Background/Aims: Previously we described insulinotropic effects of Leonurus sibiricus L. plant extracts used for diabetes mellitus treatment in Traditional Mongolian Medicine. The flavonoid quercetin and its glycoside rutin, which exert anti-diabetic properties in vivo by interfering with insulin signaling in peripheral target tissues, are constituents of these extracts. This study was performed to better understand short- and long-term effects of quercetin and rutin on beta-cells. Methods: Cell viability, apoptosis, phospho-protein abundance and insulin release were determined using resazurin, annexin-V binding assays, Western blot and ELISA, respectively. Membrane potentials (V_mem), whole-cell Ca²⁺ (ICa)- and ATP-sensitive K⁺ (IK_ATP) currents were measured by patch clamp. Intracellular Ca²⁺ (Ca_i) levels were measured by time-lapse imaging using the ratiometric Ca²⁺ indicator Fura-2. Results: Rutin, quercetin and the phosphoinositide-3-kinase (PI3K) inhibitor LY294002 caused a dose-dependent reduction in cell viability with IC₅₀ values of ∼75 µM, ∼25 µM and ∼3.5 µM, respectively. Quercetin (50 µM) significantly increased the percentage of Annexin-V+ cells within 48 hrs. The mean cell volume (MCV) of quercetin-treated cells was significantly lower. Within 2 hrs, quercetin significantly decreased basal- and insulin-stimulated Akt(T308) phosphorylation and increased Erk1/2 phosphorylation, without affecting P-Akt(S473) abundance. Basal- and glucose-stimulated insulin release were significantly stimulated by quercetin. Quercetin significantly depolarized V_mem by ∼25 mV which was prevented by the K_ATP-channel opener diazoxide, but not by the L-type ICa inhibitor nifedipine. Quercetin significantly stimulated ICa and caused a 50% inhibition of IK_ATP. The effects on V_mem, ICa and IK_ATP rapidly reached peak values and then gradually diminished to control values within ∼1 minute. With a similar time-response quercetin induced an elevation in Ca_i which was completely abolished in the absence of Ca²⁺ in the bath solution. Rutin (50 µM) did not significantly alter the percentage of Annexin-V+ cells, MCV, Akt or Erk1/2 phosphorylation, insulin secretion, or the electrophysiological behavior of INS-1 cells. Conclusion: We conclude that quercetin acutely stimulates insulin release, presumably by transient K_ATP channel inhibition and ICa stimulation. Long term application of quercetin inhibits cell proliferation and induces apoptosis, most likely by inhibition of PI3K/Akt signaling.

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1-4). In BV-2 cells, glycine (5 mM) led to a rapid Na(+)-dependent depolarization of membrane potential (V mem). Furthermore, glycine increased CV by about 9%. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13%. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na(+) with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.

Many cell types express an acid-sensitive outwardly rectifying (ASOR) anion current of an unknown function. We characterized such a current in BV-2 microglial cells and then studied its interrelation with the volume-sensitive outwardly rectifying (VSOR) Cl− current and the effect of acidosis on cell volume regulation. We used patch clamp, the Coulter method, and the pH-sensitive dye BCECF to measure Cl− currents and cell membrane potentials, mean cell volume, and intracellular pH, respectively. The ASOR current activated at pH ≤ 5.0 and displayed an I− > Cl− > gluconate− permeability sequence. When compared to the VSOR current, it was similarly sensitive to DIDS, but less sensitive to DCPIB, and insensitive to tamoxifen. Under acidic conditions, the ASOR current was the dominating Cl− conductance, while the VSOR current was apparently inactivated. Acidification caused cell swelling under isotonic conditions and prevented the regulatory volume decrease under hypotonicity. We conclude that acidification, associated with activation of the ASOR- and inactivation of the VSOR current, massively impairs cell volume homeostasis. ASOR current activation could affect microglial function under acidotoxic conditions, since acidosis is a hallmark of pathophysiological events like inflammation, stroke or ischemia and migration and phagocytosis in microglial cells are closely related to cell volume regulation.

Background/Aims: For in vitro cytotoxicity testing, discrimination of apoptosis and necrosis represents valuable information. Viability analysis performed at two different time points post treatment could serve such a purpose because the dynamics of metabolic activity of apoptotic and necrotic cells is different, i.e. a more rapid decline of cellular metabolism during necrosis whereas cellular metabolism is maintained during the entire execution phase of apoptosis. This study describes a straightforward approach to distinguish apoptosis and necrosis. Methods: A431 human epidermoid carcinoma cells were treated with different concentrations/doses of actinomycin D (Act-D), 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), Ro 31-8220, H₂O₂ and photodynamic treatment (PDT). The resazurin viability signal was recorded at 2 and 24 hrs post treatment. Apoptosis and necrosis were verified by measuring caspase 3/7 and membrane integrity. Results: Calculation of the difference curve between the 2 and 24 hrs resazurin signals yields the following information: a positive difference signal indicates apoptosis (i.e. high metabolic activity at early time points and low signal at 24 hrs post treatment) while an early reduction of the viability signal indicates necrosis. For all treatments, this dose-dependent sequence of cellular responses could be confirmed by independent assays. Conclusion: Simple and cost-effective viability analysis provides reliable information about the dose ranges of a cytotoxic agent where apoptosis or necrosis occurs. This may serve as a starting point for further in-depth characterisation of cytotoxic treatments.

Histone deacetylases (HDACs) play a key role in epigenetic mechanisms in health and disease and their dysfunction is implied in several cancer entities. Analysis of expression patterns in pancreatic neuroendocrine tumors (pNETs) indicated HDAC5 to be a potential target for future therapies. As a first step towards a possible treatment, the aim of this study was to evaluate the in vitro cellular and molecular effects of HDAC5 inhibition in pNET cells. Two pNET cell lines, BON-1 and QGP-1, were incubated with different concentrations of the selective class IIA HDAC inhibitor, LMK-235. Effects on cell viability were determined using the resazurin-assay, the caspase-assay, and Annexin-V staining. Western Blot and immunofluorescence microscopy were performed to assess the effects on HDAC5 functionality. LMK-235 lowered overall cell viability by inducing apoptosis in a dose- and time-dependent manner. Furthermore, acetylation of histone-H3 increased with higher LMK-235 concentrations, indicating functional inhibition of HDAC4/5. Immunocytochemical analysis showed that proliferative activity (phosphohistone H3 and Ki-67) decreased at highest concentrations of LMK-235 while chromogranin and somatostatin receptor 2 (SSTR2) expression increased in a dose-dependent manner. HDAC5 expression was found to be largely unaffected by LMK-235. These findings indicate LMK-235 to be a potential therapeutic approach for the development of an effective and selective pNET treatment.

Biliary tract cancer (BTC) is still a fatal disease with very poor prognosis. The lack of reliable biomarkers for early diagnosis and of effective therapeutic targets is a major demanding problem in diagnosis and management of BTC. Due to the clinically silent and asymptomatic characteristics of the tumor, most patients are diagnosed at an already advanced stage allowing only for a palliative therapeutic approach. MicroRNAs are small noncoding RNAs well known to regulate various cellular functions and pathologic events including the formation and progression of cancer. Over the last years, several studies have shed light on the role of microRNAs in BTC, making them potentially attractive therapeutic targets and candidates as biomarkers. In this review, we will focus on the role of oncogenic and tumor suppressor microRNAs and their direct targets in BTC. Furthermore, we summarize and discuss data that evaluate the diagnostic power of deregulated microRNAs as possible future biomarkers for BTC.

Skewed citation distribution is a major limitation of the Journal Impact Factor (JIF) representing an outlier-sensitive mean citation value per journal The present study focuses primarily on this phenomenon in the medical literature by investigating a total of n = 982 journals from two medical categories of the Journal Citation Report (JCR). In addition, the three highest-ranking journals from each JCR category were included in order to extend the analyses to non-medical journals. For the journals in these cohorts, the citation data (2018) of articles published in 2016 and 2017 classified as citable items (CI) were analysed using various descriptive approaches including e.g. the skewness, the Gini coefficient, and, the percentage of CI contributing 50% or 90% of the journal’s citations. All of these measures clearly indicated an unequal, skewed distribution with highly-cited articles as outliers. The %CI contributing 50% or 90% of the journal’s citations was in agreement with previously published studies with median values of 13–18% CI or 44–60% CI generating 50 or 90% of the journal’s citations, respectively. Replacing the mean citation values (corresponding to the JIF) with the median to represent the central tendency of the citation distributions resulted in markedly lower numerical values ranging from − 30 to − 50%. Up to 39% of journals showed a median citation number of zero in one medical journal category. For the two medical cohorts, median-based journal ranking was similar to mean-based ranking although the number of possible rank positions was reduced to 13. Correlation of mean citations with the measures of citation inequality indicated that the unequal distribution of citations per journal is more prominent and, thus, relevant for journals with lower citation rates. By using various indicators in parallel and the hitherto probably largest journal sample, the present study provides comprehensive up-to-date results on the prevalence, extent and consequences of citation inequality across medical and all-category journals listed in the JCR.

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