Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer.

BACKGROUND: The purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC. MATERIALS AND METHODS: Blood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay. RESULTS: Risk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1ß, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects. CONCLUSION: In conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Imaging of extracellular and intracellular ATP in pancreatic beta cells reveals correlation between glucose metabolism and purinergic signalling.

Adenosine triphosphate (ATP) is a universal energy molecule and yet cells release it and extracellular ATP is an important signalling molecule between cells. Monitoring of ATP levels outside of cells is important for our understanding of physiological and pathophysiological processes in cells/tissues. Here, we focus on pancreatic beta cells (INS-1E) and test the hypothesis that there is an association between intra- and extracellular ATP levels which depends on glucose provision. We imaged real-time changes in extracellular ATP in pancreatic beta cells using two sensors tethered to extracellular aspects of the plasma membrane (eATeam3.10, iATPSnFR1.0). Increase in glucose induced fast micromolar ATP release to the cell surface, depending on glucose concentrations. Chronic pre-treatment with glucose increased the basal ATP signal. In addition, we co-expressed intracellular ATP sensors (ATeam1.30, PercevalHR) in the same cultures and showed that glucose induced fast increases in extracellular and intracellular ATP. Glucose and extracellular ATP stimulated glucose transport monitored by the glucose sensor (FLII12Pglu-700uDelta6). In conclusion, we propose that in beta cells there is a dynamic relation between intra- and extracellular ATP that depends on glucose transport and metabolism and these processes may be tuned by purinergic signalling. Future development of ATP sensors for imaging may aid development of novel approaches to target extracellular ATP in, for example, type 2 diabetes mellitus therapy.

Associations of Serum Calprotectin, Arterial Stiffness and Long COVID Symptoms in Dalmatian Kidney Transplant Recipients.

We aimed to explore long COVID symptoms, serum calprotectin levels, and the parameters of arterial stiffness in Dalmatian kidney transplant recipients (KTRs) and their possible associations. A cross-sectional, single-center case-control study on 98 KTRs who had recovered from COVID-19 was performed. Long COVID symptoms were explored via standardized questionnaires assessing quality of life, and serum calprotectin was also measured. Out of 98 KTRs with a mean age of 62 years, 63 (64.3%) were men. Medical history, clinical and laboratory parameters, and arterial stiffness measurements were obtained for each study participant. Difficulties with mobility were present in 44.3% of the KTRs, while difficulties with self-care were present in 6.2%, difficulties with usual activities were demonstrated by 35.1%, pain in the extremities was present in 52.5%, and anxiety and depression were present in 26.8%. Our results showed significant differences regarding serum calprotectin levels in clinical manifestations of acute COVID-19 and follow-up laboratory parameters. The most significant positive predictors of the serum calprotectin value in the KTRs were respiratory insufficiency, acute kidney failure, the prescription of antihypertensives, leukocyte and neutrophil counts, the neutrophil/lymphocyte ratio and lactate dehydrogenase levels. Negative predictors were the time since COVID-19, high-density lipoprotein levels, kidney function parameters, and the lymphocyte count. To conclude, serum calprotectin has emerged as a possible promising biomarker for subclinical allograft rejection; however, further studies are needed to better understand this subject.

Synergistic effects of agonists and two-pore-domain potassium channels on secretory responses of human pancreatic duct cells Capan-1.

Mechanisms of synergistic agonist stimulation and modulation of the electrochemical driving force for anion secretion are still not fully explored in human pancreatic duct epithelial cells. The first objective of this study was therefore to test whether combined agonist stimulation augments anion transport responses in the Capan-1 monolayer model of human pancreatic duct epithelium. The second objective was to test the influence of H+,K+-ATPase inhibition on anion transport in Capan-1 monolayers. The third objective was to analyze the expression and function of K+ channels in Capan-1, which could support anion secretion and cooperate with H+,K+-ATPases in pH and potassium homeostasis. The human pancreatic adenocarcinoma cell line Capan-1 was cultured conventionally or as polarized monolayers that were analyzed by Ussing chamber electrophysiological recordings. Single-cell intracellular calcium was assayed with Fura-2. mRNA isolated from Capan-1 was analyzed by use of the nCounter assay or RT-PCR. Protein expression was assessed by immunofluorescence and western blot analyses. Combined stimulation with different physiological agonists enhanced anion transport responses compared to single agonist stimulation. The responsiveness of Capan-1 cells to histamine was also revealed in these experiments. The H+,K+-ATPase inhibitor omeprazole reduced carbachol- and riluzole-induced anion transport responses. Transcript analyses revealed abundant TASK-2, TWIK-1, TWIK-2, TASK-5, KCa3.1, and KCNQ1 mRNA expression. KCNE1 mRNA and TREK-1, TREK-2, TASK-2, and KCNQ1 protein expression were also shown. This study shows that the Capan-1 model recapitulates key physiological aspects of a bicarbonate-secreting epithelium and constitutes a valuable model for functional studies on human pancreatic duct epithelium.

An insulin hypersecretion phenotype precedes pancreatic ß cell failure in MODY3 patient-specific cells.

MODY3 is a monogenic hereditary form of diabetes caused by mutations in the transcription factor HNF1A. The patients progressively develop hyperglycemia due to perturbed insulin secretion, but the pathogenesis is unknown. Using patient-specific hiPSCs, we recapitulate the insulin secretion sensitivity to the membrane depolarizing agent sulfonylurea commonly observed in MODY3 patients. Unexpectedly, MODY3 patient-specific HNF1A+/R272C ß cells hypersecrete insulin both in vitro and in vivo after transplantation into mice. Consistently, we identified a trend of increased birth weight in human HNF1A mutation carriers compared with healthy siblings. Reduced expression of potassium channels, specifically the KATP channel, in MODY3 ß cells, increased calcium signaling, and rescue of the insulin hypersecretion phenotype by pharmacological targeting ATP-sensitive potassium channels or low-voltage-activated calcium channels suggest that more efficient membrane depolarization underlies the hypersecretion of insulin in MODY3 ß cells. Our findings identify a pathogenic mechanism leading to ß cell failure in MODY3.

COVID-19 in kidney transplant recipients; a DALMATIAN single-center experience.

INTRODUCTION: We aimed to explore COVID-19 severity, complications, and outcome predictors in the Dalmatian population of kidney transplant recipients (KTRs). METHODS: KTRs confirmed with acute COVID-19 infection until May 2021 were included and followed up for 6 months. RESULTS: Out of 50 KTRs average aged 63 years, 36 (72%) were men. Nine (18%) KTRs had no pulmonary infiltration, and twenty-nine (58%) did not require oxygen supplementation. Bilateral pulmonary infiltrates had 29 (58%) while high-flow nasal cannula or mechanical ventilation required 8 (16%) KTRs. The mortality rate was 16%. Acute kidney injury developed in 18 (36%), and acute renal replacement therapy required 2 (4%) KTRs. Nine (18%) KTRs were subsequently rehospitalized. Chronic COVID-19 syndrome reported 23 (58%) KTRs. CONCLUSIONS: D-dimers were found to be the key prognostic factor of clinical complications, emphasizing the importance of underlying thrombotic microangiopathy. Optimal immunosuppressant adjusting in KTRs with acute COVID-19 infection remains to be clarified.

Phase Angle Association with Dietary Habits and Metabolic Syndrome in Diabetic Hypertensive Patients: A Cross-Sectional Study.

Phase angle (PhA) levels are often lower than normal because both disease-specific parameters and disease-related inflammatory status, metabolic syndrome (MetS) included, can affect PhA. Therefore, the aim of this cross-sectional study was to compare body composition, metabolic profile and dietary patterns of participants with arterial hypertension (AH), type 2 diabetes mellitus (T2DM) and MetS with regard to PhA values. A total of 208 participants were included, of whom 53.6% were males. For each participant, data about body composition and anthropometric parameters, clinical and laboratory parameters, as well as food frequency questionnaire (FFQ) and Mediterranean Diet Serving Score (MDSS) were obtained. MC-780 Multi Frequency Segmental Body Mass Analyzer (Tanita) was used to assess body composition. Furthermore, waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) were calculated. The results showed that 75 (36.06%) participants had low PhA values and 133 (63.94%) had high PhA values. Participants with higher PhA values had significantly higher body fat percentage (p = 0.04), fat-free mass (kg; p < 0.001), muscle mass (kg; p < 0.001), skeletal muscle mass (% and kg; p < 0.001), sarcopenic index (SMI; p < 0.001) and mid-upper arm circumference (MUAC; p = 0.04), as well as lower fat mass percentage (p = 0.04). Regarding food frequency consumption, significantly higher intakes of red meat (p = 0.003), poultry (p = 0.02) and fast food (p = 0.003) were noticed in participants with higher PhA values. Adherence to the Mediterranean Diet (MeDi) was exceptionally low in both groups of participants, with significantly higher fish intake noticed in participants with high PhA (p = 0.03). In conclusion, our results showed that body composition could be the indicator of PhA in MetS as well as overall low adherence to the MeDi principles. These findings highlight the importance of adequate nutritional strategies and novel approaches to maintaining optimal body composition and adopting proper eating habits within the framework of one's disease.

A New, MWCNT-Based, Solid-State Thiabendazole-Selective Sensor.

Direct potentiometric measurements using solid-state sensors have a great potential for thiabendazole (TBZ) determination, considering simplicity, accuracy, and low cost. Modifying the sensing material of the sensor with multi-walled carbon nanotubes (MWCNTs) leads to improved analytical properties of the sensor. In this study, a new potentiometric solid-state sensor for TBZ determination, based on MWCNTs modified with a sulfate group, and TBZ ion as sensing material was developed. The sensor exhibited a Nernstian response for TBZ (60.4 mV/decade of activity) in a working range between 8.6 × 10-7 and 1.0 × 10-3 M. The detection limit for TBZ was 6.2 × 10-7 M. The response time of the sensor for TBZ was 8 s, and its signal drift was only 1.7 mV/h. The new sensor is applicable for direct potentiometric determination of TBZ in complex real samples, such as fruit peel. The accuracy of TBZ determination is confirmed using the standard addition method.

Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats.

AIM/HYPOTHESIS: Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. METHODS: We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. RESULTS: In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3's cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.

Mediterranean Diet Adherence and Nutritional Status in Dalmatian Diabetic Hypertensive Patients Regarding Presence of Chronic Kidney Disease-Is There Any Difference?

In recent years, the Mediterranean diet has emerged as one of the dietary patterns that could have positive effects on overall health as well in the treatment of non-communicable chronic diseases. The aim of this cross-sectional study was to determine differences in adherence to the Mediterranean diet (MeDi) and nutritional status in patients with type 2 diabetes mellitus (T2DM) and arterial hypertension (AH) regarding the presence of chronic kidney disease (CKD). Two hundred and forty-eight Dalmatian diabetic hypertensive patients (DDHP) were included, and 164 (66.1%) of them had CKD. Data about anthropometric parameters, clinical and laboratory parameters, as well as lifestyle questionnaire and Mediterranean Diet Serving Score (MDSS) were collected for each study participant. Furthermore, body composition was assessed using MC-780 Multi Frequency Segmental Body Mass Analyzer (Tanita). Body mass index (BMI) as well as waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) were calculated. Results showed that only 8.9% of DDHP were adherent to the MeDi without significant differences regarding the presence of CKD. Therefore, only 9.1% of participants with CKD were adherent to the MeDi. Dietary recommendations were received by 52.8% of DDHP and 49.4% with CKD, while only 12.8% of those with CKD were adherent to the given recommendations. The results showed that 88.3% of DDHP and 87.8% of the DDHP with CKD were overweight or obese. Statically significant lower frequency of nut intake suggested by the MeDi was found in those participants with CKD (p = 0.02). Therefore, the significant associations between adherence to each MeDi component as well as MDSS score with the development of CKD among all study subjects were not found. In conclusion, the results showed a low level of nutritional care in our region and low adherence to MeDi among DDHP. According to the results, there is an urgent need to improve nutritional care in our region, with a special focus on the MeDi for this especially vulnerable population of patients.

A brief overview of BNIP3L/NIX receptor-mediated mitophagy.

Mitophagy is a form of autophagy specialized to selectively remove mitochondria. Although the PINK1/Parkin pathway is the best described mitophagy of damaged mitochondria, receptor/mediated mitophagy seems to have a pivotal role in cellular development and specialization. The most studied mitophagy receptor BCL2/adenovirus E1B 19-kDa-interacting protein 3-like (BNIP3L/NIX) is shown to be important for the programmed removal of healthy mitochondria during terminal differentiation of erythrocytes, but its role has been proven in various cell types. Despite recent advances in our understanding of its regulation by phosphorylation and dimerization, there remain numerous questions on how BNIP3L/NIX tightly balances between cellular life and death decisions. This brief review intends to summarize ongoing dilemmas related to BNIP3L/NIX.

The P2X7 Receptor Stimulates IL-6 Release from Pancreatic Stellate Cells and Tocilizumab Prevents Activation of STAT3 in Pancreatic Cancer Cells.

Pancreatic stellate cells (PSCs) are important pancreatic fibrogenic cells that interact with pancreatic cancer cells to promote the progression of pancreatic ductal adenocarcinoma (PDAC). In the tumor microenvironment (TME), several factors such as cytokines and nucleotides contribute to this interplay. Our aim was to investigate whether there is an interaction between IL-6 and nucleotide signaling, in particular, that mediated by the ATP-sensing P2X7 receptor (P2X7R). Using human cell lines of PSCs and cancer cells, as well as primary PSCs from mice, we show that ATP is released from both PSCs and cancer cells in response to mechanical and metabolic cues that may occur in the TME, and thus activate the P2X7R. Functional studies using P2X7R agonists and inhibitors show that the receptor is involved in PSC proliferation, collagen secretion and IL-6 secretion and it promotes cancer cell migration in a human PSC-cancer cell co-culture. Moreover, conditioned media from P2X7R-stimulated PSCs activated the JAK/STAT3 signaling pathway in cancer cells. The monoclonal antibody inhibiting the IL-6 receptor, Tocilizumab, inhibited this signaling. In conclusion, we show an important mechanism between PSC-cancer cell interaction involving ATP and IL-6, activating P2X7 and IL-6 receptors, respectively, both potential therapeutic targets in PDAC.

Differences in the Pattern of Non-Recreational Sharing of Prescription Analgesics among Patients in Rural and Urban Areas.

INTRODUCTION: This study aimed to analyze differences in sharing of prescription analgesics between rural and urban populations. METHODS: We surveyed 1000 participants in outpatient family medicine settings in Croatia. We used a 35-item questionnaire to analyze patients' characteristics, pain intensity, prescription analgesic sharing behavior, and perception of risks regarding sharing prescription medications. RESULTS: Prescription analgesic sharing was significantly more frequent in the rural (64%) than in the urban population 55% (p = 0.01). Participants from rural areas more commonly asked for verbal or written information than those from urban areas when taking others' prescription analgesics (p < 0.001) or giving such analgesics (p < 0.001). Participants from rural areas more commonly informed their physician about such behavior compared to those from urban areas (p < 0.01), and they were significantly more often asked about such behavior by their physician (p < 0.01). Perceptions about risks associated with sharing prescription medication were similar between rural and urban populations. CONCLUSIONS: There are systematic differences in the frequency of prescription analgesics and associated behaviors between patients in family medicine who live in rural and urban areas. Patients from rural areas were more prone to share prescription analgesics. Future studies should examine reasons for differences in sharing prescription analgesics between rural and urban areas.

Dimerization of mitophagy receptor BNIP3L/NIX is essential for recruitment of autophagic machinery.

Mitophagy is a conserved intracellular catabolic process responsible for the selective removal of dysfunctional or superfluous mitochondria to maintain mitochondrial quality and need in cells. Here, we examine the mechanisms of receptor-mediated mitophagy activation, with the focus on BNIP3L/NIX mitophagy receptor, proven to be indispensable for selective removal of mitochondria during the terminal differentiation of reticulocytes. The molecular mechanisms of selecting damaged mitochondria from healthy ones are still very obscure. We investigated BNIP3L dimerization as a potentially novel molecular mechanism underlying BNIP3L-dependent mitophagy. Forming stable homodimers, BNIP3L recruits autophagosomes more robustly than its monomeric form. Amino acid substitutions of key transmembrane residues of BNIP3L, BNIP3LG204A or BNIP3LG208V, led to the abolishment of dimer formation, resulting in the lower LC3A-BNIP3L recognition and subsequently lower mitophagy induction. Moreover, we identified the serine 212 as the main amino acid residue at the C-terminal of BNIP3L, which extends to the intermembrane space, responsible for dimerization. In accordance, the phosphomimetic mutation BNIP3LS212E leads to a complete loss of BNIP3L dimerization. Thus, the interplay between BNIP3L phosphorylation and dimerization indicates that the combined mechanism of LIR phosphorylation and receptor dimerization is needed for proper BNIP3L-dependent mitophagy initiation and progression.Abbreviations: AMBRA1: autophagy and beclin 1 regulator 1; Baf A1: bafilomycin A1; BH3: BCL2 homology 3; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CoCl2: cobalt (II) chloride; FKBP8: FKBP prolyl isomerase 8; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; GST: glutathione S-transferase; IMM: inner mitochondrial membrane; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OMM: outer mitochondrial membrane; PHB2: prohibitin 2; PI: propidium iodide; PINK1: PTEN induced kinase 1; TM: transmembrane domain; TOMM20: translocase of outer mitochondrial membrane 20.

Purinergic Signaling in Pancreas-From Physiology to Therapeutic Strategies in Pancreatic Cancer.

The purinergic signaling has an important role in regulating pancreatic exocrine secretion. The exocrine pancreas is also a site of one of the most serious cancer forms, the pancreatic ductal adenocarcinoma (PDAC). Here, we explore how the network of purinergic and adenosine receptors, as well as ecto-nucleotidases regulate normal pancreatic cells and various cells within the pancreatic tumor microenvironment. In particular, we focus on the P2X7 receptor, P2Y2 and P2Y12 receptors, as well as A2 receptors and ecto-nucleotidases CD39 and CD73. Recent studies indicate that targeting one or more of these candidates could present new therapeutic approaches to treat pancreatic cancer. In pancreatic cancer, as much as possible of normal pancreatic function should be preserved, and therefore physiology of purinergic signaling in pancreas needs to be considered.

Proton Pump Inhibitors Reduce Pancreatic Adenocarcinoma Progression by Selectively Targeting H+, K+-ATPases in Pancreatic Cancer and Stellate Cells.

Pancreatic duct cells are equipped with acid/base transporters important for exocrine secretion. Pancreatic ductal adenocarcinoma (PDAC) cells may utilize such transporters to acidify extracellular tumor microenvironment, creating a niche favoring cell proliferation, fibrosis and resistance to chemotherapy-all contributing to the notoriously bad prognosis of this disease. Here, we report that gastric and non-gastric H+, K+-ATPases (coded by ATP4A and ATP12A) are overexpressed in human and murine pancreatic cancer and that we can target them specifically with proton pump inhibitors (PPIs) and potassium-competitive acid blockers (P-CABs) in in vitro models of PDAC. Focusing on pantoprazole, we show that it significantly reduced human cancer cell proliferation by inhibiting cellular H+ extrusion, increasing K+ conductance and promoting cyclin D1-dependent cell cycle arrest and preventing STAT3 activation. Pantoprazole also decreased collagen secretion from pancreatic stellate cells. Importantly, in vivo studies show that pantoprazole treatment of tumor-bearing mice reduced tumor size, fibrosis and expression of angiogenic markers. This work provides the first evidence that H+, K+-ATPases contribute to PDAC progression and that these can be targeted by inhibitors of these pumps, thus proving a promising therapeutic strategy.

AATF and SMARCA2 are associated with thyroid volume in Hashimoto's thyroiditis patients.

Thyroid volume of Hashimoto's thyroiditis (HT) patients varies in size over the course of disease and it may reflect changes in biological function of thyroid gland. Patients with subclinical hypothyroidism predominantly have increased thyroid volume whereas patients with more pronounced hypothyroidism have smaller thyroid volumes. Suggested mechanism for thyroid atrophy is thyrocyte death due to apoptosis. We performed the first genome-wide association study (GWAS) of thyroid volume in two groups of HT patients, depending on levothyroxine (LT4) therapy, and then meta-analysed across. Study included 345 HT patients in total and 6 007 322 common autosomal genetic variants. Underlying hypothesis was that genetic components that are involved in regulation of thyroid volume display their effect in specific pathophysiologic conditions of thyroid gland of HT patients. We additionally performed immunohistochemical analysis using thyroid tissues and analysed differences in expression levels of identified proteins and apoptotic marker between HT patients and controls. We found genome-wide significant association of two loci, both involved in apoptosis, with thyroid volume of HT patients: rs7212416 inside apoptosis-antagonizing transcription factor AATF (P = 8.95 × 10-9) and rs10738556 near chromatin-remodeling SMARCA2 (P = 2.83 × 10-8). In immunohistochemical analysis we observed that HT patients with homozygous AATF risk genotypes have decreased AATF expression (0.46-fold, P < 0.0001) and increased apoptosis (3.99-fold, P = 0.0001) in comparison to controls. HT patients with heterozygous SMARCA2 genotypes have decreased SMARCA2 expression, albeit without reaching statistical significance (1.07-fold, P = 0.5876), and significantly increased apoptosis (4.11-fold, P < 0.0001). By two lines of evidence we show that two highly plausible genetic loci, AATF and SMARCA2, may be involved in determining the thyroid volume of HT patients. The results of our study significantly add to the current knowledge of disturbed biological mechanisms in thyroid gland of HT patients.

Update of P2Y receptor pharmacology: IUPHAR Review 27.

Eight G protein-coupled P2Y receptor subtypes respond to extracellular adenine and uracil mononucleotides and dinucleotides. P2Y receptors belong to the δ group of rhodopsin-like GPCRs and contain two structurally distinct subfamilies: P2Y1 , P2Y2 , P2Y4 , P2Y6 , and P2Y11 (principally Gq protein-coupled P2Y1 -like) and P2Y12-14 (principally Gi protein-coupled P2Y12 -like) receptors. Brain P2Y receptors occur in neurons, glial cells, and vasculature. Endothelial P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors induce vasodilation, while smooth muscle P2Y2 , P2Y4 , and P2Y6 receptor activation leads to vasoconstriction. Pancreatic P2Y1 and P2Y6 receptors stimulate while P2Y13 receptors inhibits insulin secretion. Antagonists of P2Y12 receptors, and potentially P2Y1 receptors, are anti-thrombotic agents, and a P2Y2 /P2Y4 receptor agonist treats dry eye syndrome in Asia. P2Y receptor agonists are generally pro-inflammatory, and antagonists may eventually treat inflammatory conditions. This article reviews recent developments in P2Y receptor pharmacology (using synthetic agonists and antagonists), structure and biophysical properties (using X-ray crystallography, mutagenesis and modelling), physiological and pathophysiological roles, and present and potentially future therapeutic targeting.

The Vacuolar H+ ATPase α3 Subunit Negatively Regulates Migration and Invasion of Human Pancreatic Ductal Adenocarcinoma Cells.

Increased metabolic acid production and upregulation of net acid extrusion render pH homeostasis profoundly dysregulated in many cancers. Plasma membrane activity of vacuolar H+ ATPases (V-ATPases) has been implicated in acid extrusion and invasiveness of some cancers, yet often on the basis of unspecific inhibitors. Serving as a membrane anchor directing V-ATPase localization, the a subunit of the V0 domain of the V-ATPase (ATP6V0a1-4) is particularly interesting in this regard. Here, we map the regulation and roles of ATP6V0a3 in migration, invasion, and growth in pancreatic ductal adenocarcinoma (PDAC) cells. a3 mRNA and protein levels were upregulated in PDAC cell lines compared to non-cancer pancreatic epithelial cells. Under control conditions, a3 localization was mainly endo-/lysosomal, and its knockdown had no detectable effect on pHi regulation after acid loading. V-ATPase inhibition, but not a3 knockdown, increased HIF-1 expression and decreased proliferation and autophagic flux under both starved and non-starved conditions, and spheroid growth of PDAC cells was also unaffected by a3 knockdown. Strikingly, a3 knockdown increased migration and transwell invasion of Panc-1 and BxPC-3 PDAC cells, and increased gelatin degradation in BxPC-3 cells yet decreased it in Panc-1 cells. We conclude that in these PDAC cells, a3 is upregulated and negatively regulates migration and invasion, likely in part via effects on extracellular matrix degradation.