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Background: Autoimmune pancreatitis is characterized by diffuse swelling of the pancreas and a high serum immunoglobulin (Ig) G4 concentration. Histopathologically, dense infiltration of lymphocytes and IgG4-positive plasma cells with fibrosis are seen in the pancreas. Although allergic diseases complicating autoimmune pancreatitis have been reported, the clinical features of bronchial asthma complicated by autoimmune pancreatitis remain unclear. Case Summary: We report three cases of bronchial asthma preceding the onset of type 1 autoimmune pancreatitis by 3 months to 30 years. All three cases were males with high serum IgG, IgG4, and IgE concentrations. The radioallergosorbent tests were positive for common allergens such as mites and house dust. One case had a pulmonary manifestation that proved to be an inflammatory pseudotumor of the lung with an accumulation of IgG4-positive plasma cells. The asthma symptom was ameliorated by oral prednisolone therapy for autoimmune pancreatitis, and when the corticosteroid doses were reduced, asthma became worse in all three cases. Discussion: It is possible that atopy and increased Th2 cell activity are related to a higher coincidence of IgG4-related diseases such as type 1 autoimmune pancreatitis. Because the present cases are few in number, further studies are necessary. [ABSTRACT FROM AUTHOR]

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T. [ABSTRACT FROM AUTHOR]

Aberrant HCO3- transport is a hallmark of cystic fibrosis (CE) and is associated with aberrant Cl-dependent HCO3- transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3- current by CFTR cannot account for CFTR-activated HCO3- transport and that CFTR does not activate AE1-AE4. In contrast, CFTR markedly activates Cl- and OH-/HCO3- transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform-specific stoichiometries. DRA activity occurred at a Cl-/HCO3- ratio ⩾2. SLC26A6 activity is voltage regulated and occurred at HCO3-/Cl- ⩾2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and GS51D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3- transport (one SLC26 transporter-electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain-electroneutral transport), the CE-associated aberrant HCO3- transport, and reveal a new function of CFTR with clinical implications for CE and congenital chloride diarrhea. [ABSTRACT FROM AUTHOR]

Presents a study that identified water transport pathways in the pancreatic duct cells of rats. Examination of the osmotic water permeability of isolated interlobular ducts; Effects of basolateral and luminal mercuric chloride on fluid secretion and water permeability; Analysis of aquaporins in whole pancreatic and isolated interlobular duct cells.

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl- has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3-) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl-/HCO3- exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ~60%, impaired CFTR-mediated HCO3-/Cl- transport activity to 50-60%, and impaired CFTR-coupled Cl-/HCO3- exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese. [ABSTRACT FROM AUTHOR]

In order to apply fecal pancreatic elastase for follow-up of exocrine pancreatic function in chronic pancreatitis and cystic fibrosis, we examined the sensitivity, specificity, and long-term variability of a new polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Patients with definite chronic pancreatitis ( n = 23), probable or possible chronic pancreatitis ( n = 14), autoimmune pancreatitis ( n = 7), or acute pancreatitis ( n = 11), and 51 healthy subjects and 11 healthy infants participated in this study. Pancreatic function was graded as normal ( n = 3), mild ( n = 18), moderate ( n = 9), or severe ( n = 18) exocrine insufficiency on the basis of secretin tests. Fecal pancreatic elastase was measured by a new ELISA. Fecal pancreatic elastase concentration in control subjects varied widely, with a median of 478 µg/g. The specificity of this test was 90.2% with a cutoff value of >200 µg/g. The sensitivities were 60.9% for detecting definite chronic pancreatitis, 76.5% for calcifying pancreatitis, 71.4% for autoimmune pancreatitis, and 7.1% for probable or possible chronic pancreatitis. The sensitivities were 16.7% for mild, 12.5% for moderate, and 72.2% for severe exocrine pancreatic insufficiency. Forty patients were reexamined after a median interval of 347 days. The fecal pancreatic elastase levels between the first and second tests were not significantly different. Two infants, 4.5 and 5 months old, had abnormally low values, but after a median of 304 days all infants showed normal levels (median, 444 µg/g). Fecal pancreatic elastase is a reproducible marker for severe exocrine pancreatic insufficiency. This test is valuable for longitudinal follow-up of exocrine pancreatic function. [ABSTRACT FROM AUTHOR]

Motivation: The analysis of RNA-Seq data from individual differentiating cells enables us to reconstruct the differentiation process and the degree of differentiation (in pseudo-time) of each cell. Such analyses can reveal detailed expression dynamics and functional relationships for differentiation. To further elucidate differentiation processes, more insight into gene regulatory networks is required. The pseudo-time can be regarded as time information and, therefore, single-cell RNASeq data are time-course data with high time resolution. Although time-course data are useful for inferring networks, conventional inference algorithms for such data suffer from high time complexity when the number of samples and genes is large. Therefore, a novel algorithm is necessary to infer networks from single-cell RNA-Seq during differentiation. Results: In this study, we developed the novel and efficient algorithm SCODE to infer regulatory networks, based on ordinary differential equations. We applied SCODE to three single-cell RNASeq datasets and confirmed that SCODE can reconstruct observed expression dynamics. We evaluated SCODE by comparing its inferred networks with use of a DNaseI-footprint based network. The performance of SCODE was best for two of the datasets and nearly best for the remaining dataset. We also compared the runtimes and showed that the runtimes for SCODE are significantly shorter than for alternatives. Thus, our algorithm provides a promising approach for further singlecell differentiation analyses. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells (hPSCs) have the capacity to differentiate into essentially all cell types in the body. Such differentiation can be directed to specific cell types by appropriate cell culture conditions or overexpressing lineage-defining transcription factors (TFs). Especially, for the activation of myogenic program, early studies have shown the effectiveness of enforced expression of TFs associated with myogenic differentiation, such as PAX7 and MYOD1. However, the efficiency of direct differentiation was rather low, most likely due to chromatin features unique to hPSCs, which hinder the access of TFs to genes involved in muscle differentiation. Indeed, recent studies have demonstrated that ectopic expression of epigenetic-modifying factors such as a histone demethylase and an ATP-dependent remodeling factor significantly enhances myogenic differentiation from hPSCs. In this article, we review the recent progress for in vitro generation of skeletal muscles from hPSCs through forced epigenetic and transcriptional manipulation. [ABSTRACT FROM AUTHOR]

Key points The ductal system of the pancreas secretes large volumes of alkaline fluid containing HCO3− concentrations as high as 140 m m during hormonal stimulation., A computational model has been constructed to explore the underlying ion transport mechanisms. Parameters were estimated by fitting the model to experimental data from guinea-pig pancreatic ducts., The model was readily able to secrete 140 m m HCO3−. Its capacity to do so was not dependent upon special properties of the cystic fibrosis transmembrane conductance regulator (CFTR) anion channels and solute carrier family 26 member A6 (SLC26A6) anion exchangers., We conclude that the main requirement for secreting high HCO3− concentrations is to minimize the secretion of Cl− ions., These findings help to clarify the mechanism responsible for pancreatic HCO3− secretion, a vital process that prevents the formation of protein plugs and viscous mucus in the ducts, which could otherwise lead to pancreatic disease., Abstract A computational model of guinea-pig pancreatic duct epithelium was developed to determine the transport mechanism by which HCO3− ions are secreted at concentrations in excess of 140 m m. Parameters defining the contributions of the individual ion channels and transporters were estimated by least-squares fitting of the model predictions to experimental data obtained from isolated ducts and intact pancreas under a range of experimental conditions. The effects of cAMP-stimulated secretion were well replicated by increasing the activities of the basolateral Na+-HCO3− cotransporter (NBC1) and apical Cl−/HCO3− exchanger (solute carrier family 26 member A6; SLC26A6), increasing the basolateral K+ permeability and apical Cl− and HCO3− permeabilities (CFTR), and reducing the activity of the basolateral Cl−/HCO3− exchanger (anion exchanger 2; AE2). Under these conditions, the model secreted ∼140 m m HCO3− at a rate of ∼3 nl min−1 mm−2, which is consistent with experimental observations. Alternative 1:2 and 1:1 stoichiometries for Cl−/HCO3− exchange via SLC26A6 at the apical membrane were able to support a HCO3−-rich secretion. Raising the HCO3−/Cl− permeability ratio of CFTR from 0.4 to 1.0 had little impact upon either the secreted HCO3− concentration or the volume flow. However, modelling showed that a reduction in basolateral AE2 activity by ∼80% was essential in minimizing the intracellular Cl− concentration following cAMP stimulation and thereby maximizing the secreted HCO3− concentration. The addition of a basolateral Na+-K+-2Cl− cotransporter (NKCC1), assumed to be present in rat and mouse ducts, raised intracellular Cl− and resulted in a lower secreted HCO3− concentration, as is characteristic of those species. We conclude therefore that minimizing the driving force for Cl− secretion is the main requirement for secreting 140 m m HCO3−. [ABSTRACT FROM AUTHOR]

Harnessing epigenetic regulation is crucial for the efficient and proper differentiation of pluripotent stem cells (PSCs) into desired cell types. Histone H3 lysine 27 trimethylation (H3K27me3) functions as a barrier against cell differentiation through the suppression of developmental gene expression in PSCs. Here, we have generated human PSC (hPSC) lines in which genome-wide reduction of H3K27me3 can be induced by ectopic expression of the catalytic domain of the histone demethylase JMJD3 (called JMJD3c). We found that transient, forced demethylation of H3K27me3 alone triggers the upregulation of mesoendodermal genes, even when the culture conditions for the hPSCs are not changed. Furthermore, transient and forced expression of JMJD3c followed by the forced expression of lineage-defining transcription factors enabled the hPSCs to activate tissue-specific genes directly. We have also shown that the introduction of JMJD3c facilitates the differentiation of hPSCs into functional hepatic cells and skeletal muscle cells. These results suggest the utility of the direct manipulation of epigenomes for generating desired cell types from hPSCs for cell transplantation therapy and platforms for drug screenings. [ABSTRACT FROM AUTHOR]

Osteoprotegerin (OPG), a decoy receptor for receptor activator of NF-κB ligand (RANKL), antagonizes RANKL’s osteoclastogenic function in bone. We previously demonstrated that systemic administration of lipopolysaccharide (LPS) to mice elevates OPG levels and reduces RANKL levels in peripheral blood. Here, we show that mice infected with Salmonella, Staphylococcus, Mycobacteria or influenza virus also show elevated serum OPG levels. We then asked whether OPG upregulation following microbial invasion had an effect outside of bone. To do so, we treated mice with LPS and observed OPG production in pancreas, especially in β-cells of pancreatic islets. Insulin release following LPS administration was enhanced in mice lacking OPG, suggesting that OPG inhibits insulin secretion under acute inflammatory conditions. Consistently, treatment of MIN6 pancreatic β-cells with OPG decreased their insulin secretion following glucose stimulation in the presence of LPS. Finally, our findings suggest that LPS-induced OPG upregulation is mediated in part by activator protein (AP)-1 family transcription factors, particularly Fos proteins. Overall, we report that acute microbial infection elevates serum OPG, which maintains β-cell homeostasis by restricting glucose-stimulated insulin secretion, possibly preventing microbe-induced exhaustion of β-cell secretory capacity. [ABSTRACT FROM AUTHOR]

Freezing is usually used for preservation and storage of biological samples; however, this process may have some adverse effects such as cell membrane damage. Aquaporin (AQP), a water channel protein, has been suggested to play some roles for cryopreservation although its molecular mechanism remains unclear. Here we show that membrane damage caused by ultra-quick freezing is rescued by the expression of AQP4. We next examine if the expression of AQP combined with ultra-quick freezing can be used to select cells efficiently under freezing conditions where most cells are died. CHO cells stably expressing AQP4 were exclusively selected from mixed cell cultures. Having identified the increased expression of AQP4 during ES cell differentiation into neuro-ectoderm using bioinformatics, we confirmed the improved survival of differentiated ES cells with AQP4 expression. Finally we show that CHO cells transiently transfected with Endothelin receptor A and Aqp4 were also selected and concentrated by multiple cycles of freezing/thawing, which was confirmed with calcium imaging in response to endothelin. Furthermore, we found that the expression of AQP enables a reduction in the amount of cryoprotectants for freezing, thereby decreasing osmotic stress and cellular toxicity. Taken together, we propose that this simple but efficient and safe method may be applicable to the selection of mammalian cells for applications in regenerative medicine as well as cell-based functional assays or drug screening protocols. [ABSTRACT FROM AUTHOR]

There is urgent need for biomarkers that provide early detection of pancreatic ductal adenocarcinoma (PDAC) as well as discrimination of autoimmune pancreatitis, as current clinical approaches are not suitably accurate for precise diagnosis. We used mass spectrometry to analyze protein profiles of more than 300 plasma specimens obtained from PDAC, noncancerous pancreatic diseases including autoimmune pancreatitis patients and healthy subjects. We obtained 1063 proteomic signals from 160 plasma samples in the training cohort. A proteomic signature consisting of 7 mass spectrometry signals was used for construction of a proteomic model for detection of PDAC patients. Using the test cohort, we confirmed that this proteomic model had discrimination power equal to that observed with the training cohort. The overall sensitivity and specificity for detection of cancer patients were 82.6% and 90.9%, respectively. Notably, 62.5% of the stage I and II cases were detected by our proteomic model. We also found that 100% of autoimmune pancreatitis patients were correctly assigned as noncancerous individuals. In the present paper, we developed a proteomic model that was shown able to detect early-stage PDAC patients. In addition, our model appeared capable of discriminating patients with autoimmune pancreatitis from those with PDAC. [ABSTRACT FROM AUTHOR]

Aquaporin 12 (AQP12) is the most recently identified member of the mammalian AQP family and is specifically expressed in pancreatic acinar cells. In vitro expression studies have revealed that AQP12 is localized at intracel- lular sites. To determine the physiological roles of AQP12 in the pancreas, we generated knockout mice for this gene (AQP12-KO). No obvious differences were observed under normal conditions between wild-type (WT) and AQP12-KO mice in terms of growth, blood chemistry, pancreatic fluid content, or histology. However, when we induced pancreatitis through the administration of a cholecystokinin-8 (CCK-8) analog, the AQP12-KO mice showed more severe patholog- ical damage to this organ than WT mice. Furthermore, when we analyzed exocytosis in the pancreatic acini using a two-photon excitation imaging method, the results revealed larger exocytotic vesicles (vacuoles) in the acini of AQP12-KO mice at a high CCK-8 dose (100 nM). From these results, we conclude that AQP12 may function in the mechanisms that control the proper secretion of pancreatic fluid following rapid and intense stimulation. [ABSTRACT FROM AUTHOR]

Cellular mechanisms underlying the impairment of pancreatic fluid and electrolyte secretion in diabetes were examined using interlobular ducts isolated from rat pancreas. Fluid secretion was assessed by monitoring changes in luminal volume. HCO uptake across the basolateral membrane was estimated from the recovery of intracellular pH following an acid load. Exposure to high glucose concentrations inhibited fluid secretion and reduced the rate of basolateral HCO uptake in secretin-stimulated ducts isolated from normal rats. In ducts isolated from streptozotocin-treated diabetic rats, fluid secretion and basolateral HCO uptake were also severely impaired but could be largely reversed by incubation in normal-glucose solutions. Sodium-dependent glucose cotransporter 1 (SGLT1), glucose transporter (GLUT)1, GLUT2, and GLUT8 transcripts were detected by reverse transcriptase polymerase chain reaction in isolated ducts. Raising the luminal glucose concentration in microperfused ducts caused a depolarization of the membrane potential, consistent with the presence of SGLT1 at the apical membrane. Unstimulated ducts filled with high-glucose solutions lost luminal fluid by a phlorizin-sensitive mechanism, indicating that pancreatic ducts are capable of active glucose reabsorption from the lumen via SGLT1. In ducts exposed to high glucose concentrations, continuous glucose diffusion to the lumen and active reabsorption via SGLT1 would lead to elevation of intracellular Na+ concentration and sustained depolarization of the apical membrane. These two factors would tend to inhibit the basolateral uptake and apical efflux of Cl− and HCO and could therefore account for the impaired fluid and electrolyte secretion that is observed in diabetes. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate the feasibility and safety of endoscopic ultrasonography (EUS)-guided trucut biopsy (TCB) for diagnosis of autoimmune pancreatitis (AIP). Fourteen patients with suspected AIP based on imaging studies underwent both EUS-guided fine-needle aspiration (FNA) and EUS-TCB for diagnosis of AIP and exclusion of pancreatic cancer (PC). According to the revised Japanese clinical diagnostic criteria, AIP was diagnosed in eight while the remaining six patients had pancreatitis of other etiologies. Pathologically, AIP was defined as lymphoplasmacytic sclerosing pancreatitis (LPSP), and sub-divided into two types: definite LPSP (d-LPSP) showing fulspectrum of LPSP and probable LPSP (p-LPSP) without obliterative phlebitis or abundant (>10 cells/hpf) IgG4-positive plasmacytes infiltration. PC was excluded in all patients. EUS-FNA resulted in three of eight patients with AIP were reported as p-LPSP, one was reported as normal, and 4 were inconclusive. One of six with non-autoimmune pancreatitis was diagnosed as p-LPSP on EUS-FNA, one as idiopathic chronic pancreatitis (ICP) and four were inconclusive. By using EUS-TCB, all AIP patients were diagnosed as LPSP (4 d-LPSP and 4 p-LPSP). Of the six patients with non-autoimmune pancreatitis, three were diagnosed as LPSP (1 d-LPSP and 2 p-LPSP) and three showed ICP on TCB. No complications were identified in any patient with either EUS-FNA or TCB. EUS-TCB is a safe and accurate procedure for obtaining a histological diagnosis in patients with suspected AIP. EUS-TCB can serve as a rescue technique in cases of AIP lacking typical findings. [ABSTRACT FROM AUTHOR]

Pancreatic duct epithelium secretes a HCO3--rich fluid by a mechanism dependent on cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. However, the exact role of CFTR remains unclear. One possibility is that the HCO3- permeability of CFTR provides a pathway for apical HCO3- efflux during maximal secretion. We have therefore attempted to measure electrodiffusive fluxes of HCO3- induced by changes in membrane potential across the apical membrane of interlobular ducts isolated from the guinea pig pancreas. This was done by recording the changes in intracellular pH (pHi) that occurred in luminally perfused ducts when membrane potential was altered by manipulation of bath K+ concentration. Apical HCO3- fluxes activated by cyclic AMP were independent of Cl- and luminal Na+, and substantially inhibited by the CFTR blocker, CFTRinh-172. Furthermore, comparable HCO3- fluxes observed in ducts isolated from wild-type mice were absent in ducts from cystic fibrosis (SF) mice. To estimate the HCO3- permeability of the apical membrane under physiological conditions, guinea pig ducts were luminally perfused with a solution containing 125 mM HCO3- and 24 mM Cl- in the presence of 5% CO2. From the changes in pHi, membrane potential, and buffering capacity, the flux and electrochemical gradient of HCO3- across the apical membrane were determined and used to calculate the HCO~1 permeability. Our estimate of ~0.1 μm sec-1 for the apical HCO3- permeability of guinea pig duct cells under these conditions is close to the value required to account for observed rates of HCO3- secretion. This suggests that CFTR functions as a HCO3- channel in pancreatic duct cells, and that it provides a significant pathway for HCO3- transport across the apical membrane. [ABSTRACT FROM AUTHOR]

The SLC26 transporters are a family of mostly luminal Cl-- and HCO3- transporters. The transport mechanism and the Cl-/HCO3- stoichiometry are not known for any member of the family. To address these questions, we simultaneously measured the HCO3- and Cl- fluxes and the current or membrane potential of slc26a3 and slc26a6 expressed in Xenopus laevis oocytes and the current of the transporters expressed in human embryonic kidney 293 cells. slc26a3 mediates a coupled 2Cl-/1HCO3- exchanger. The membrane potential modulated the apparent affinity for extracellular Cl-- of Cl-/HCO3- exchange by slc26a3. Interestingly, the replacement of Cl-- with NO3- or SCN- uncoupled the transport, with large NO3- and SCN- currents and low HCO3- transport. An apparent uncoupled current was also developed during the incubation of slc26a3-expressing oocytes in HCO3-buffered Cl---free media. These findings were used to develop a turnover cycle for Cl-- and HCO3- transport by slc26a3. Cl-- and HCP3- flux measurements revealed that slc26a6 mediates a 1Cl-/2HCO3- exchange. Accordingly, holding the membrane potential at 40 and -100 mV accelerated and inhibited, respectively, Cl---mediated HCO3- influx, and holding the membrane potential at -100 mV increased HCO3--mediated Cl-- influx. These findings indicate that slc26a6 functions as a coupled 1Cl-/2HCO3- exchanger. The significance of isoform-specific Cl-- and HCO3- transport stoichiometry by slc26a3 and slc26a6 is discussed in the context of diseases of epithelial Cl-- absorption and HCO3- secretion. [ABSTRACT FROM AUTHOR]

Ethanol strongly augments secretin-stimulated, but not acetylcholine (ACh)-stimulated, fluid secretion from pancreatic duct cells. To understand its mechanism of action, we examined the effect of short-chain n-alcohols on fluid secretion and intracellular Ca2+ concentration ([Ca2+]i) in guinea pig pancreatic ducts. Fluid secretion was measured by monitoring the luminal volume of isolated interlobular ducts. [Ca2+]i was estimated using fura-2 microfluorometry. Methanol and ethanol at 0.3-10 mM concentrations significantly augmented fluid secretion and induced a transient elevation of [Ca2+]i in secretin- or dibutyryl adenosine 3′,5′-cyclic monophosphate (DBcAMP)-stimulated ducts. However, they failed to affect fluid secretion and [Ca2+]i in unstimulated and ACh-stimulated ducts. In contrast, propanol and butanol at 0.3-10 mM concentrations significantly reduced fluid secretion and decreased [Ca2+ ]i in unstimulated ducts and in ducts stimulated with secretin, DBcAMP, or ACh. Both stimulatory and inhibitory effects of n-alcohols completely disappeared after their removal from the perfusate. Propanol and butanol inhibited the plateau phase, but not the initial peak, of [Ca2+]i response to ACh as well as the [Ca2+]i elevation induced by thapsigargin, suggesting that they inhibit Ca2+ influx. Removal of extracellular Ca2+ reduced [Ca2+]i in duct cells and completely abolished secretin-stimulated fluid secretion. In conclusion, there is a distinct cutoff point between ethanol (C2) and propanol (C3) in their effects on fluid secretion and [Ca2+]i in duct cells. Short-chain n-alcohols appear to affect pancreatic ductal fluid secretion by activating or inhibiting the plasma membrane Ca2+ channel. [ABSTRACT FROM AUTHOR]

Background: Transplantation of pancreatic β cells generated in vitro from pluripotent stem cells (hPSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) has been proposed as an alternative therapy for diabetes. Though many differentiation protocols have been developed for this purpose, lentivirus-mediated forced expression of transcription factors (TF)—PDX1 and NKX6.1—has been at the forefront for its relatively fast and straightforward approach. However, considering that such cells will be used for therapeutic purposes in the future, it is desirable to develop a procedure that does not leave any footprint on the genome, as any changes of DNAs could potentially be a source of unintended, concerning effects such as tumorigenicity. In this study, we attempted to establish a novel protocol for rapid and footprint-free hESC differentiation into a pancreatic endocrine lineage by using synthetic mRNAs (synRNAs) encoding PDX1 and NKX6.1. We also tested whether siPOU5F1, which reduces the expression of pluripotency gene POU5F1 (also known as OCT4), can enhance differentiation as reported previously for mesoderm and endoderm lineages. Methods: synRNA-PDX1 and synRNA-NKX6.1 were synthesized in vitro and were transfected five times to hESCs with a lipofection reagent in a modified differentiation culture condition. siPOU5F1 was included only in the first transfection. Subsequently, cells were seeded onto a low attachment plate and aggregated by an orbital shaker. At day 13, the degree of differentiation was assessed by quantitative RT-PCR (qRT-PCR) and immunohistochemistry for endocrine hormones such as insulin, glucagon, and somatostatin. Results: Both PDX1 and NKX6.1 expression were detected in cells co-transfected with synRNA-PDX1 and synRNA-NKX6.1 at day 3. Expression levels of insulin in the transfected cells at day 13 were 450 times and 14 times higher by qRT-PCR compared to the levels at day 0 and in cells cultured without synRNA transfection, respectively. Immunohistochemically, pancreatic endocrine hormones were not detected in cells cultured without synRNA transfection but were highly expressed in cells transfected with synRNA-PDX1, synRNA-NKX6.1, and siPOU5F1 at as early as day 13. Conclusions: In this study, we report a novel protocol for rapid and footprint-free differentiation of hESCs to endocrine cells. [ABSTRACT FROM AUTHOR]

The mechanisms of immunoactivation by salt are now becoming clearer. However, those of immunosuppression remain unknown. Since clinical evidence indicates that salt protects proximal tubules from injury, we investigated mechanisms responsible for salt causing immunosuppression in proximal tubules. We focused on cytokine-related gene expression profiles in kidneys of mice fed a high salt diet using microarray analysis and found that both an interferon gamma (IFNγ) inducible chemokine, chemokine (C-X-C motif) ligand 9 (CXCL9), and receptor, CXCR3, were suppressed. We further revealed that a high salt concentration suppressed IFNγ inducible chemokines in HK2 proximal tubular cells. Finally, we demonstrated that a high salt concentration decreased IFNGR1 expression in the basolateral membrane of HK2 cells, leading to decreased phosphorylation of activation sites of Janus kinase 1 (JAK1) and Signal Transducers and Activator of Transcription 1 (STAT1), activators of chemokines. JAK inhibitor canceled the effect of a high salt concentration on STAT1 and chemokines, indicating that the JAK1-STAT1 signaling pathway is essential for this mechanism. In conclusion, a high salt concentration suppresses IFNγ-JAK1-STAT1 signaling pathways and chemokine expressions in proximal tubules. This finding may explain how salt ameliorates proximal tubular injury and offer a new insight into the linkage between salt and immunity. [ABSTRACT FROM AUTHOR]

Efficient differentiation of human pluripotent stem cells (hPSCs) into neurons is paramount for disease modeling, drug screening, and cell transplantation therapy in regenerative medicine. In this manuscript, we report the capability of five transcription factors (TFs) toward this aim: NEUROG1, NEUROG2, NEUROG3, NEUROD1, and NEUROD2. In contrast to previous methods that have shortcomings in their speed and efficiency, a cocktail of these TFs as synthetic mRNAs can differentiate hPSCs into neurons in 7 days, judged by calcium imaging and electrophysiology. They exhibit motor neuron phenotypes based on immunostaining. These results indicate the establishment of a novel method for rapid, efficient, and footprint-free differentiation of functional neurons from hPSCs. [ABSTRACT FROM AUTHOR]