Non-physiological Concentrations Research Articles

Cardiovascular and Metabolic Benefits of Extra Virgin Olive Oil Phenolic Compounds: Mechanistic Insights from In Vivo Studies.

Extra virgin olive oil (EVOO) represents a significant source of monounsaturated fatty acids (MUFA) and vitamin E, but it is also considered a functional food, due to the content of peculiar bioactive molecules, such as phenolic compounds, being able to modulate various processes related to aging and the most common metabolic and degenerative diseases. A lot of experimental research has focused on some of these components, but in most cases, the studies were performed in vitro testing compounds at non-physiological concentrations and achieving results that cannot easily be translated in vivo. Recent clinical studies demonstrated that in vivo these compounds are able to regulate physiological functions and prevent several pathological events including metabolic and cardiovascular diseases (CVDs), which represent the main causes of death worldwide. This review aims to sum up the major evidence on the beneficial effects of EVOO phenolic compounds in vivo on these pathologies, describing and evaluating the efficacy in relation to the mechanisms of diseases of the whole phenolic fraction and some of its specific components.

#957 The immunometabolic profile of human peritoneal macrophages during peritoneal dialysis and its relation to systemic cardiovascular effects

Abstract Background and Aims Patients with end-stage kidney disease (ESKD) treated with peritoneal dialysis (PD) exhibit a high risk of heart failure (HF). This largely concerns HF with a preserved ejection fraction (HFpEF). Uremic toxins, oxidative stress and systemic inflammation related to ESKD contribute to this HFpEF phenotype, which may be aggravated by the daily administration of glucose-rich PD-fluids. These induce insulin resistance and formation of advanced glycaemic end products, resulting in microvascular injury and arteriosclerosis. A hallmark of HFpEF is the presence of profibrotic cardiac macrophages that aggravate fibrosis. In the peritoneum, the non-physiological high glucose concentrations trigger local oxidative stress, fibrosis and inflammation, processes alike those seen in HFpEF. While the role of macrophages in HFpEF is established, the involvement of human peritoneal macrophages (HPMs) in PD-induced peritoneal injury is largely unknown. We hypothesize that HPMs, chronically exposed to the glucose-rich fluids, exhibit an inflammation-driven immunometabolic profile. This may contribute to cardiovascular (CV)-risk in PD-treated patients. We present preliminary results on the isolation of HPMs from PD effluent (PDE) and a detailed protocol to establish the immunometabolic profile of HPMs. These profiles will subsequently be related to cardiac function (left ventricular global longitudinal strain (LV-GLS), an echocardiographic measure of HFpEF) and parameters of systemic inflammation. Method In 15 adult PD-treated patients we will prospectively perform cardiac echography, draw a blood sample and collect PDE for HPM isolation. To obtain control HPMs a peritoneal saline flush will be collected from 5 adult patients with non-metastasized colon cancer during a routine diagnostic laparoscopy. HPMs will be isolated from the PDE and saline by centrifugation, followed by freezing for storage. We compared fresh and thawed cells of a PDE-sample to evaluate the impact of storage on HPM phenotype and functionality, respectively using flow cytometry and measurement of IL-6 production, with and without a lipopolysaccharide (LPS) challenge. In-depth HPM characterization will be performed by measuring glucose and lactate, conducting metabolic flux analysis, assessing glycolysis and mitochondrial characteristics using Seahorse®, and employing cytometry to evaluate reactive oxygen species and metabolic fuel preference. A phenotypical profile will be established using fluorescent probes and antibodies. To assess functionality, HPMs will be exposed in vitro to “M1” and “M2” inducers and we will use human monocyte-derived macrophages from healthy adult controls as a comparison. We will relate the immunometabolic, phenotypical and functional HPM characteristics to LV-GLS, blood cytokine (obtained with Bio-Plex multiplex) and blood immune profiles (obtained with cytometry) in PD-treated patients. Furthermore, we will compare the HPM characteristics of the PD-treated patients with the control group. Results Preliminary results of 6 patients show that we can isolate HPMs from the PDE with a yield of >5M cells. The phenotype and functionality of HPMs was evaluated both directly after isolation and after a freeze-thaw cycle. Flow cytometry showed high percentages of CD14+HLA-DR+CD64+ macrophages in both fresh and biobanked HPMs. IL-6 production was observed in both fresh and biobanked HPMs even without LPS-stimulation (Figure). Conclusion By successfully isolating and storing HPMs from the easily accessible PDE, we optimized the conditions to assess their immunometabolic characteristics. The proposed protocol not only aims to provide valuable insights into HPM phenotype and functionality, but also allows us to relate these to cardiac function and systemic inflammation. With this strategy, we aim to gain insight in the immunometabolic effects of PD on peritoneal immune cells and how these relate to CV-effects.

Oxygen and HIF1α-dependent SDF1 expression in primary astrocytes.

In the naturally hypoxic in utero fetal environment of preterm infants, oxygen and oxygen-sensitive signaling pathways play an important role in brain development, with hypoxia-inducible factor-1α (HIF1α) being an important regulator. Early exposure to nonphysiological high oxygen concentrations by birth in room can induce HIF1α degradation and may affect neuronal and glial development. This involves the dysregulation of astroglial maturation and function, which in turn might contribute to oxygen-induced brain injury. In this study, we investigated the effects of early high oxygen exposure on astroglial maturation and, specifically, on astroglial stromal cell-derived factor 1 (SDF1) expression in vivo and in vitro. In our neonatal mouse model of hyperoxia preterm birth brain injury in vivo, high oxygen exposure affected astroglial development and cortical SDF1 expression. These results were further supported by reduced Sdf1 expression, impaired proliferation, decreased total cell number, and altered expression of astroglial markers in astrocytes in primary cultures grown under high oxygen conditions. Moreover, to mimic the naturally hypoxic in utero fetal environment, astroglial Sdf1 expression was increased after low oxygen exposure in vitro, which appears to be regulated by HIF1α activity. Additionally, the knockdown of Hif1α revealed HIF1α-dependent Sdf1 expression in vitro. Our results indicate HIF1α and oxygen-dependent chemokine expression in primary astrocytes and highlight the importance of oxygen conditions for brain development.

Purine Nucleotide Alterations in Tumoral Cell Lines Maintained with Physiological Levels of Folic Acid.

Most cancer cells have an increased synthesis of purine nucleotides to fulfil their enhanced division rate. The de novo synthesis of purines requires folic acid in the form of N10-formyltetrahydrofolate (10-formyl-THF). However, regular cell culture media contain very high, non-physiological concentrations of folic acid, which may have an impact on cell metabolism. Using cell culture media with physiological levels of folic acid (25 nM), we uncover purine alterations in several human cell lines. HEK293T, Jurkat, and A549 cells accumulate 5'-aminoimidazole-4-carboxamide ribonucleotide (ZMP), an intermediary of the de novo biosynthetic pathway, at physiological levels of folic acid, but not with the artificially high levels (2200 nM) present in regular media. Interestingly, HEK293T and Jurkat cells do not accumulate high levels of ZMP when AICAr, the precursor of ZMP, is added to medium containing 2200 nM folate; instead, ATP levels are increased, suggesting an enhanced de novo synthesis. On the other hand, HeLa and EHEB cells do not accumulate ZMP at physiological levels of folic acid, but they do accumulate in medium containing AICAr plus 2200 nM folate. Expression of SLC19A1, which encodes the reduced folate carrier (RFC), is increased in HEK293T and Jurkat cells compared with HeLa and EHEB, and it is correlated with the total purine nucleotide content at high levels of folic acid or with ZMP accumulation at physiological levels of folic acid. In conclusion, tumoral cell lines show a heterogenous response to folate changes in the media, some of them accumulating ZMP at physiological levels of folic acid. Further research is needed to clarify the ZMP downstream targets and their impact on cell function.

Urolithin A Alleviates Colitis in Mice by Improving Gut Microbiota Dysbiosis, Modulating Microbial Tryptophan Metabolism, and Triggering AhR Activation

Urolithin A (UroA) is a microbial metabolite derived from ellagitannins and ellagic acid with good bioavailability. In this study, we explored the anticolitis activity of UroA and clarified the mechanism by 16S rDNA sequencing and metabonomics. UroA alleviated dextran sulfate sodium (DSS)-induced colitis in mice, characterized by a decreased disease activity index, increased colon length, and improved colonic histopathological lesions, along with inhibited phosphorylation of the mitogen-activated protein kinase signaling pathway. In addition, UroA improved gut microbiota dysbiosis and modulated the microbiota metabolome. Furthermore, targeted metabolomics focused on tryptophan catabolites showed that UroA significantly increased the production of indole-3-aldehyde (IAld) and subsequently led to increased colonic expression of aryl hydrocarbon receptor (AhR) and promoted the serum content of IL-22 in mice with colitis. Collectively, our data identified a novel anticolitis mechanism of UroA by improving gut microbiota dysbiosis, modulating microbial tryptophan metabolism, promoting IAld production, and triggering AhR/IL-22 axis activation. However, a limitation noted in this study is that these beneficial effects of UroA were found at 50 μM in vitro and 20 mg/kg in vivo, which were nonphysiological concentrations.

Modern possibilities and prospects in the treatment of adrenal insufficiency

Adrenal insufficiency (AI) is a life-threatening disease characterized by a decrease in the production of hormones by the adrenal cortex and requires lifelong replacement therapy with glucocorticoids (GCs) and, in some cases, mineralocorticoids (MCs). The number of individuals with primary and secondary AI in Europe is estimated at 20-50 per 100 thousand people, the increase in the number of cases of this disease in the world may be partly due to the rapid increase in the frequency of use of GC in various clinical strategies, including in oncological diseases.In AI, hormone replacement therapy is vital, but long-term use of GC may be associated with various adverse effects, especially at non-physiological concentrations of GC. This review provides a brief overview of the current therapeutic possibilities of AI hormone therapy, which contributes to the prevention of the development of adrenal crises, as well as promising opportunities for more effective imitation of the physiological profile of cortisol, aimed at preventing the development of undesirable effects of therapy. Several different forms of AI are currently available, differing in profile, duration of action, and route of administration. The most preferred currently in the treatment of AI are GCs with a short half-life, which provides a more manageable action profile and a lower risk of side effects. Promising substitution therapy options include modified-release hydrocortisone preparations, as well as new methods of drug administration using an insulin pump, which will better mimic the physiological effects of endogenous cortisol.

Drugs against metabolic diseases as potential senotherapeutics for aging-related respiratory diseases.

Recent advances in aging research have provided novel insights for the development of senotherapy, which utilizes cellular senescence as a therapeutic target. Cellular senescence is involved in the pathogenesis of various chronic diseases, including metabolic and respiratory diseases. Senotherapy is a potential therapeutic strategy for aging-related pathologies. Senotherapy can be classified into senolytics (induce cell death in senescent cells) and senomorphics (ameliorate the adverse effects of senescent cells represented by the senescence-associated secretory phenotype). Although the precise mechanism has not been elucidated, various drugs against metabolic diseases may function as senotherapeutics, which has piqued the interest of the scientific community. Cellular senescence is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF), which are aging-related respiratory diseases. Large-scale observational studies have reported that several drugs, such as metformin and statins, may ameliorate the progression of COPD and IPF. Recent studies have reported that drugs against metabolic diseases may exert a pharmacological effect on aging-related respiratory diseases that can be different from their original effect on metabolic diseases. However, high non-physiological concentrations are needed to determine the efficacy of these drugs under experimental conditions. Inhalation therapy may increase the local concentration of drugs in the lungs without exerting systemic adverse effects. Thus, the clinical application of drugs against metabolic diseases, especially through an inhalation treatment modality, can be a novel therapeutic approach for aging-related respiratory diseases. This review summarizes and discusses accumulating evidence on the mechanisms of aging, as well as on cellular senescence and senotherapeutics, including drugs against metabolic diseases. We propose a developmental strategy for a senotherapeutic approach for aging-related respiratory diseases with a special focus on COPD and IPF.

Alternate atxA and acpA dependent response of Bacillus anthracis to serum, HCO3- and CO2.

Bacillus anthracis overcomes host immune responses by producing capsule and secreting toxins. Production of these virulence factors in response to entering the host environment was shown to be regulated by atxA, the major virulence regulator, known to be activated by HCO3- and CO2. While toxin production is regulated directly by atxA, capsule production is independently mediated by two regulators; acpA and acpB. In addition, it was demonstrated that acpA has at least two promotors, one of them shared with atxA. We used a genetic approach to study capsule and toxin production under different conditions. Unlike previous works utilizing NBY, CA or R-HCO3- medium under CO2 enriched conditions, we used a sDMEM-based medium. Thus, toxin and capsule production can be induced in ambient or CO2 enriched atmosphere. Using this system, we could differentiate between induction by 10% NRS, 10% CO2 or 0.75% HCO3-. In response to high CO2, capsule production is induced by acpA based response in an atxA-independent manner, with little to no toxin (protective antigen PA) production. atxA based response is activated in response to serum independently of CO2, inducing toxin and capsule production in an acpA or acpB dependent manner. HCO3- was also found to activate atxA based response, but in non-physiological concentrations. Our findings may help explain the first stages of inhalational infection, in which spores germinating in dendritic cells require protection (by encapsulation) without affecting cell migration to the draining lymph-node by toxin secretion.

High phenylalanine concentrations induce demyelination and microglial activation in mouse cerebellar organotypic slices

Phenylketonuria (PKU) is an inborn error of metabolism. Mutations in the enzyme phenylalanine hydroxylase (PAH)-encoding gene lead to a decreased metabolism of the amino acid phenylalanine (Phe). The deficiency in PAH increases Phe levels in blood and brain. Accumulation of Phe can lead to delayed development, psychiatric problems and cognitive impairment. White matter (WM) damage is a neuropathological hallmark of PKU and can be seen even in early detected and treated PKU patients. The mechanisms linking high Phe concentrations to WM abnormalities remain unclear. We tested the effects of high Phe concentrations on myelin in three in vitro models of increasing complexity: two simple cell culture models and one model that preserves local brain tissue architecture, a cerebellar organotypic slice culture prepared from postnatal day (P) 8 CD-1 mice. Various Phe concentrations (0.1–10 mM) and durations of exposure were tested. We found no toxic effect of high Phe in the cell culture models. On the contrary, the treatment promoted the maturation of oligodendrocytes, particularly at the highest, non-physiological Phe concentrations. Exposure of cerebellar organotypic slices to 2.4 mM Phe for 21 days in vitro (DIV), but not 7 or 10 DIV, resulted in a significant decrease in myelin basic protein (MBP), calbindin-stained neurites, and neurites co-stained with MBP. Following exposure to a toxic concentration of Phe, a switch to the control medium for 7 days did not lead to remyelination, while very active remyelination was seen in slices following demyelination with lysolecithin. An enhanced number of microglia, displaying an activated type morphology, was seen after exposure of the slices to 2.4 mM Phe for 10 or 21 DIV. The results suggest that prolonged exposure to high Phe concentrations can induce microglial activation preceding significant disruption of myelin.

Occurrence of GHB in blood and urine specimens from victims of alleged sexual assault or blackout in Western Switzerland

Although much has been written on the subject of drug facilitated sexual assault (DFSA) or victim of blackout, no epidemiological study has yet been conducted in Switzerland to evaluate the occurrence of GHB and other drugs in this context. GHB is frequently mentioned on social networks and in the media as the most regularly used substance in DFSA, but often without being based on scientific studies. In order to have an assessment of the situation, political authorities asked our laboratory to systematically screen for GHB in samples obtained in case of DSFA or blackout, and driving under the influence of drugs (DUID), with the aim to assess the occurrence of voluntary or involuntary GHB use in Western Switzerland. All DFSA, blackout and DUID cases received in 2021 in our laboratory were routinely tested for the presence of GHB at non-physiological concentrations by enzymatic assay with a urine cut-off of 10 mg equivalent GHB/L. All positive cases were confirmed by gas chromatography coupled to mass spectrometry (GC-MS) using a cut-off of 6 mg/L in urine and 1.5 mg/L in whole blood. All DFSA and blackout cases were also analyzed by GC-MS, to ensure the absence of false negative cases. When requested by the justice, other substances such as ethanol, benzodiazepines, cannabinoids, cocaine, amphetamines, opioids, antidepressant, and other psychoactive substances were screening in urine and/or blood, by immunoassays, head-space gas chromatography coupled to flame ionization detector (HS-GC-FID), GC-MS, and/or liquid chromatography coupled to tandem mass spectrometry or high resolution mass spectrometry (LC-MS/MS; LC-HRMS (orbitrap)). 60 DFSA and blackout cases and 633 DUID cases were included in the study. Women predominated over men in the DFSA and blackout group (77%), while men predominated over women in the DUID group (90%). Median age in the DFSA and blackout group was 23, and median age in the DUID group was 30. The median delay to obtain urine after the event was 12 hours in case of DFSA and blackout, and 2 hours for DUID cases. The median delay to obtain whole blood after the event was 12 hours in case of DFSA and blackout, and 1.5 hours for DUID cases. The presence of GHB at concentration above the cut-off was detected in 1 DFSA and blackout case (130 mg/L in blood and 730 mg/L in urine), and in 3 DUID cases (78 mg/L, not detected, and not detected in blood, and 660 mg/L, 8.6 mg/L, and 25 mg/L in urine, respectively). These cases concerned only men. For these 4 cases, other substances were also detected. Thus, for the DFSA and blackout case, methamphetamine, methylphenidate, zolpidem and pregabaline were detected, and in the DUID cases, methamphetamine was detected in one case, ethanol, cocaine and cannabis were detected in another case, and 3-MMC was detected in the last case. When analyzed, ethanol was detected in 48% of DFSA and blackout cases, cannabinoids in 17%, benzodiazepines and Z-drugs in 10%, antidepressant in 9.8%, cocaine in 6.5%, amphetamines in 6.5%, methylphenidate in 4.8%, and opioids in 2.1%. Median concentration of ethanol after back calculation was 2.07 g/kg in DFSA and blackout cases. The results obtained, showing a very limited voluntary or involuntary use of GHB, are comparable to those obtained in other international studies. For the first time in Switzerland, a study was conducted for the evaluation of the occurrence of GHB in blood and urine specimens from victims of alleged sexual assault or blackout, and DUID cases. The results showed that GHB is very rarely consumed and used in DSFA or blackout cases, in comparison to other psychoactive drugs.

Mitochondrial dysfunction associated with ascorbate synthesis in plants

Mitochondria are the major organelles of energy production; however, active mitochondria can decline their energetic role and show a dysfunctional status. Mitochondrial dysfunction was induced by high non-physiological level of L-galactone-1,4-lactone (L-GalL), the precursor of ascorbate (AsA), in plant mitochondria. The dysfunction induced by L-GalL was associated with the fault in the mitochondrial electron partition and reactive oxygen species (ROS) over-production. Using mitochondria from RNAi-plant lines harbouring silenced L-galactone-1,4-lactone dehydrogenase (L-GalLDH) activity, it was demonstrated that such dysfunction is dependent on this enzyme activity. The capacity of alternative respiration was strongly decreased by L-GalL, probably mediated by redox-inactivation of the alternative oxidase (AOX) enzyme. Although, alternative respiration was shown to be the key factor that helps support AsA synthesis in dysfunctional mitochondria. Experiments with respiratory inhibitors showed that ROS formation and mitochondrial dysfunction were more associated with the decline in the activities of COX (cytochrome oxidase) and particularly AOX than with the lower activities of respiratory complexes I and III. The application of high L-GalL concentrations induced proteomic changes that indicated alterations in proteins related to oxidative stress and energetic status. However, supra-optimal L-GalL concentration was not deleterious for plants. Instead, the L-GalLDH activity could be positive. Indeed, it was found that wild type plants performed better growth than L-GalLDH-RNAi plants in response to high non-physiological L-GalL concentrations.

Paper-Based Multiplexed Colorimetric Device for the Simultaneous Detection of Salivary Biomarkers.

In this study, we describe a monolithic and fully integrated paper-based device for the simultaneous detection of three prognostic biomarkers in saliva. The pattern of the proposed multiplexed device is designed with a central sample deposition zone and three identical arms, each containing a pre-treatment and test zone. Its one-step fabrication is realized by CO2 laser cutting, providing remarkable parallelization and rapidity (ca. 5 s/device). The colorimetric detection is based on the sensitive and selective target-induced reshaping of plasmonic multibranched gold nanoparticles, which exhibit a clear spectral shift (and blue-to-pink color change) in case of non-physiological concentrations of the three salivary biomarkers. A rapid and multiplexed naked-eye or smartphone-based readout of the colorimetric response is achieved within 10 min. A prototype kit for POCT testing is also reported, providing robustness and easy handling of the device.

Preparation of Asymmetric Vesicles with Trapped CsCl Avoids Osmotic Imbalance, Non-Physiological External Solutions, and Minimizes Leakage.

The natural asymmetry of cellular membranes influences their properties. In recent years, methodologies for preparing asymmetric vesicles have been developed that rely on cyclodextrin-catalyzed exchange of lipids between donor lipid multilamellar vesicles and acceptor lipid unilamellar vesicles, and the subsequent separation of the, now asymmetric, acceptor vesicles from the donors. Isolation is often accomplished by preloading acceptor vesicles with a high concentration of sucrose, typically 25% (w/w), and separating from donor and cyclodextrin by sucrose gradient centrifugation. We found that when the asymmetric vesicles prepared using methyl-α-cyclodextrin exchange were dispersed under hypotonic conditions using physiological salt solutions, there was enhanced leakage of an entrapped probe, 6-carboxyfluorescein. Studies with symmetric vesicles showed this was due to osmotic pressure and was specific to hypotonic solutions. Inclusion of cholesterol partly reduced leakage but did not completely eliminate it. To avoid having to use hypotonic conditions or to suspend vesicles at nonphysiological solute concentrations to minimize leakage, a method for preparing asymmetric vesicles using acceptor vesicle-entrapped CsCl at a physiological ion concentration (100 mM) was developed. Asymmetric vesicles prepared with the entrapped CsCl protocol were highly resistant to 6-carboxyfluorescein leakage out of the vesicles.

Inflammatory Cytokine Profile and Plasticity of Brain and Spinal Microglia in Response to ATP and Glutamate.

Microglia are the primary cells in the central nervous system that identify and respond to injury or damage. Such a perturbation in the nervous system induces the release of molecules including ATP and glutamate that act as damage-associated molecular patterns (DAMPs). DAMPs are detected by microglia, which then regulate the inflammatory response in a manner sensitive to their surrounding environment. The available data indicates that ATP and glutamate can induce the release of pro inflammatory factors TNF (tumor necrosis factor), IL-1β (interleukin 1 beta), and NO (nitric oxide) from microglia. However, non-physiological concentrations of ATP and glutamate were often used to derive these insights. Here, we have compared the response of spinal cord microglia (SM) relative to brain microglia (BM) using physiologically relevant concentrations of glutamate and ATP that mimic injured conditions in the central nervous system. The data show that ATP and glutamate are not significant modulators of the release of cytokines from either BM or SM. Consistent with previous studies, spinal microglia exhibited a general trend toward reduced release of inflammatory cytokines relative to brain-derived microglia. Moreover, we demonstrate that the responses of microglia to these DAMPs can be altered by modifying the biochemical milieu in their surrounding environment. Preconditioning brain derived microglia with media from spinal cord derived mixed glial cultures shifted their release of IL-1ß and IL-6 to a less inflammatory phenotype consistent with spinal microglia.

Magnesium Influences Membrane Fusion during Myogenesis by Modulating Oxidative Stress in C2C12 Myoblasts.

Magnesium (Mg) is essential to skeletal muscle where it plays a key role in myofiber relaxation. Although the importance of Mg in the mature skeletal muscle is well established, little is known about the role of Mg in myogenesis. We studied the effects of low and high extracellular Mg in C2C12 myogenic differentiation. Non-physiological Mg concentrations induce oxidative stress in myoblasts. The increase of reactive oxygen species, which occurs during the early phase of the differentiation process, inhibits myoblast membrane fusion, thus impairing myogenesis. Therefore, correct Mg homeostasis, also maintained through a correct dietary intake, is essential to assure the regenerative capacity of skeletal muscle fibers.

The Ca2+ channel CatSper is not activated by cAMP/PKA signaling but directly affected by chemicals used to probe the action of cAMP and PKA

The sperm-specific Ca2+ channel CatSper (cation channel of sperm) controls the influx of Ca2+ into the flagellum and, thereby, the swimming behavior of sperm. A hallmark of human CatSper is its polymodal activation by membrane voltage, intracellular pH, and oviductal hormones. Whether CatSper is also activated by signaling pathways involving an increase of cAMP and ensuing activation of PKA is, however, a matter of controversy. To shed light on this question, we used kinetic ion-sensitive fluorometry, patch-clamp recordings, and optochemistry to study transmembrane Ca2+ flux and membrane currents in human sperm from healthy donors and from patients that lack functional CatSper channels. We found that human CatSper is neither activated by intracellular cAMP directly nor indirectly by the cAMP/PKA-signaling pathway. Instead, we show that nonphysiological concentrations of cAMP and membrane-permeable cAMP analogs used to mimic the action of intracellular cAMP activate human CatSper from the outside via a hitherto-unknown extracellular binding site. Finally, we demonstrate that the effects of common PKA inhibitors on human CatSper rest predominantly, if not exclusively, on off-target drug actions on CatSper itself rather than on inhibition of PKA. We conclude that the concept of an intracellular cAMP/PKA-activation of CatSper is primarily based on unspecific effects of chemical probes used to interfere with cAMP signaling. Altogether, our findings solve several controversial issues and reveal a novel ligand-binding site controlling the activity of CatSper, which has important bearings on future studies of cAMP and Ca2+ signaling in sperm.

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides

BackgroundApproximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3′,5′-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced.ResultsTo quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity.ConclusionsWe found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.

Assessment of tobacco heating system 2.4 on osteogenic differentiation of mesenchymal stem cells and primary human osteoblasts compared to conventional cigarettes.

BACKGROUNDCigarette smoking (CS) is the most common method of consuming tobacco. Deleterious effects on bone integrity, increased incidence of fractures, and delayed fracture healing are all associated with CS. Over 150 of the 6500 molecular species contained in cigarette smoke and identified as toxic compounds are inhaled by CS and, via the bloodstream, reach the skeletal system. New technologies designed to develop a reduced-risk alternative for smokers are based on electronic nicotine delivery systems, such as e-cigarettes and tobacco heating systems (THS). THS are designed to heat tobacco instead of burning it, thereby reducing the levels of harmful toxic compounds released.AIMTo examine the effects of THS on osteoprogenitor cell viability and function compared to conventional CS.METHODSHuman immortalized mesenchymal stem cells (n = 3) and primary human pre-osteoblasts isolated from cancellous bone samples from BG Unfall Klinik Tübingen (n = 5) were osteogenically differentiated in vitro with aqueous extracts generated from either the THS 2.4 “IQOS” or conventional “Marlboro” cigarettes for up to 21 d. Cell viability was analyzed using resazurin conversion assay (mitochondrial activity) and calcein-AM staining (esterase activity). Osteogenic differentiation and bone cell function were evaluated using alkaline phosphatase (AP) activity, while matrix formation was analyzed through alizarin red staining. Primary cilia structure was examined by acetylated α-tubulin immunofluorescent staining. Free radical production was evaluated with 2’,7’-dichlorofluorescein-diacetate assay.RESULTSOur data clearly show that THS is significantly less toxic to bone cells than CS when analyzed by mitochondrial and esterase activity (P < 0.001). No significant differences in cytotoxicity between the diverse flavors of THS were observed. Harmful effects from THS on bone cell function were observed only at very high, non-physiological concentrations. In contrast, extracts from conventional cigarettes significantly reduced the AP activity (by two-fold) and matrix mineralization (four-fold) at low concentrations. Additionally, morphologic analysis of primary cilia revealed no significant changes in the length of the organelle involved in osteogenesis of osteoprogenitor cells, nor in the number of ciliated cells following THS treatment. Assessment of free radical production demonstrated that THS induced significantly less oxidative stress than conventional CS in osteoprogenitor cells.CONCLUSIONTHS was significantly less harmful to osteoprogenitor cells during osteogenesis than conventional CS. Additional studies are required to confirm whether THS is a better alternative for smokers to improve delays in bone healing following fracture.

HYPOXIA AND REPRODUCTIVE HEALTH: Oxygen and development of the human placenta.

Development of the human placenta takes place in contrasting oxygen concentrations at different stages of gestation, from ~20 mmHg during the first trimester rising to ~60 mmHg at the start of the second trimester before gradually declining to ~40 mmHg at term. In view of these changes, the early placenta has been described as 'hypoxic'. However, placental metabolism is heavily glycolytic, supported by the rich supply of glucose from the endometrial glands, and there is no evidence of energy compromise. On the contrary, the trophoblast is highly proliferative, with the physiological low-oxygen environment promoting maintenance of stemness in progenitor populations. These conditions favour the formation of the cytotrophoblastic shell that encapsulates the conceptus and interfaces with the endometrium. Extravillous trophoblast cells on the outer surface of the shell undergo an epithelial-mesenchymal transition and acquire invasive potential. Experimental evidence suggests that these changes may be mediated by the higher oxygen concentration present within the placental bed. Interpreting in vitro data is often difficult, however, due to the use of non-physiological oxygen concentrations and trophoblast-like cell lines or explant models. Trophoblast is more vulnerable to hyperoxia or fluctuating levels of oxygen than to hypoxia, and some degree of placental oxidative stress likely occurs in all pregnancies towards term. In complications of pregnancy, such as early-onset pre-eclampsia, malperfusion generates high levels of oxidative stress, causing release of factors that precipitate the maternal syndrome. Further experiments are required using genuine trophoblast progenitor cells and physiological concentrations to fully elucidate the pathways by which oxygen regulates placental development.

Testing Pollen Tube Proteins for In Vivo Binding to Phosphatidic Acid by n-Butanol Treatment and Confocal Microscopy.

The general role of cellular membranes is to provide a barrier and to generate separate reaction spaces. However, additional functions of membrane domains enriched in certain classes of lipids have been discovered, which represent an important area of ongoing research. Such membrane domains can be found in cells at different size scales (e.g., nanodomains, microdomains), represent membrane regions with special physical properties and play important roles in the direct or indirect propagation of signaling processes. Domain formation within the plasma membrane (PM) does not only involve the accumulation of specific lipids, but also the recruitment of specific transmembrane or PM-associated peripheral proteins. Phosphatidic acid (PA) is increasingly recognized as an important signaling lipid and component of PM domains. This lipid is involved in the regulation not only of biotic or abiotic stress responses, but also of pollen tube tip growth and of other forms of polar cell expansion. Although many PA-binding proteins have been characterized, a conserved PA interaction motif could not be identified in these proteins. Consequently, protein binding to PA cannot be predicted based on sequence analysis, but has to be biochemically tested using lipid strip or liposome assays. Although these assays are often informative, they are generally based on the use of artificial model membranes, which compared to natural membranes contain fewer lipid types often at non-physiological concentrations. In this chapter, we describe an alternative in vivo assay that can be employed to analyze protein binding to PA at the PM of normally elongating tobacco pollen tubes. This assay is based on the use of n-butanol (n-ButOH), which inhibits phospholipase D (PLD) and thereby blocks a major biosynthetic pathway that generates PA within the PM from substrates like phosphatidylcholine (PC) or phosphatidylethanolamine (PE). PLD inhibition reduces the PA content of the PM and consequently the level of PM association of PA-binding proteins, which can be analyzed using fluorescence microscopy. Methods enabling n-ButOH treatment of cultured tobacco pollen tubes expressing YFP-tagged PA-binding proteins as well as the quantitative determination of the PM association of these proteins are described.