulated in CFTR inhibitor-treated or DF508 islets, as compared to the controls or wildtypes, respectively. Dihydrotestosterone (DHT)-induced PCOS rats exhibited significantly lower fasting glucagon levels with higher CFTR expression in α cells compared to that of controls. Treatment of mouse islets or αTC1-9 cells with DHT enhanced CFTR expression and reduced the levels of glucagon and PC2. The inhibitory effect of DHT on glucagon production was blocked by CFTR inhibitors in mouse islets, and mimicked by overexpressing CFTR in αTC1-9 cells with reduced phosphorylation of the cAMP/Ca2+ response element binding protein (p-CREB), a key transcription factor for glucagon and PC2. These results revealed a previously undefined role of CFTR in suppressing glucagon production in α-cells, defects in which may contribute to glucose metabolic disorder seen in CF and PCOS.