Search

Objective: Several studies have attempted to identify genetic determinants of clinical response to opioids administered during labor or after cesarean section. However, their results were often contrasting. A systematic review and meta-analysis was conducted to quantitatively assess the association between gene polymorphisms and clinical outcomes of opioid administration in the treatment of labor pain and post-cesarean pain., Methods: A comprehensive search was performed up to December 2023 using PubMed, Web of Knowledge, Cochrane Library, and OpenGrey databases. The clinical endpoints of interest were pain score after opioid treatment, total opioid consumption, patient's analgesic satisfaction, and incidence of opioid side effects. Random-effects meta-analyses were conducted when data were available in at least three studies., Results: Twenty-six studies enrolling 7765 patients were included in the systematic review. Overall, a total of 12 candidate polymorphic genes (OPRM1, COMT, CYP2D6, CYP3A4, ABCB1, ABCC3, UGT2B7, CGRP, OPRK1, OPRD1, KCNJ6, KCNJ9) were considered by the included studies, among which the most investigated variant was OPRM1 rs1799971. Overall pooled results indicated that individuals carrying the G allele of OPRM1 rs1799971 required higher opioid doses for pain management in comparison to rs1799971 AA subjects (standardized mean difference: 0.26; 95% CI: 0.09-0.44; P = 0.003). Such an association was confirmed in the subgroups of patients with labor pain and post-cesarean pain., Conclusion: The present meta-analysis provides strong evidence of an association between OPRM1 rs1799971 and opioid dose requirement for relief of labor pain or post-cesarean pain. However, given the insufficient evidence for other polymorphic gene variants, large studies are still needed to investigate the impact of genetic variability on the efficacy and safety of opioid medications for relief of labor pain and post-cesarean pain (INPLASY Registration No. 202410040)., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

IP 3 receptor (IP 3 R)-mediated Ca 2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca 2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinson's disease (PD). The dependence of Ca 2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically encoded Ca 2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of ~20 nm is optimal for Ca 2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP 3 R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced, which correlated with a reduction of mitochondrial Ca 2+ uptake. Stabilization of the ER-mitochondrial interaction at 20 nm, but not at 10 nm, fully rescued mitochondrial Ca 2+ uptake in PD astrocytes. Our work determines with precision the optimal distance for Ca 2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function., (© 2024. The Author(s).)

Bitter taste receptors (TAS2Rs) expressed in extraoral tissues represent a whole-body sensory system, whose role and mechanisms could be of interest for the identification of new therapeutic targets. It is known that TAS2R46s in pre-contracted airway smooth muscle cells increase mitochondrial calcium uptake, leading to bronchodilation, and that several SNPs have been identified in its gene sequence. There are very few reports on the structure-function analysis of TAS2Rs. Thus, we delved into the subject by using mutagenesis and in silico studies. We generated a cellular model that expresses native TAS2R46 to evaluate the influence of the four most common SNPs on calcium fluxes following the activation of the receptor by its specific ligand absinthin. Then, docking studies were conducted to correlate the calcium flux results to the structural mutation. The analysed SNPs differently modulate the TAS2R46 signal cascade according to the altered protein domain. In particular, the SNP in the sixth transmembrane domain of the receptors did not modulate calcium homeostasis, while the SNPs in the sequence coding for the fourth transmembrane domain completely abolished the mitochondrial calcium uptake. In conclusion, these results indicate the fourth transmembrane domain of TAS2R46 is critical for the intrinsic receptor activity.

Bitter taste receptors (TAS2Rs) are not only responsible for taste perception in the oral cavity, but are spread throughout the body, generating a widespread chemosensory system. In humans, 25 subtypes have been identified and are differentially expressed in tissues and organs, including in the immune system. In fact, several TAS2R subtypes have been detected in neutrophils, lymphocytes, B and T cells, NK cells, and monocytes/macrophages, in which they regulate various protective functions of the innate immune system. Given its recognized anti-inflammatory and antioxidant activity, and the generally protective role of bitter taste receptors, in this work, we studied TAS2R46's potential in the protection of human monocyte/macrophage DNA from stress-induced damage. Through both direct and indirect assays and a single-cell gel electrophoresis assay, we demonstrated that absinthin, a specific TAS2R46 agonist, counteracts the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS) and reduces DNA damage in both cell types. Even though the release of ROS from monocytes/macrophages is fundamental for contrast pathogen agents, supraphysiological ROS production impairs their function, finally leading to cell death. Our results highlight TAS2R46 as a novel player involved in the protection of monocytes and macrophages from oxidative stress damage, while simultaneously supporting their antimicrobial activity.

Alzheimer's disease (AD) represents an urgent yet unmet challenge for modern society, calling for exploration of innovative targets and therapeutic approaches. Astrocytes, main homeostatic cells in the CNS, represent promising cell-target. Our aim was to investigate if deletion of the regulatory CaNB1 subunit of calcineurin in astrocytes could mitigate AD-related memory deficits, neuropathology, and neuroinflammation. We have generated two, acute and chronic, AD mouse models with astrocytic CaNB1 ablation (ACN-KO). In the former, we evaluated the ability of β-amyloid oligomers (AβOs) to impair memory and activate glial cells once injected in the cerebral ventricle of conditional ACN-KO mice. Next, we generated a tamoxifen-inducible astrocyte-specific CaNB1 knock-out in 3xTg-AD mice (indACNKO-AD). CaNB1 was deleted, by tamoxifen injection, in 11.7-month-old 3xTg-AD mice for 4.4 months. Spatial memory was evaluated using the Barnes maze; β-amyloid plaques burden, neurofibrillary tangle deposition, reactive gliosis, and neuroinflammation were also assessed. The acute model showed that ICV injected AβOs in 2-month-old wild type mice impaired recognition memory and fostered a pro-inflammatory microglia phenotype, whereas in ACN-KO mice, AβOs were inactive. In indACNKO-AD mice, 4.4 months after CaNB1 depletion, we found preservation of spatial memory and cognitive flexibility, abolishment of amyloidosis, and reduction of neurofibrillary tangles, gliosis, and neuroinflammation. Our results suggest that ACN is crucial for the development of cognitive impairment, AD neuropathology, and neuroinflammation. Astrocyte-specific CaNB1 deletion is beneficial for both the abolishment of AβO-mediated detrimental effects and treatment of ongoing AD-related pathology, hence representing an intriguing target for AD therapy., (© 2024 Wiley Periodicals LLC.)

Macrophages, key players in the innate immune system, showcase remarkable adaptability. Derived from monocytes, these phagocytic cells excel in engulfing and digesting pathogens and foreign substances as well as contributing to antigen presentation, initiating and regulating adaptive immunity. Macrophages are highly plastic, and the microenvironment can shaper their phenotype leading to numerous distinct polarized subsets, exemplified by the two ends of the spectrum: M1 (classical activation, inflammatory) and M2 (alternative activation, anti-inflammatory). RNA sequencing (RNA-Seq) has revolutionized molecular biology, offering a comprehensive view of transcriptomes. Unlike microarrays, RNA-Seq detects known and novel transcripts, alternative splicing, and rare transcripts, providing a deeper understanding of genome complexity. Despite the decreasing costs of RNA-Seq, data consolidation remains limited, hindering noise reduction and the identification of authentic signatures. Macrophages polarization is routinely ascertained by qPCR to evaluate those genes known to be characteristic of M1 or M2 skewing. Yet, the choice of these genes is literature- and experience-based, lacking therefore a systematic approach. This manuscript builds on the significant increase in deposited RNA-Seq datasets to determine an unbiased and robust murine M1 and M2 polarization profile. We now provide a consolidated list of global M1 differentially expressed genes (i.e. robustly modulated by IFN-γ, LPS, and LPS+ IFN-γ) as well as consolidated lists of genes modulated by each stimulus (IFN-γ, LPS, LPS+ IFN-γ, and IL-4)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Colombo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bitter taste receptors are involved not only in taste perception but in various physiological functions as their anatomical location is not restricted to the gustatory system. We previously demonstrated expression and activity of the subtype hTAS2R46 in human airway smooth muscle and broncho-epithelial cells, and here we show its expression and functionality in human skeletal muscle cells. Three different cellular models were used: micro-dissected human skeletal tissues, human myoblasts/myotubes and human skeletal muscle cells differentiated from urine stem cells of healthy donors. We used qPCR, immunohistochemistry and immunofluorescence analysis to evaluate gene and protein hTAS2R46 expression. In order to explore receptor activity, cells were incubated with the specific bitter ligands absinthin and 3ß-hydroxydihydrocostunolide, and calcium oscillation and relaxation were evaluated by calcium imaging and collagen assay, respectively, after a cholinergic stimulus. We show, for the first time, experimentally the presence and functionality of a type 2 bitter receptor in human skeletal muscle cells. Given the tendentially protective role of the bitter receptors starting from the oral cavity and following also in the other ectopic sites, and given its expression already at the myoblast level, we hypothesize that the bitter receptor can play an important role in the development, maintenance and in the protection of muscle tissue functions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Talmon, Massara, Quaregna, De Battisti, Boccafoschi, Lecchi, Puppo, Bettega Cajandab, Salamone, Bovio, Boldorini, Riva, Pollastro and Fresu.)

Calcineurin (CaN), a Ca 2+ /calmodulin-activated serine/threonine phosphatase, acts as a Ca 2+ -sensitive switch regulating cellular functions through protein dephosphorylation and activation of gene transcription. In astrocytes, the principal homeostatic cells in the CNS, over-activation of CaN is known to drive pathological transcriptional remodelling, associated with neuroinflammation in diseases such as Alzheimer's disease, epilepsy and brain trauma. Recent reports suggest that, in physiological conditions, the activity of CaN in astrocytes is transcription-independent and is required for maintenance of basal protein synthesis rate and activation of astrocytic Na + /K + pump thereby contributing to neuronal functions such as neuronal excitability and memory formation. In this contribution we overview the role of Ca 2+ and CaN signalling in astroglial pathophysiology focusing on the emerging physiological role of CaN in astrocytes. We propose a model for the context-dependent switch of CaN activity from the post-transcriptional regulation of cell proteostasis in healthy astrocytes to the CaN-dependent transcriptional activation in neuroinflammation-associated diseases., (© 2022. The Author(s).)

It is well documented that patients affected by rheumatoid arthritis (RA) have distinct susceptibility to the different biologic DMARDs available on the market, probably because of the many facets of the disease. Monocytes are deeply involved in the pathogenesis of RA and we therefore evaluated and compared the transcriptomic profile of monocytes isolated from patients on treatment with methotrexate alone or in combination with tocilizumab, anti-TNFα or abatacept and from healthy donors. Whole-genome transcriptomics yielded a list of regulated genes by Rank Product statistics and DAVID was then used for functional annotation enrichment analysis. Last, data were validated by qRT-PCR. Abatacept, tocilizumab and anti-TNFa cohorts were separately compared with methotrexate, leading to the identification of 78, 6, and 436 differentially expressed genes, respectively. The upper-most ranked genes were related to inflammatory processes and immune responses. Such an approach draws the genomic profile of monocytes in treated RA patients and lays the basis for finding gene signature for tailored therapeutic choices., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Talmon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bitter taste receptors (TAS2Rs) have recently arisen as a potential drug target for asthma due to their localization in airway cells. These receptors are expressed in all cell types of the respiratory system comprising epithelial, smooth muscle and immune cells; however, the expression pattern of the subtypes is different in each cell type and, accordingly, so is their role, for example, anti-inflammatory or bronchodilator. The most challenging aspect in studying TAS2Rs has been the identification of the downstream signaling cascades. Indeed, TAS2R activation leads to canonical IP3-dependent calcium release from the ER, but, alongside, there are other mechanisms that differ according to the histological localization. In this review, we summarize the current knowledge on the cytosolic calcium modulation downstream of TAS2R activation in the epithelial, smooth muscle and immune cells of the airway system.

Recent advancements in regenerative medicine have enhanced the development of biomaterials as multi-functional dressings, capable of accelerating wound healing and addressing the challenge of chronic wounds. Hydrogels obtained from decellularized tissues have a complex composition, comparable to the native extracellular environment, showing highly interesting characteristics for wound healing applications. In this study, a bovine pericardium decellularized extracellular matrix (dECM) hydrogel was characterized in terms of macromolecules content, and its immunomodulatory, angiogenic and wound healing potential has been evaluated. The polarization profile of human monocytes-derived macrophages seeded on dECM hydrogel was assessed by RT-qPCR. Angiogenic markers expression has been evaluated by Western blot and antibody array on cell lysates derived from endothelial cells cultured on dECM hydrogel, and a murine in vivo model of hindlimb ischemia was used to evaluate the angiogenic potential. Fibroblast migration was assessed by a transwell migration assay, and an in vivo murine wound healing model treated with dECM hydrogels was also used. The results showed a complex composition, of which the major component is collagen type I. The dECM hydrogel is biocompatible, able to drive M2 phenotype polarization, stimulate the expression of angiogenic markers in vitro, and prevent loss of functionality in hindlimb ischemia model. Furthermore, it drives fibroblast migration and shows ability to facilitate wound closure in vivo, demonstrating its great potential for regenerative applications.

Muscular diseases are characterized by a wide genetic diversity and the Ca 2+ -signalling machinery is often perturbed. Its characterization is therefore pivotal and requires appropriate cellular models. Muscle biopsies are the best approach but are invasive for the patient and difficult to justify if the biopsy is not for diagnostic purposes. To circumvent this, interest is mounting in urine-derived stem cells that can be differentiated into skeletal muscle cells. In the present study, we isolated stem cells from urine (USC) samples of healthy donors and differentiated them by MyoD lentiviral vector transduction into skeletal muscle cells (USC-SkMC). As expected, USCs and USC-SkMCs are characterized by a radically different pattern of expression of stem and skeletal muscle markers. Characterization of cells in the present manuscript focused on Ca 2+ -signalling. Undifferentiated and differentiated cells differed in the expression of key proteins involved in Ca 2+ -homeostasis and also displayed different Ca 2+ -responses to external stimuli, confirming that during differentiation there was a transition from a non-excitable to an excitable phenotype. In USCs, the main mechanism of calcium entry was IP 3 dependent, suggesting a major involvement of receptor-operated Ca 2+ entry. Indeed, U-73122 (a PLC inhibitor) significantly inhibited the Ca 2+ increase triggered by ATP both in calcium and calcium-free conditions. In USC-SkMCs both store- and receptor-operated calcium entry were active. Furthermore, a caffeine challenge led to Ca 2+ release both in the presence or absence of extracellular calcium, which was inhibited by ryanodine, suggesting the presence and functionality of ryanodine receptors in USC-SkMCs. Lastly, the voltage-operated calcium channels are operative in USC-SkMCs, unlike in USCs, since stimulation with high concentration of KCl induced a significant calcium transient, partially reversed by verapamil. Our data therefore support the use of skeletal muscle cells derived from USCs as an easily amenable tool to investigate Ca 2+ -homeostasis, in particular in those (neuro)muscular diseases that lack valid alternative models., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Several contributions of circulating microvesicles (MVs) to the endothelial dysfunction have been reported in the past; a head-to-head comparison of platelet- and monocyte-derived MVs has however never been performed. To this aim, we assessed the involvement of these MVs in vessel damage related processes, i.e., oxidative stress, inflammation, and leukocyte-endothelial adhesion. Platelets and monocytes isolated from healthy subjects (HS, n = 15) were stimulated with TRAP-6 and LPS to release MVs that were added to human vascular endothelial cell (hECV) culture to evaluate superoxide anion production, inflammatory markers (IL-6, TNF α , NF-κB mRNA expression), and hECV adhesiveness. The effects of the MVs-induced from HS were compared to those induced by MVs spontaneously released from cells of patients with ST-segment elevation myocardial infarction (STEMI, n = 7). MVs released by HS-activated cells triggered a threefold increase in oxidative burst in a concentration-dependent manner. Only MVs released from monocytes doubled IL-6, TNF α , and NF-κB mRNA expression and monocyte-endothelial adhesion. Interestingly, the effects of the MVs isolated from STEMI-monocytes were not superimposable to previous ones except for adhesion to hECV. Conversely, MVs released from STEMI-platelets sustained both redox state and inflammatory phenotype. These data provide evidence that MVs released from activated and/or pathologic platelets and monocytes differently affect endothelial behavior, highlighting platelet-MVs as causative factors of impaired endothelial function in the acute phase of STEMI.

Cardiovascular diseases (CVDs), mainly ischemic heart disease (IHD) and stroke, are the leading cause of global mortality and major contributors to disability worldwide. Despite their heterogeneity, almost all CVDs share a common feature: the endothelial dysfunction. This is defined as a loss of functionality in terms of anti-inflammatory, anti-thrombotic and vasodilatory abilities of endothelial cells (ECs). Endothelial function is greatly ensured by the mechanotransduction of shear forces, namely, endothelial wall shear stress (WSS). Low WSS is associated with endothelial dysfunction, representing the primary cause of atherosclerotic plaque formation and an important factor in plaque progression and remodeling. In this work, the role of factors released by ECs subjected to different magnitudes of shear stress driving the functionality of downstream endothelium has been evaluated. By means of a microfluidic system, HUVEC monolayers have been subjected to shear stress and the conditioned media collected to be used for the subsequent static culture. The results demonstrate that conditioned media retrieved from low shear stress experimental conditions (LSS-CM) induce the downregulation of endothelial nitric oxide synthase (eNOS) expression while upregulating peripheral blood mononuclear cell (PBMC) adhesion by means of higher levels of adhesion molecules such as E-selectin and ICAM-1. Moreover, LSS-CM demonstrated a significant angiogenic ability comparable to the inflammatory control media (TNFα-CM); thus, it is likely related to tissue suffering. We can therefore suggest that ECs stimulated at low shear stress (LSS) magnitudes are possibly involved in the paracrine induction of peripheral endothelial dysfunction, opening interesting insights into the pathogenetic mechanisms of coronary microvascular dysfunction.

In order to identify a suitable alternative to non-steroidal anti-inflammatory drugs (NSAIDs) we aimed to develop derivatives of vortioxetine, a multimodal anti-depressive drug that has been shownpreviously to be endowed withanti-inflammatory activity in human monocytes/macrophages. Vortioxetine (1) was synthesized in good yield and different alkyl and aryl derivatives were prepared based on their structural diversity and easy availability. The compounds were tested on human monocytes isolated from healthy donors for theireffect on superoxide anion production and cytokine gene expression, and for COX-1/2 gene expression and activity modulation. Moreover, a docking study was performed to predict the interactions between the synthesized compounds and COX-1 and COX-2. Correlating experimental biological data to the molecular modelling studies, it emerged that among the novel compounds, 6 was endowed of antioxidant and anti-COX-1 activity, vortioxetine and 3 were good antioxidants and mild anti-COX-1/2 inhibitors, while 7 was a good anti-COX-1/2 inhibitor and 11 was more specific versus COX-2., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A series of analogues of Amb639752, a novel diacylglycerol kinase (DGK) inhibitor recently discovered by us via virtual screening, have been tested. The compounds were evaluated as DGK inhibitors on α, θ, and ζ isoforms, and as antagonists on serotonin receptors. From these assays emerged two novel compounds, namely 11 and 20 , which with an IC 50 respectively of 1.6 and 1.8 µM are the most potent inhibitors of DGKα discovered to date. Both compounds demonstrated the ability to restore apoptosis in a cellular model of X-linked lymphoproliferative disease as well as the capacity to reduce the migration of cancer cells, suggesting their potential utility in preventing metastasis. Finally, relying on experimental biological data, molecular modelling studies allow us to set a three-point pharmacophore model for DGK inhibitors.

Bitter taste receptors (hTAS2R) are expressed ectopically in various tissues, raising the possibility of a pharmacological exploitation. This seems of particular relevance in airways, since hTAS2Rs are involved in the protection of the aerial tissues from infections and in bronchodilation. The bis-guaianolide absinthin ( 1 ), one of the most bitter compounds known, targets the hTAS2R46 bitter receptor. Absinthin ( 1 ), an unstable compound, readily turns into anabsinthin ( 2 ) with substantial retention of the bitter properties, and this compound was used as a starting material to explore the chemical space around the bis-guaianolide bitter pharmacophore. Capitalizing on the chemoselective opening of the allylic lactone ring, the esters 3 and 4 , and the nor -azide 6 were prepared and assayed on human bronchoepithelial (BEAS-2B) cells expressing hTAS2R46. Anti-inflammatory activity was evaluated by measuring the expression of MUC5AC, iNOS, and cytokines, as well as the production of superoxide anion, qualifying the methyl ester 3 as the best candidate for additional studies.

Cardiovascular diseases represent the leading cause of death in developed countries. Modern surgical methods show poor efficiency in the substitution of small-diameter arteries (<6 mm). Due to the difference in mechanical properties between the native artery and the substitute, the behavior of the vessel wall is a major cause of inefficient substitutions. The use of decellularized scaffolds has shown optimal prospects in different applications for regenerative medicine. The purpose of this work was to obtain polylysine-enriched vascular substitutes, derived from decellularized porcine femoral and carotid arteries. Polylysine acts as a matrix cross-linker, increasing the mechanical resistance of the scaffold with respect to decellularized vessels, without altering the native biocompatibility and hemocompatibility properties. The biological characterization showed an excellent biocompatibility, while mechanical tests displayed that the Young's modulus of the polylysine-enriched matrix was comparable to native vessel. Burst pressure test demonstrated strengthening of the polylysine-enriched matrix, which can resist to higher pressures with respect to native vessel. Mechanical analyses also show that polylysine-enriched vessels presented minimal degradation compared to native. Concerning hemocompatibility, the performed analyses show that polylysine-enriched matrices increase coagulation time, with respect to commercial Dacron vascular substitutes. Based on these findings, polylysine-enriched decellularized vessels resulted in a promising approach for vascular substitution., (Copyright © 2020 Fusaro, Calvo Catoira, Ramella, Sacco Botto, Talmon, Fresu, Hidalgo-Bastida and Boccafoschi.)

Background: Methylxanthines are important pharmacological agents in the treatment of asthma and of chronic obstructive pulmonary diseases. The present study was designed to compare the ability of doxofylline and theophylline to modulate inflammatory pathways in human monocytes., Methods: Monocytes isolated from healthy anonymous human buffy coats were treated with doxofylline or theophylline in the presence of phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS), and their phenotype, the oxidative burst, cytokine expression and release, cAMP production, and protein kinase C (PKC) activity were evaluated., Results: Doxofylline and theophylline did not have overlapping effects on human monocytes. While sharing some common characteristics, they differed significantly in their selectivity. Theophylline affected LPS- above PMA-induced cellular responsivity, while doxofylline behaved in the opposite manner. Furthermore, when testing PKC activity, we found an inhibitory effect of doxofylline but not of theophylline, at equimolar doses., Conclusions: In conclusion, our data support the growing hypothesis that doxofylline does not have a superimposable mechanism of action compared to theophylline, and this may both explain some differences in the risk/benefit ratio and may direct studies to tailor therapy for patients., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Type 2 taste receptors (TAS2R) are G protein-coupled receptors first described in the gustatory system, but have also been shown to have extraoral localizations, including airway smooth muscle (ASM) cells, in which TAS2R have been reported to induce relaxation. TAS2R46 is an unexplored subtype that responds to its highly specific agonist absinthin. Here, we first demonstrate that, unlike other bitter-taste receptor agonists, absinthin alone (1 μm) in ASM cells does not induce Ca 2+ signals but reduces histamine-induced cytosolic Ca 2+ increases. To investigate this mechanism, we introduced into ASM cells aequorin-based Ca 2+ probes targeted to the cytosol, subplasma membrane domain, or the mitochondrial matrix. We show that absinthin reduces cytosolic histamine-induced Ca 2+ rises and simultaneously increases Ca 2+ influx into mitochondria. We found that this effect is inhibited by the potent human TAS2R46 (hTAS2R46) antagonist 3β-hydroxydihydrocostunolide and is no longer evident in hTAS2R46-silenced ASM cells, indicating that it is hTAS2R46-dependent. Furthermore, these changes were sensitive to the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxy)phenyl-hydrazone (FCCP); the mitochondrial calcium uniporter inhibitor KB-R7943 (carbamimidothioic acid); the cytoskeletal disrupter latrunculin; and an inhibitor of the exchange protein directly activated by cAMP (EPAC), ESI-09. Similarly, the β2 agonist salbutamol also could induce Ca 2+ shuttling from cytoplasm to mitochondria, suggesting that this new mechanism might be generalizable. Moreover, forskolin and an EPAC activator mimicked this effect in HeLa cells. Our findings support the hypothesis that plasma membrane receptors can positively regulate mitochondrial Ca 2+ uptake, adding a further facet to the ability of cells to encode complex Ca 2+ signals., (© 2019 Talmon et al.)

As part of an effort to identify druggable diacylglycerol kinase alpha (DGKα) inhibitors, we used an in-silico approach based on chemical homology with the two commercially available DGKα inhibitors R59022 and R59949. Ritanserin and compound AMB639752 emerged from the screening of 127 compounds, showing an inhibitory activity superior to the two commercial inhibitors, being furthermore specific for the alpha isoform of diacylglycerol kinase. Interestingly, AMB639752 was also devoid of serotoninergic activity. The ability of both ritanserin and AMB639752, by inhibiting DGKα in intact cells, to restore restimulation induced cell death (RICD) in SAP deficient lymphocytes was also tested. Both compounds restored RICD at concentrations lower than the two previously available inhibitors, indicating their potential use for the treatment of X-linked lymphoproliferative disease 1 (XLP-1), a rare genetic disorder in which DGKα activity is deregulated., (Copyright © 2018 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Abdominal aortic aneurysm (AAA) is a focal dilatation of the aorta, caused by both genetic and environmental factors. Although vascular endothelium plays a key role in AAA progression, the biological mechanisms underlying the mechanical stress involvement are only partially understood. In this study, we developed an in vitro model to characterize the role of mechanical stress as a potential trigger of endothelial deregulation in terms of inflammatory response bridging between endothelial cells (ECs), inflammatory cells, and matrix remodeling. In AAA patients, data revealed different degrees of calcification, inversely correlated with wall stretching and also with inflammation and extracellular matrix degradation. In order to study the role of mechanical stimulation, endothelial cell line (EA.hy926) has been cultured in healthy (10% strain) and pathological (5% strain) dynamic conditions using a bioreactor. In presence of tumor necrosis factor alpha (TNF-α), high levels of matrix metalloproteinase-9 (MMP-9) expression and inflammation are obtained, while mechanical stimulation significantly counteracts the TNF-α effects. Moreover, physiological deformation also plays a significant role in the control of the oxidative stress. Overall our findings indicate that, due to wall calcification, in AAA there is a significant change in terms of decreased wall stretching.

The discovery of taste receptors hTAS2Rs expression in extra oral tissue, especially in the gastrointestinal tract and in the respiratory system, has endowed bitter receptors of functionalities that exceed the simple perception of taste and flavour. In particular, stimulation of hTAS2Rs by bitter agents in the airway smooth muscle triggers bronchodilation of possible pharmacological relevance. To study the receptor localization in pulmonary smooth muscle cells and to investigate their biological response to hTAS2R38 activation, we have developed a fluorescent probe for hTAS2R38 starting from the sesquiterpene lactone costunolide, available in multigram amounts from Artemisia umbelliformis Lam. The N-methylanthranilate-containing probe demonstrated a very low cytotoxicity compared to the natural product toward human airway smooth muscle cells and epithelial bronchial cells, but fully retained its binding to hTAS2R38, making it possible the fluorescent detection of cells expressing this bitter receptor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Purpose: A crosstalk between the immune system and depression has been postulated, with monocytes/macrophages and cytokines having a key role in this interaction. In this study, we examined whether vortioxetine, a multimodal anti-depressive drug, was endowed with anti-inflammatory and antioxidative activity, leading to immunomodulatory effects on human monocytes and macrophages., Experimental Approach: Human monocytes were isolated from buffy coats and used as such or differentiated into M1 and M2 macrophages. Cells were treated with vortioxetine before or after differentiation, and their responsiveness was evaluated. This included oxy-radical and TNFα production, TNFα and PPARγ gene expression and NF-κB translocation., Key Results: Vortioxetine significantly reduced the PMA-induced oxidative burst in monocytes and in macrophages (M1 and M2), causing a concomitant shift of macrophages from the M1 to the M2 phenotype, demonstrated by a significant decrease in the expression of the surface marker CD86 and an increase in CD206. Moreover, treatment of monocytes with vortioxetine rendered macrophages derived from this population less sensitive to PMA, as it reduced the oxidative burst, NF-kB translocation, TNFα release and expression while inducing PPARγ gene expression. FACS analysis showed a significant decrease in the CD14 + /CD16 + /CD86 + M1 population., Conclusions and Implications: These results demonstrate that in human monocytes/macrophages, vortioxetine has antioxidant activity and anti-inflammatory effects driving the polarization of macrophages towards their alternative phenotype. These findings suggest that vortioxetine, alongside its antidepressive effect, may have immunomodulatory properties., (© 2017 The British Pharmacological Society.)

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol.

Background: Intrauterine infusions have been widely used for the treatment of endometritis in the mare. Nevertheless, their consequences on endocrine and endometrial molecular aspects are unknown. We studied the effect of a 1% povidone-iodine solution intrauterine infusion on progesterone levels, endometrial histology and estrogen (ERα) and progesterone (PR) receptor distribution by immunohistochemistry., Methods: Fourteen healthy mares were used in this study. Estruses were synchronized and seven mares were treated with intrauterine infusions at days 0 and 2 post ovulation of two consecutive estrous cycles. Uterine biopsy samples were taken on days 6 and 15 post ovulation., Results: The treatment did not induce an inflammatory response indicating endometritis, neither affected the ERα. However, it reduced the percentage of PR positive cells (PPC) on day 6 (deep glandular epithelium, control: 95.7 vs. infused: 61.5, P < 0.05). Treated mares tended to have lower progesterone levels on day 2 (3.9 ng/ml vs. 6.6 ng/ml, P = 0.07), and higher levels on day 15 compared with controls (4.4 ng/ml vs. 1.3 ng/ml, P = 0.07)., Conclusion: a 1% povidone-iodine infusion during days 0 and 2 post ovulation in healthy mares did not induce histological changes indicating endometritis, but altered progesterone concentrations and reduced the expression of endometrial PR at day 6 without affecting the ERα. These changes could reduce embryo survival.

Cyclooxygenase (COX) enzymes are molecular targets of nonsteroidal anti-inflammatory drugs (NSAIDs), the most used medication worldwide. However, the COX enzymes are not the sole molecular targets of NSAIDs. Recently, we showed that two NSAIDs, diclofenac and meclofenamate, also act as openers of Kv7.2/3 K(+) channels underlying the neuronal M-current. Here we designed new derivatives of diphenylamine carboxylate to dissociate the M-channel opener property from COX inhibition. The carboxylate moiety was derivatized into amides or esters and linked to various alkyl and ether chains. Powerful M-channel openers were generated, provided that the diphenylamine moiety and a terminal hydroxyl group are preserved. In transfected CHO cells, they activated recombinant Kv7.2/3 K(+) channels, causing a hyperpolarizing shift of current activation as measured by whole-cell patch-clamp recording. In sensory dorsal root ganglion and hippocampal neurons, the openers hyperpolarized the membrane potential and robustly depressed evoked spike discharges. They also decreased hippocampal glutamate and GABA release by reducing the frequency of spontaneous excitatory and inhibitory post-synaptic currents. In vivo, the openers exhibited anti-convulsant activity, as measured in mice by the maximal electroshock seizure model. Conversion of the carboxylate function into amide abolished COX inhibition but preserved M-channel modulation. Remarkably, the very same template let us generating potent M-channel blockers. Our results reveal a new and crucial determinant of NSAID-mediated COX inhibition. They also provide a structural framework for designing novel M-channel modulators, including openers and blockers.

This paper attempts to clarify Minuchin's concept of family boundaries and to modify the original definition to one based on social systems concepts that are neutral with respect to pathology. The authors distinguish between logical and physical conceptualizations of "boundary" and differentiate the broad metaphor of family boundaries into proximity (i.e., interpersonal boundaries) and hierarchy (i.e., generational boundaries). Assuming that the nature of a relationship is reflected by the extent of overlap or sharing of interpersonal territories, Wood (14) adapted Goffman's (2) concept of territorial preserve to encompass six types of territories family members may share to a greater or lesser extent, reflecting the family's pattern of proximity. The family's hierarchy is reflected by who is "in charge," parents or children. We explore the process of boundary reorganization in families in transition, offering a nonstatic and health-oriented view of boundary permeability. Using the anthropological concept of "rite of passage," we suggest an alternative view of stressful transitional events such as marriage and divorce. We illustrate with examples the reorganization of proximity and hierarchy that occurs during periods of transition.