Physiological concentrations of cyanide stimulate mitochondrial Complex IV and enhance cellular bioenergetics.

In mammalian cells, cyanide is viewed as a cytotoxic agent, which exerts its effects through inhibition of mitochondrial Complex IV (Cytochrome C oxidase [CCOx]). However, the current report demonstrates that cyanide's effect on CCOx is biphasic; low (nanomolar to low-micromolar) concentrations stimulate CCOx activity, while higher (high-micromolar) concentrations produce the "classic" inhibitory effect. Low concentrations of cyanide stimulated mitochondrial electron transport and elevated intracellular adenosine triphosphate (ATP), resulting in the stimulation of cell proliferation. The stimulatory effect of cyanide on CCOx was associated with the removal of the constitutive, inhibitory glutathionylation on its catalytic 30- and 57-kDa subunits. Transfer of diluted Pseudomonas aeruginosa (a cyanide-producing bacterium) supernatants to mammalian cells stimulated cellular bioenergetics, while concentrated supernatants were inhibitory. These effects were absent with supernatants from mutant Pseudomonas lacking its cyanide-producing enzyme. These results raise the possibility that cyanide at low, endogenous levels serves regulatory purposes in mammals. Indeed, the expression of six putative mammalian cyanide-producing and/or -metabolizing enzymes was confirmed in HepG2 cells; one of them (myeloperoxidase) showed a biphasic regulation after cyanide exposure. Cyanide shares features with "classical" mammalian gasotransmitters NO, CO, and H2S and may be considered the fourth mammalian gasotransmitter.

Meta-analysis of metabolites involved in bioenergetic pathways reveals a pseudohypoxic state in Down syndrome.

Clinical observations and preclinical studies both suggest that Down syndrome (DS) may be associated with significant metabolic and bioenergetic alterations. However, the relevant scientific literature has not yet been systematically reviewed. The aim of the current study was to conduct a meta-analysis of metabolites involved in bioenergetics pathways in DS to conclusively determine the difference between DS and control subjects. We discuss these findings and their potential relevance in the context of pathogenesis and experimental therapy of DS. Articles published before July 1, 2020, were identified by using the search terms "Down syndrome" and "metabolite name" or "trisomy 21" and "metabolite name". Moreover, DS-related metabolomics studies and bioenergetics literature were also reviewed. 41 published reports and associated databases were identified, from which the descriptive information and the relevant metabolomic parameters were extracted and analyzed. Mixed effect model revealed the following changes in DS: significantly decreased ATP, CoQ10, homocysteine, serine, arginine and tyrosine; slightly decreased ADP; significantly increased uric acid, succinate, lactate and cysteine; slightly increased phosphate, pyruvate and citrate. However, the concentrations of AMP, 2,3-diphosphoglycerate, glucose, and glutamine were comparable in the DS vs. control populations. We conclude that cells of subjects with DS are in a pseudo-hypoxic state: the cellular metabolic and bio-energetic mechanisms exhibit pathophysiological alterations that resemble the cellular responses associated with hypoxia, even though the supply of the cells with oxygen is not disrupted. This fundamental alteration may be, at least in part, responsible for a variety of functional deficits associated with DS, including reduced exercise difference, impaired neurocognitive status and neurodegeneration.

Biological noise and positional effects influence cell stemness.

Biological (or cellular) noise is the random quantitative variability of proteins and other molecules in individual, genetically identical cells. As the result of biological noise in the levels of some transcription factors that determine a cell's differentiation status, differentiated cells may dedifferentiate to a stem cell state given a sufficiently long time period. Here, to provide direct evidence supporting this hypothesis, we used a live-cell monitoring system based on enhanced green fluorescent protein (eGFP) expression to continuously assess the "stemness" of individual human and murine malignant mesothelioma cells over a period of up to 3 months. Re-expression of the transcription factors, the top hierarchical stemness markers Sox2 (SRY-box 2) and Oct4 (octamer-binding transcription factor), monitored as cell eGFP expression was observed in a subpopulation of differentiated eGFP(-) malignant mesothelioma cells. However, we found that this transition was extremely rare. Of note, when it did occur, neighboring cells that were not direct descendants of a newly emerged eGFP(+) stem cell were more likely than non-neighboring cells to also become an eGFP(+) stem cell. This observation suggested a positional effect and led to a clustered "mosaic" reappearance of eGFP(+) stem cells. Moreover, stem cells reappeared even in cell cultures derived from one single differentiated eGFP(-) cell. On the basis of our experimental in vitro and in vivo findings, we developed a tumor growth model to predict the clustered localization of cancer stem cells within a tumor mass.

Extending the Mathematical Palette for Developmental Pattern Formation: Piebaldism.

Here, we present a theoretical investigation with potential insights on developmental mechanisms. Three biological factors, consisting of two diffusing factors and a cell-autonomous immobile transcription factor are combined with different feedback mechanisms. This results in four different situations or fur patterns. Two of them reproduce classical Turing patterns: (1) regularly spaced spots, (2) labyrinth patterns or straight lines with an initial slope in the activation of the transcription factor. The third situation does not lead to patterns, but results in different homogeneous color tones. Finally, the fourth one sheds new light on the possible mechanisms leading to the formation of piebald patterns exemplified by the random patterns on the fur of some cows' strains and Dalmatian dogs. Piebaldism is usually manifested as white areas of fur, hair, or skin due to the absence of pigment-producing cells in those regions. The distribution of the white and colored zones does not reflect the classical Turing patterns. We demonstrate that these piebald patterns are of transient nature, developing from random initial conditions and relying on a system's bistability. We show numerically that the presence of a cell-autonomous factor not only expands the range of reaction diffusion parameters in which a pattern may arise, but also extends the pattern-forming abilities of the reaction-diffusion equations.

Using data on snus use in Sweden to compare different modelling approaches to estimate the population health impact of introducing a smoke-free tobacco product.

BACKGROUND: We have developed an approach for modelling the health impact of introducing new smoke-free tobacco products. We wished to compare its estimates with those of alternative approaches, when applied to snus, used in Sweden for many years. METHODS: Modelling was restricted to men aged 30-79 years for 1980-2009 and to four smoking-related diseases. Mortality data were extracted for Sweden and other European countries. Published data provided Swedish prevalence estimates for combinations of never/former/current smoking and snus use, and smoking prevalence estimates for other European countries. Approach 1 compares mortality in Sweden and in other countries with a smoking prevalence similar to Sweden's prevalence of combined smoking/snus use. Approaches 2 and 3 compare mortality in Sweden with hypothetical mortality had snus users smoked. Approach 3 uses our health impact model, individuals starting with the tobacco prevalence of Sweden in 1980. Tobacco histories during 30-year follow-up were then estimated using transition probabilities, with risk derived using a negative exponential model. Approach 2 uses annual tobacco prevalence estimates coupled with estimates of relative risk of current and former smokers regardless of history. The main applications of Approaches 2 and 3 assume that only smoking affects mortality, though sensitivity analyses using Approach 3 allow for risk to vary in snus users and dual users. RESULTS: Using Approach 2, estimated mortality increases in Sweden in 1980-2009 had snus not been introduced were: lung cancer 8786; COPD 1781; IHD 10,409; stroke 1720. The main Approach 3 estimates were similar (7931, 1969; 12,501; 1901). They decreased as risk in snus users and dual users increased. Approach 1 estimates differed wildly (77,762, 32,538; 77,438; 76,946), remaining very different following correction for differences between Sweden and the comparison countries in non-smoking-related disease mortality. CONCLUSIONS: Approach 1 is unreliable, accounting inadequately for non-tobacco factors affecting mortality. Approaches 2 and 3 provide reasonably similar approximate estimates of the mortality increase had snus not been available, but have differing advantages and disadvantages. Only Approach 3 considers tobacco history, but develops histories using tobacco transition probabilities, which is possibly less reliable than using estimated tobacco prevalences at each follow-up year.

Regulation of calretinin in malignant mesothelioma is mediated by septin 7 binding to the CALB2 promoter.

BACKGROUND: The calcium-binding protein calretinin (gene name: CALB2) is currently considered as the most sensitive and specific marker for the diagnosis of malignant mesothelioma (MM). MM is a very aggressive tumor strongly linked to asbestos exposure and with no existing cure so far. The mechanisms of calretinin regulation, as well as its distinct function in MM are still poorly understood. METHODS: We searched for transcription factors binding to the CALB2 promoter and modulating calretinin expression. For this, DNA-binding assays followed by peptide shotgun-mass spectroscopy analyses were used. CALB2 promoter activity was assessed by dual-luciferase reporter assays. Furthermore, we analyzed the effects of CALB2 promoter-binding proteins by lentiviral-mediated overexpression or down-regulation of identified proteins in MM cells. The modulation of expression of such proteins by butyrate was determined by subsequent Western blot analysis. Immunohistochemical analysis of embryonic mouse lung tissue served to verify the simultaneous co-expression of calretinin and proteins interacting with the CALB2 promoter during early development. Finally, direct interactions of calretinin with target proteins were evidenced by co-immunoprecipitation experiments. RESULTS: Septin 7 was identified as a butyrate-dependent transcription factor binding to a CALB2 promoter region containing butyrate-responsive elements (BRE) resulting in decreased calretinin expression. Accordingly, septin 7 overexpression decreased calretinin expression levels in MM cells. The regulation was found to operate bi-directionally, i.e. calretinin overexpression also decreased septin 7 levels. During murine embryonic development calretinin and septin 7 were found to be co-expressed in embryonic mesenchyme and undifferentiated mesothelial cells. In MM cells, calretinin and septin 7 colocalized during cytokinesis in distinct regions of the cleavage furrow and in the midbody region of mitotic cells. Co-immunoprecipitation experiments revealed this co-localization to be the result of a direct interaction between calretinin and septin 7. CONCLUSIONS: Our results demonstrate septin 7 not only serving as a "cytoskeletal" protein, but also as a transcription factor repressing calretinin expression. The negative regulation of calretinin by septin 7 and vice versa sheds new light on mechanisms possibly implicated in MM formation and identifies these proteins as transcriptional regulators and putative targets for MM therapy.

Putative cancer stem cells may be the key target to inhibit cancer cell repopulation between the intervals of chemoradiation in murine mesothelioma.

BACKGROUND: Cancer cell repopulation during chemotherapy or radiotherapy is a major factor limiting the efficacy of treatment. Cancer stem cells (CSC) may play critical roles during this process. We aim to demonstrate the role of mesothelioma stem cells (MSC) in treatment failure and eventually to design specific target therapies against MSC to improve the efficacy of treatment in malignant mesothelioma. METHODS: Murine mesothelioma AB12 and RN5 cells were used to compare tumorigenicity in mice. The expression of CSC-associated genes was evaluated by quantitative real-time PCR in both cell lines treated with chemo-radiation. Stemness properties of MSC-enriched RN5-EOS-Puro2 cells were characterized with flow cytometry and immunostaining. A MSC-specific gene profile was screened by microarray assay and confirmed thereafter. Gene Ontology analysis of the selected genes was performed by GOMiner. RESULTS: Tumor growth delay of murine mesothelioma AB12 cells was achieved after each cycle of cisplatin treatment, however, tumors grew back rapidly due to cancer cell repopulation between courses of chemotherapy. Strikingly, a 10-times lower number of irradiated cells in both cell lines led to a similar tumor incidence and growth rate as with untreated cells. The expression of CSC-associated genes such as CD24, CD133, CD90 and uPAR was dramatically up-regulated, while others did not change significantly after chemoradiation. Highly enriched MSC after selection with puromycin displayed an increasing GFP-positive population and showed typical properties of stemness. Comparatively, the proportion of MSC significantly increased after RN5-EOS parental cells were treated with either chemotherapy, γ-ray radiation, or a combination of the two, while MSC showed more resistance to the above treatments. A group of identified genes are most likely MSC-specific, and major pathways related to regulation of cell growth or apoptosis are involved. Upregulation of the gene transcripts Tnfsf18, Serpinb9b, Ly6a, and Nppb were confirmed. CONCLUSION: Putative MSC possess the property of stemness showing more resistance to chemoradiation, suggesting that MSC may play critical roles in cancer cell repopulation. Further identification of selected genes may be used to design novel target therapies against MSC, so as to eliminate cancer cell repopulation in mesothelioma.

Endogenous TRPV1 stimulation leads to the activation of the inositol phospholipid pathway necessary for sustained Ca2+ oscillations.

Sensory neuron subpopulations as well as breast and prostate cancer cells express functional transient receptor potential vanilloid type 1 (TRPV1) ion channels; however little is known how TRPV1 activation leads to biological responses. Agonist-induced activation of TRPV1 resulted in specific spatiotemporal patterns of cytoplasmic Ca2+ signals in breast and prostate cancer-derived cells. Capsaicin (CAPS; 50µM) evoked intracellular Ca2+ oscillations and/or intercellular Ca2+ waves in all cell lines. As evidenced in prostate cancer Du 145 cells, oscillations were largely dependent on the expression of functional TRPV1 channels in the plasma membrane, phospholipase C activation and on the presence of extracellular Ca2+ ions. Concomitant oscillations of the mitochondrial matrix Ca2+ concentration resulted in mitochondria energization evidenced by increased ATP production. CAPS-induced Ca2+ oscillations also occurred in a subset of sensory neurons, yet already at lower CAPS concentrations (1µM). Stimulation of ectopically expressed TRPV1 channels in CAPS-insensitive NIH-3T3 cells didn't provoke CAPS-triggered Ca2+ oscillations; rather it resulted in low-magnitude, long-lasting elevations of the cytosolic Ca2+ concentration. This indicates that sole TRPV1 activation is not sufficient to generate Ca2+ oscillations. Instead the initial TRPV1-mediated signal leads to the activation of the inositol phospholipid pathway. This in turn suffices to generate a biologically relevant frequency-modulated Ca2+ signal.

Activation of endogenous TRPV1 fails to induce overstimulation-based cytotoxicity in breast and prostate cancer cells but not in pain-sensing neurons.

Vanilloids including capsaicin and resiniferatoxin are potent transient receptor potential vanilloid type 1 (TRPV1) agonists. TRPV1 overstimulation selectively ablates capsaicin-sensitive sensory neurons in animal models in vivo. The cytotoxic mechanisms are based on strong Na(+) and Ca(2+) influx via TRPV1 channels, which leads to mitochondrial Ca(2+) accumulation and necrotic cell swelling. Increased TRPV1 expression levels are also observed in breast and prostate cancer and derived cell lines. Here, we examined whether potent agonist-induced overstimulation mediated by TRPV1 might represent a means for the eradication of prostate carcinoma (PC-3, Du 145, LNCaP) and breast cancer (MCF7, MDA-MB-231, BT-474) cells in vitro. While rat sensory neurons were highly vanilloid-sensitive, normal rat prostate epithelial cells were resistant in vivo. We found TRPV1 to be expressed in all cancer cell lines at mRNA and protein levels, yet protein expression levels were significantly lower compared to sensory neurons. Treatment of all human carcinoma cell lines with capsaicin didn't lead to overstimulation cytotoxicity in vitro. We assume that the low vanilloid-sensitivity of prostate and breast cancer cells is associated with low expression levels of TRPV1, since ectopic TRPV1 expression rendered them susceptible to the cytotoxic effect of vanilloids evidenced by plateau-type Ca(2+) signals, mitochondrial Ca(2+) accumulation and Na(+)- and Ca(2+)-dependent membrane disorganization. Moreover, long-term monitoring revealed that merely the ectopic expression of TRPV1 stopped cell proliferation and often induced apoptotic processes via strong activation of caspase-3 activity. Our results indicate that specific targeting of TRPV1 function remains a putative strategy for cancer treatment.

The Effect of Gap Junctional Coupling on the Spatiotemporal Patterns of Ca2+ Signals and the Harmonization of Ca2+-Related Cellular Responses.

Calcium ions (Ca2+) are important mediators of a great variety of cellular activities e.g. in response to an agonist activation of a receptor. The magnitude of a cellular response is often encoded by frequency modulation of Ca2+ oscillations and correlated with the stimulation intensity. The stimulation intensity highly depends on the sensitivity of a cell to a certain agonist. In some cases, it is essential that neighboring cells produce a similar and synchronized response to an agonist despite their different sensitivity. In order to decipher the presumed function of Ca2+ waves spreading among connecting cells, a mathematical model was developed. This model allows to numerically modifying the connectivity probability between neighboring cells, the permeability of gap junctions and the individual sensitivity of cells to an agonist. Here, we show numerically that strong gap junctional coupling between neighbors ensures an equilibrated response to agonist stimulation via formation of Ca2+ phase waves, i.e. a less sensitive neighbor will produce the same or similar Ca2+ signal as its highly sensitive neighbor. The most sensitive cells within an ensemble are the wave initiator cells. The Ca2+ wave in the cytoplasm is driven by a sensitization wave front in the endoplasmic reticulum. The wave velocity is proportional to the cellular sensitivity and to the strength of the coupling. The waves can form different patterns including circular rings and spirals. The observed pattern depends on the strength of noise, gap junctional permeability and the connectivity probability between neighboring cells. Our simulations reveal that one highly sensitive region gradually takes the lead within the entire noisy system by generating directed circular phase waves originating from this region.

Routes of Ca2+ Shuttling during Ca2+ Oscillations: FOCUS ON THE ROLE OF MITOCHONDRIAL Ca2+ HANDLING AND CYTOSOLIC Ca2+ BUFFERS.

In some cell types, Ca(2+) oscillations are strictly dependent on Ca(2+) influx across the plasma membrane, whereas in others, oscillations also persist in the absence of Ca(2+) influx. We observed that, in primary mesothelial cells, the plasmalemmal Ca(2+) influx played a pivotal role. However, when the Ca(2+) transport across the plasma membrane by the "lanthanum insulation method" was blocked prior to the induction of the serum-induced Ca(2+) oscillations, mitochondrial Ca(2+) transport was found to be able to substitute for the plasmalemmal Ca(2+) exchange function, thus rendering the oscillations independent of extracellular Ca(2+). However, in a physiological situation, the Ca(2+)-buffering capacity of mitochondria was found not to be essential for Ca(2+) oscillations. Moreover, brief spontaneous Ca(2+) changes were observed in the mitochondrial Ca(2+) concentration without apparent changes in the cytosolic Ca(2+) concentration, indicating the presence of a mitochondrial autonomous Ca(2+) signaling mechanism. In the presence of calretinin, a Ca(2+)-buffering protein, the amplitude of cytosolic spikes during oscillations was decreased, and the amount of Ca(2+) ions taken up by mitochondria was reduced. Thus, the increased calretinin expression observed in mesothelioma cells and in certain colon cancer might be correlated to the increased resistance of these tumor cells to proapoptotic/pronecrotic signals. We identified and characterized (experimentally and by modeling) three Ca(2+) shuttling pathways in primary mesothelial cells during Ca(2+) oscillations: Ca(2+) shuttled between (i) the endoplasmic reticulum (ER) and mitochondria, (ii) the ER and the extracellular space, and (iii) the ER and cytoplasmic Ca(2+) buffers.

Characterization and modeling of Ca2+ oscillations in mouse primary mesothelial cells.

Brief changes in the cytosolic and intra-organellar Ca2+ concentration serve as specific signals for various physiological processes. In mesothelial cells lining the surface of internal organs and the walls of body cavities, a re-entry in the cell cycle (G0-G1 transition) evoked by serum re-administration induces long-lasting Ca2+ oscillations with a slowly decreasing frequency. Individual mesothelial cells show a wide range of different oscillatory patterns within a single, supposedly homogenous cell population. Changes in the cytoplasmic Ca2+ concentration (ccyt) show baseline oscillatory patterns i.e., discrete Ca2+ transients starting from a constant basal ccyt level. The ER Ca2+ concentration (cER) displays a sawtooth wave at a semi-depleted ER state; the minimum level is reached just briefly after the maximal value for ccyt. These oscillations depend on plasmalemmal Ca2+ influx and on the inositol trisphosphate concentration [InsP3]; the Ca2+ influx is a crucial determinant of the oscillation frequency. Partial blocking of SERCA pumps modifies the oscillation frequency in both directions, i.e. increasing it in some cells and lowering it in others. Current mathematical models for Ca2+ oscillations mostly fail to reproduce two experimentally observed phenomena: the broad range of interspike intervals and constant basal ccyt levels between two Ca2+ spikes. Here we developed a new model based on--and fitted to--Ca2+ recordings of ccyt and cER recorded in primary mouse mesothelial cells. The model allowed for explaining many features of experimentally observed Ca2+ oscillations. We consider this model to be suitable to simulate various types of InsP3 receptor-based baseline Ca2+ oscillations.

Overexpression or absence of calretinin in mouse primary mesothelial cells inversely affects proliferation and cell migration.

BACKGROUND: The Ca(2+)-binding protein calretinin is currently used as a positive marker for identifying epithelioid malignant mesothelioma (MM) and reactive mesothelium, but calretinin's likely role in mesotheliomagenesis remains unclear. Calretinin protects immortalized mesothelial cells in vitro from asbestos-induced cytotoxicity and thus might be implicated in mesothelioma formation. To further investigate calretinin's putative role in the early steps of MM generation, primary mesothelial cells from calretinin knockout (CR-/-) and wildtype (WT) mice were compared. METHODS: Primary mouse mesothelial cells from WT and CR-/- mice were investigated with respect to morphology, marker proteins, proliferation, cell cycle parameters and mobility in vitro. Overexpression of calretinin or a nuclear-targeted variant was achieved by a lentiviral expression system. RESULTS: CR-/- mice have a normal mesothelium and no striking morphological abnormalities compared to WT animals were noted. Primary mouse mesothelial cells from both genotypes show a typical "cobblestone-like" morphology and express mesothelial markers including mesothelin. In cells from CR-/- mice in vitro, we observed more giant cells and a significantly decreased proliferation rate. Up-regulation of calretinin in mesothelial cells of both genotypes increases the proliferation rate and induces a cobblestone-like epithelial morphology. The length of the S/G2/M phase is unchanged, however the G1 phase is clearly prolonged in CR-/- cells. They are also much slower to close a scratch in a confluent cell layer (2D-wound assay). In addition to a change in cell morphology, an increase in proliferation and mobility is observed, if calretinin overexpression is targeted to the nucleus. Thus, both calretinin and nuclear-targeted calretinin increase mesothelial cell proliferation and consequently, speed up the scratch-closure time. The increased rate of scratch closure in WT cells is the result of two processes: an increased proliferation rate and augmented cell mobility of the border cells migrating towards the empty space. CONCLUSIONS: We hypothesize that the differences in proliferation and mobility between WT and CR-/- mesothelial cells are the likely result from differences in their developmental trajectories. The mechanistic understanding of the function of calretinin and its putative implication in signaling pathways in normal mesothelial cells may help understanding its role during the processes that lead to mesothelioma formation and could possibly open new avenues for mesothelioma therapy, either by directly targeting calretinin expression or indirectly by targeting calretinin-mediated downstream signaling.

Mechanism of capsaicin receptor TRPV1-mediated toxicity in pain-sensing neurons focusing on the effects of Na(+)/Ca(2+) fluxes and the Ca(2+)-binding protein calretinin.

Transient receptor potential vanilloid subtype 1 (TRPV1) receptor is a pain-sensing, ligand-gated, non-selective cation channel expressed in peripheral sensory neurons. Prolonged activation of TRPV1 by capsaicin leads to cell swelling and formation of membrane blebs in rat dorsal root ganglion (DRG) neurons. Similar results were obtained in NIH3T3 fibroblast cells stably expressing TRPV1. Here, we assessed the contribution of Ca(2+) and Na(+) ions to TRPV1-mediated changes. Cell swelling was caused by a substantial influx of extracellular Na(+) via TRPV1 channels, causing concomitant transport of water. In the absence of extracellular Na(+), the membrane blebbing was completely inhibited, but Ca(2+) influx did not change under these conditions. Na(+) influx was modulated by the intracellular Ca(2+) concentration ([Ca(2+)]i). Elevation of [Ca(2+)]i by ionomycin sensitized/activated TRPV1 channels causing cell swelling in TRPV1-positive cells. In the absence of extracellular Ca(2+), capsaicin caused only little increase in [Ca(2+)]i indicating that the increase in [Ca(2+)]i observed after capsaicin application is derived essentially from extracellular Ca(2+) and not from internal Ca(2+) stores. In the absence of extracellular Ca(2+) also the process of cell swelling was considerably slower. Calretinin is a Ca(2+) buffer protein, which is expressed in a subset of TRPV1-positive neurons. Calretinin decreased the amplitude, but slowed down the decay of Ca(2+) signals evoked by ionomycin. Cells co-expressing TRPV1 and calretinin were less sensitive to TRPV1-mediated, capsaicin-induced volume increases. In TRPV1-expressing NIH3T3 cells, calretinin decreased the capsaicin-induced Ca(2+) and Na(+) influx. Swelling and formation of membrane blebs resulted in impaired plasma membrane integrity finally leading to cell death. Our results hint towards a mechanistic explanation for the apoptosis-independent capsaicin-evoked neuronal loss and additionally reveal a protective effect of calretinin; we propose that the Ca(2+)-buffering capacity of calretinin reduces the susceptibility of calretinin-expressing DRG neurons against cell swelling/death caused by overstimulation of TRPV1 channels. This article is part of a Special Issue entitled:12th European Symposium on Calcium.

Role of the cystathionine ß-synthase / H2S pathway in the development of cellular metabolic dysfunction and pseudohypoxia in down syndrome.

BACKGROUND: Overexpression of the transsulfuration enzyme cystathionine-ß-synthase (CBS), and overproduction of its product, hydrogen sulfide (H2S) are recognized as potential pathogenetic factors in Down syndrome (DS). The purpose of the study was to determine how the mitochondrial function and core metabolic pathways are affected by DS and how pharmacological inhibition of CBS affects these parameters. METHODS: 8 human control and 8 human DS fibroblast cell lines have been subjected to bioenergetic and fluxomic and proteomic analysis with and without treatment with a pharmacological inhibitor of CBS. RESULTS: DS cells exhibited a significantly higher CBS expression than control cells, and produced more H2S. They also exhibited suppressed mitochondrial electron transport and oxygen consumption and suppressed Complex IV activity, impaired cell proliferation and increased ROS generation. Inhibition of H2S biosynthesis with aminooxyacetic acid reduced cellular H2S, improved cellular bioenergetics, attenuated ROS and improved proliferation. 13C glucose fluxomic analysis revealed that DS cells exhibit a suppression of the Krebs cycle activity with a compensatory increase in glycolysis. CBS inhibition restored the flux from glycolysis to the Krebs cycle and reactivated oxidative phosphorylation. Proteomic analysis revealed no CBS-dependent alterations in the expression level of the enzymes involved in glycolysis, oxidative phosphorylation and the pentose phosphate pathway. DS was associated with the dysregulation of several components of the autophagy network; CBS inhibition normalized several of these parameters. CONCLUSIONS: Increased H2S generation in DS promotes pseudohypoxia and contributes to cellular metabolic dysfunction by causing a shift from oxidative phosphorylation to glycolysis.

Early prediction of acute necrotizing pancreatitis by artificial intelligence: a prospective cohort-analysis of 2387 cases.

Pancreatic necrosis is a consistent prognostic factor in acute pancreatitis (AP). However, the clinical scores currently in use are either too complicated or require data that are unavailable on admission or lack sufficient predictive value. We therefore aimed to develop a tool to aid in necrosis prediction. The XGBoost machine learning algorithm processed data from 2387 patients with AP. The confidence of the model was estimated by a bootstrapping method and interpreted via the 10th and the 90th percentiles of the prediction scores. Shapley Additive exPlanations (SHAP) values were calculated to quantify the contribution of each variable provided. Finally, the model was implemented as an online application using the Streamlit Python-based framework. The XGBoost classifier provided an AUC value of 0.757. Glucose, C-reactive protein, alkaline phosphatase, gender and total white blood cell count have the most impact on prediction based on the SHAP values. The relationship between the size of the training dataset and model performance shows that prediction performance can be improved. This study combines necrosis prediction and artificial intelligence. The predictive potential of this model is comparable to the current clinical scoring systems and has several advantages over them.

Meta-analysis of gene expression patterns in Down syndrome highlights significant alterations in mitochondrial and bioenergetic pathways.

Individuals with Down syndrome (DS) have an extra copy of chromosome 21. Clinical observations and preclinical studies both suggest that DS is associated with altered bioenergetic pathways. Several studies have reported that differentially expressed genes in DS are located not only on chromosome 21 but also on all other chromosomes. Numerous sets of microarray and RNA-seq data are publicly accessible through the Gene Expression Omnibus. We have conducted a meta-analysis on differentially expressed genes between DS and control subjects. Data deposited before July 1, 2020, were identified by using the search terms "Down syndrome" or "trisomy 21" and "human". Gene expression data were analyzed and normalized for each study. The mixed effect model was used to identify the differentially expressed genes. We conclude that in DS more than 60% of the genes located on chromosome 21 are significantly upregulated and none of them are downregulated. In addition, a significant dysregulation of genes occurs on all other chromosomes as well. Several of the upregulated genes in DS encode for important components of various bioenergetic pathways, for instance PFKL and ACLY. Genes involved in oxidative phosphorylation are mostly downregulated in DS. The gene expression alterations are consistent with the development of significant metabolic disturbances ("pseudohypoxia") in DS cells, which may explain some of the well-known functional defects (ranging from neuronal dysfunction to reduced exercise tolerance) associated with DS.

Resiniferatoxin mediated ablation of TRPV1+ neurons removes TRPA1 as well.

OBJECTIVES: Resiniferatoxin, the most potent agonist of inflammatory pain/vanilloid receptor/cation channel (TRPV1) can be used for neuron subtype specific ablation of pain generating cells at the level of the peripheral nervous system by Ca(2+)-excytotoxicity. Molecular neurosurgery is an emerging technology either to alleviate severe pain in cancer or treat/prevent different local neuropathies. Our aim was determining sensory modalities that may be lost after resiniferatoxin treatment. METHODS: Newborn or adult mice were treated with resiniferatoxin, then changes in chemical and heat sensitivity were correlated with alterations of the cell composition of sensory ganglions. RESULTS: Only mice treated at adult age became less sensitive to heat stimuli, while both treatment groups lost sensitivity to specific vanilloid agonists of TRPV1 and, interestingly, to allyl-isothiocyanate, a selective agonist of TRPA1. Our in vivo and post mortem analytical results confirmed that TRPV1 and TRPA1 function together and resiniferatoxin-mediated neurosurgery removes both sensor molecules. DISCUSSION: In adult mice resiniferatoxin causes: i) desensitization to heat and ii) sensitization to cold. Cold hyperalgesia, an imbalance in thermosensation, might be conferred by a prominent cold receptor that is expressed in surviving resiniferatoxin-resistant sensory neurons and compensates for pain signals lost with TRPA1 and TRPV1 double positive cells in the peripheral nervous system.

ADP-Ribose and oxidative stress activate TRPM8 channel in prostate cancer and kidney cells.

Activation of TRPM8 channel through oxidative stress may induce Ca2+ and pro-apoptotic signals in prostate cancer and kidney cells. The aim of this study was to evaluate activation of TRPM8 can increase apoptosis and oxidative stress in the prostate cancer (Du145M8), TRPM8 knock out (Du 145M8KO), transfected (HEK293TM8) and non-transfected human kidney (HEK293) cells. Intracellular Ca2+ responses to TRPM8 activation were increased in the Du145M8 and HEK293TM8 cells from coming cumene hydrogen peroxide (CHPx), menthol, ADP-Ribose (ADPR), but not in the HEK293 and Du 145M8KO cells. The intracellular Ca2+ responses to both ADPR and CHPx were totally inhibited by the thiol cycle antioxidant glutathione, and TRPM8 blockers (N-(p-amylcinnamoyl)anthranilic acid and capsazepine). Apoptosis, Annexin V, mitochondrial membrane depolarization, intracellular ROS, caspase 3 and 9 values were increased through TRPM8 activation in the Du 145M8 but not in the Du 145M8KO and non-transfected HEK293 cells by CHPx and hydrogen peroxide. In conclusion, apoptotic and oxidant effects on the cells were increased activation of TRPM8 by oxidative stress and ADPR. Activation of TRPM8 through oxidative stress and ADPR in the cells could be used as an effective strategy in the treatment of prostate cancer cells.

The phytohormone forchlorfenuron decreases viability and proliferation of malignant mesothelioma cells in vitro and in vivo.

Malignant mesothelioma (MM) is one of the most aggressive cancer types with a patient's life expectancy of typically less than one year upon diagnosis. The urgency of finding novel therapeutic approaches to treat mesothelioma is evident. Here we tested the effect of the plant-growth regulator forchlorfenuron (FCF), an inhibitor of septin function(s) in mammalian cells, on the viability and proliferation of MM cell lines, as well as other tumor cell lines derived from lung, prostate, colon, ovary, cervix and breast. Exposure to FCF strongly inhibited proliferation of human and mouse (most efficiently epithelioid) MM cells and all other tumor cells in a concentration-dependent manner and led to cell cycle arrest and cell death. The role of septin 7 (SEPT7), a presumably essential target of FCF in MM cells was confirmed by an shRNA strategy. FCF was robustly inhibiting tumor cell growth in vitro at low micromolar (IC50: ≈20-60µM) concentrations and more promisingly also in vivo. Initial experiments with FCF analogous revealed the importance of FCF's chloride group for efficient cell growth inhibition. FCF's rather low systemic toxicity might warrant for an extended search for other related and possibly more potent FCF analogues to target MM and putatively other septin-dependent tumors.