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Chronic kidney disease (CKD) is thus deemed to a global health problem. Renal fibrosis, characterized by accumulation of extracellular matrix (ECM) components in the kidney, is considered a common pathway leading to CKD. Regulator of calcineurin1 (RCAN1), identified as a competitive endogenous inhibitor of the phosphatase calcineurin, participates in ECM deposition in various organs. However, the role of RCAN1 in renal fibrosis remains unclear. Here, unilateral ureteral obstruction (UUO), a well-known model to induce renal fibrosis in vivo, was performed on mice for a week. To overexpress RCAN1.4 in vivo, recombinant adeno-associated virus 9-packed RCAN1.4 over-expression plasm was employed in mice kidney. Lentivirus-packed RCAN1.4 over-expression plasm was employed to transfer into HK-2 and NRK-49F cells in vitro. The results indicated that RCAN1.4 expression was impaired both in UUO-induced renal fibrosis in vivo and TGF-β1-induced renal fibrosis in vitro. However, knocking in of RCAN1.4 suppressed the production of extracellular matrix (ECM) both in vivo and in vitro. Furthermore, in vitro, the apoptosis-related proteins, including the ratio of Bax/Bcl-2 and cleaved-caspase3, were elevated in cells transfected with RCAN1.4 overexpression plasmid. In addition, we found that RCAN1.4 could rugulated NFAT2 nuclear distribution by inhibiting calcineurin pathway. So overexpression of RCAN1.4 could reverse renal fibrosis, attenuate ECM related protein accumulation, promote apoptosis of myofibroblast via inhibiting Calcineurin/NFAT2 signaling pathway. Taken together, our study demonstrated that targeting RCAN1.4 may be therapeutic efficacy in renal fibrosis.

Ca2+ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca2+ signaling inhibitors in natural resources using a yeast-based screening method and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li+ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure–activity relationships of bakkenolides contained in P. japonicus as inhibitors of interleukin-2 production in human T cells.

Brain aging and dementia are current problems that must be solved. The levels of imidazoline 2 receptors (I(2)-IRs) are increased in the brain in Alzheimer’s disease (AD) and other neurodegenerative diseases. We tested the action of the specific and selective I(2)-IR ligand B06 in a mouse model of accelerated aging and AD, the senescence-accelerated mouse prone 8 (SAMP8) model. Oral administration of B06 for 4 weeks improved SAMP8 mouse behavior and cognition and reduced AD hallmarks, oxidative stress, and apoptotic and neuroinflammation markers. Likewise, B06 regulated glial excitatory amino acid transporter 2 and N-methyl-d aspartate 2A and 2B receptor subunit protein levels. Calcineurin (CaN) is a phosphatase that controls the phosphorylation levels of cAMP response element-binding (CREB), apoptotic mediator BCL-2-associated agonist of cell death (BAD) and GSK3β, among other molecules. Interestingly, B06 was able to reduce the levels of the CaN active form (CaN A). Likewise, CREB phosphorylation, BAD gene expression, and other factors were modified after B06 treatment. Moreover, phosphorylation of a target of CaN, nuclear factor of activated T-cells, cytoplasmic 1 (NFAT(C1)), was increased in B06-treated mice, impeding the transcription of genes related to neuroinflammation and neural plasticity. In summary, this I(2) imidazoline ligand can exert its beneficial effects on age-related conditions by modulating CaN pathway action and affecting several molecular pathways, playing a neuroprotective role in SAMP8 mice. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00281-2) contains supplementary material, which is available to authorized users.

BackgroundIn recent decades, Candida glabrata has emerged as a frequent cause of life-threatening fungal infection. In C. glabrata, echinocandin resistance is associated with mutations in FKS1/FKS2 (β-1,3-glucan synthase). The calmodulin/calcineurin pathway is implicated in response to antifungal stress and calcineurin gene disruption specifically reverses Fks2-mediated resistance of clinical isolates.ObjectivesWe evaluated the impact of calmodulin inhibition by fluphenazine in two caspofungin-resistant C. glabrata isolates.MethodsC. glabrata isolates were identified by ITS1/ITS4 (where ITS stands for internal transcribed spacer) sequencing and the echinocandin target FKS1/FKS2 genes were sequenced. Susceptibility testing of caspofungin in the presence of fluphenazine was performed by a modified CLSI microbroth dilution method. The effect of the fluphenazine/caspofungin combination on heat stress (37°C or 40°C), oxidative stress (0.2 and 0.4 mM menadione) and biofilm formation (polyurethane catheter) was analysed. A Galleria mellonella model using blastospores (1 × 109 cfu/mL) was developed to evaluate the impact of this combination on larval survival.ResultsF659del was found in the FKS2 gene of both resistant strains. In these clinical isolates, fluphenazine increased susceptibility to caspofungin and reduced their thermotolerance. Furthermore, the fluphenazine/caspofungin combination significantly impaired biofilm formation in an in vitro polyurethane catheter model. All these features participated in the increasing survival of infected G. mellonella after combination treatment in comparison with caspofungin alone.ConclusionsIn a repurposing strategy, our findings confirm that calmodulin could provide a relevant target in life-threatening fungal infectious diseases.

Almost 30 years after the detection of chronic interstitial nephritis in agricultural communities (CINAC) its etiology remains unknown. To help define this we examined 34 renal biopsies from Sri Lanka, El Salvador, India and France of patients with chronic kidney disease 2-3 and diagnosed with CINAC by light and electron microscopy. In addition to known histopathology, we identified a unique constellation of proximal tubular cell findings including large dysmorphic lysosomes with a light-medium electron-dense matrix containing dispersed dark electron-dense non-membrane bound "aggregates". These aggregates associated with varying degrees of cellular/tubular atrophy, apparent cell fragment shedding and no-weak proximal tubular cell proliferative capacity. Identical lysosomal lesions, identifiable by electron microscopy, were observed in 9% of renal transplant implantation biopsies, but were more prevalent in six month (50%) and 12 month (67%) protocol biopsies and in indication biopsies (76%) of calcineurin inhibitor treated transplant patients. The phenotype was also found associated with nephrotoxic drugs (lomustine, clomiphene, lithium, cocaine) and in some patients with light chain tubulopathy, all conditions that can be directly or indirectly linked to calcineurin pathway inhibition or modulation. One hundred biopsies of normal kidneys, drug/toxin induced nephropathies, and overt proteinuric patients of different etiologies to some extent could demonstrate the light microscopic proximal tubular cell changes, but rarely the electron microscopic lysosomal features. Rats treated with the calcineurin inhibitor cyclosporine for four weeks developed similar proximal tubular cell lysosomal alterations, which were absent in a dehydration group. Overall, the finding of an identical proximal tubular cell (lysosomal) lesion in CINAC and calcineurin inhibitor nephrotoxicity in different geographic regions suggests a common paradigm where CINAC patients undergo a tubulotoxic mechanism similar to calcineurin inhibitor nephrotoxicity.

Multi-drug resistance is becoming an increasingly severe clinical challenge not only among pathogenic bacteria but among fungal pathogens as well. Drug design is inherently more challenging for the eukaryotic fungi due to their closer evolutionary similarity to humans. The recent rapid expansion in invasive infections throughout the world by Candida auris are of particular concern due to a substantial mortality rate, comparatively facile transmission, and an increasing level of resistance to all three of the major classes of anti-fungal drugs. One promising avenue for the development of an alternative class of anti-fungal agents currently under investigation is for drugs against the FK506-binding protein FKBP12 which, when bound to that drug, inhibits the fungal calcineurin signaling pathway with a resultant diminution in virulence. The specific challenge to this approach is that the homologous human calcineurin pathway functions in controlling the tissue immunity response, so that drug selectivity for the fungal pathway must be designed. To facilitate such efforts, we report the nearly complete backbone and sidechain resonances for the FKBP12 proteins of both Candida auris and clinically significant Candida glabrata fungi.

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungus-selective, nonimmunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of the Mucor circinelloides FKBP12 bound to FK506 and of the Aspergillus fumigatus, M. circinelloides, and human FKBP12 proteins bound to the FK506 analog APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic, and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes. The work presented here, aimed at the rational design of more effective calcineurin inhibitors, indeed suggests that modifications to the APX879 scaffold centered around the C15, C16, C18, C36, and C37 positions provide the potential to significantly enhance fungal selectivity. IMPORTANCE Invasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, and in silico methodologies, we pinpoint regions of the FK506 scaffold and a less immunosuppressive analog, APX879, centered around the C15 to C18 and C36 to C37 positions that could be altered with selective extensions and/or deletions to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

Monothiol glutaredoxins are important regulators of iron homeostasis that play conserved roles in the sensing and trafficking of iron-sulfur clusters. We previously characterized the role of the monothiol glutaredoxin Grx4 in iron homeostasis, the interaction with the iron regulator Cir1, and virulence in Cryptococcus neoformans. This important fungal pathogen causes cryptococcal meningoencephalitis in immunocompromised individuals worldwide. Here, we demonstrate that Grx4 is required for proliferation at elevated temperatures (both 37°C and 39°C) and under stress conditions. In particular, the grx4Δ mutant was hypersensitive to SDS, calcofluor white (CFW), and caffeine, suggesting that Grx4 is required for membrane and cell wall integrity (CWI). In this context, we found that Grx4 regulated the phosphorylation of the Mpk1 mitogen-activated protein kinase (MAPK) of the CWI pathway in cells grown at elevated temperature or upon treatment with CFW, caffeine, or SDS. The grx4Δ mutant also displayed increased sensitivity to FK506 and cyclosporin A, two inhibitors of the calcineurin pathway, indicating that Grx4 may influence growth at higher temperatures in parallel with calcineurin signaling. Upon thermal stress or calcium treatment, loss of Grx4 also caused partial mis-localization of Crz1, the transcription factor that is a calcineurin substrate. The phenotypes of the grx4Δ, crz1Δ, and cna1Δ (calcineurin) mutants suggest shared contributions to the regulation of temperature, cell wall, and other stresses. In summary, we show that Grx4 is also a key regulator of the responses to a variety of stress conditions in addition to its roles in iron homeostasis in C. neoformans.

As a potentially toxic heavy metal, Cadmium (Cd) can cause endoplasmic reticulum and oxidative stress, and thus lead to cell death. To explore the mechanisms of Cd toxicity, we investigated the UPRE-lacZ expression, the intracellular reactive oxygen species (ROS) and cell death in the 151 Cd-sensitive mutants of Saccharomyces cerevisiae in response to Cd stress. We identified 101 genes regulating UPRE-lacZ expression were involved in preventing ROS production and/or cell death from increasing to high levels, while mutants for 72 genes caused both elevated ROS production and cell death, indicating the Cd-induced ROS production and cell death are mediated by UPR. Genes involved in cell wall integrity (CWI) pathway, vacuolar protein sorting (VPS) and vacuolar transport, calcium/calcineurin pathway and PHO pathways were all required for the Cd-induced UPR, intracellular ROS and cell death. To conclude, this study highlights the importance of Cd-induced UPR, intracellular ROS levels and cell death that may play crucial roles in Cd-induced toxicity.

Several examples of transcription factors that show stochastic, unsynchronized pulses of nuclear localization have been described. Here we show that under constant calcium stress, nuclear localization pulses of the transcription factor Crz1 follow stochastic variations in cytosolic calcium concentration. We find that the size of the stochastic calcium bursts is positively correlated with the number of subsequent Crz1 pulses. Based on our observations, we propose a simple stochastic model of how the signaling pathway converts a constant external calcium concentration into a digital number of Crz1 pulses in the nucleus, due to the time delay from nuclear transport and the stochastic decoherence of individual Crz1 molecule dynamics. We find support for several additional predictions of the model and suggest that stochastic input to nuclear transport may produce noisy digital responses to analog signals in other signaling systems.

Short-chain fatty acids (SCFAs), metabolites of colonic bacterial fermentation of complex carbohydrates, are closely related to the release of gut hormones. In this study, we examined the involvement of transient receptor potential ankyrin 1 (TRPA1) in SCFA-induced increase in intracellular calcium ([Ca2+ ]i ) and its impact on gut hormone secretion using naturally TRPA1 expressing intestinal secretin tumour cell-1 (STC-1) cell line. Individual SCFAs and their physiological mix enhanced calcium influx in TRPA1-dependent manner. SCFA mix also significantly increased membrane expression of TRPA1. Gene expression studies revealed that SCFA mix elevated the expression of genes involved in calcium-activated calcineurin pathway in TRPA1-dependent manner and cAMP-regulated transcriptional co-activators (CRTC) pathway independent to TRPA1. Genes representing synaptic vesicular exocytosis and gut hormone precursors were significantly elevated with SCFA mix treatment. Treatment with TRPA1 antagonist HC-030031 markedly reduced these effects. The release of gut hormones was elevated with 10 mm SCFA mix in TRPA1 dependent manner. Our in vivo prebiotic study results suggested presence of an environment conducive to increase in gut hormone secretion. Overall, our findings provide an evidence for the possible role of TRPA1 in SCFA-induced increase in gut hormone secretion, hence another mechanism of action for prebiotics.

Background: Chronic kidney disease (CKD) is thus deemed to a global health problem. Renal fibrosis, characterized by accumulation of extracellular matrix (ECM) components in the kidney, is considered a common pathway leading to CKD. Regulator of calcineurin1 (RCAN1), identified as a competitive endogenous inhibitor of the phosphatase calcineurin, participates in ECM deposition in various organs. However, the role of RCAN1 in renal fibrosis remains unclear. Methods: Unilateral ureteral obstruction (UUO), a well-known model to induce renal fibrosis, was performed on mice for a week. To overexpress RCAN1.4 in vivo, Recombinant adeno-associated virus (rAAV) 9-packed RCAN1.4 over-expression plasm was employed in mice kidney. Lentivirus-packed RCAN1.4 over-expression plasm was employed to transfer into HK-2 and NRK-49F cells in vitro. Hematoxylin and eosin (H & E) were performed to assess histopathological kidney injury, Sirus-red and Masson’s trichrome staining were carried out for observing collagen deposition. Western blotting was also used to determine the expression levels. Results: RCAN1 isoform 4 (RCAN1.4) expression was impaired after suffering UUO-induced renal fibrosis in vivo and TGF-β1-induced renal fibrosis in vitro. Furthermore, knocking in of RCAN1.4 in mice kidney, transfected with rAAV9-packed RCAN1.4 over-expression plasm, reduced the expression levels of hallmarks of EMT. In vitro, the apoptosis-related proteins, including the ratio of Bax/Bcl-2 and cleaved-caspase3, were elevated in cells transfected with RCAN1.4 overexpression plasmid. Moreover, over-expression of RCAN1.4 recovered the renal function and attenuated ECM deposition. In addition, RCAN1.4 regulated NFAT2 nuclear distribution by inhibiting calcineurin pathway. Conclusions: The expression of RCAN1.4 was attenuated both in UUO-induced fibrotic kidney of mouse and in TGF-β1-induced fibrotic model in vitro. Moreover, overexpression of RCAN1.4 could reverse renal fibrosis, attenuate ECM related protein accumulation, promote apoptosis of myofibroblast via inhibiting CaN/NFAT2 signaling pathway. Taken together, our study demonstrated that targeting RCAN1.4 may be therapeutic efficacy in renal fibrosis Funding: This study was supported by the National Natural Science Foundation of China (No.82000639) and Innovation Project of Medical School of Wuhan University (TFZZ2018017). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: This experiment was authorized by the Ethics Committee of Renmin Hospital of Wuhan University, and the procedures were carried out adhere to the principles of Animal Care of Wuhan University (Wuhan, China).

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial.ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload.Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signalling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/-) mice and absent in homozygous (GqI-/-) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and α-skeletal actin (α-SA) was increased 48 hours after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and α-SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKIIδ levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels.ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but are not required for LVH induced by mechanical pressure overload (TAC); the latter is mediated by activation of the CaMKII-HDAC4-MEF2 pathway.

Intracellular calcium (Ca2+) is crucial for signal transduction inCryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signalling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+-binding mediated by the EF-hand domains of the Ca2+sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+homeostasis under stress and non-stress conditions, as thencs1Δmutant is sensitive to a high Ca2+concentration and has an elevated basal Ca2+level that correlates with increased expression of the Ca2+transporter genes,CCH1andMID1. Furthermore,NCS1expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast toSaccharomyces cerevisiae,NCS1expression inC. neoformansis regulated by the calcineurin pathway via the transcription factor Crz1, asNCS1expression is reduced by FK506 treatment andCRZ1deletion. Moreover, thencs1Δmutant shares a high temperature and high Ca2+sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ andcam1Δ) and theNCS1promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1-deficency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data shows that Ncs1 plays distinct roles in Ca2+sensing inC. neoformansdespite widespread functional conservation of Ncs1 and other regulators of Ca2+homeostasis.ImportanceCryptococcus neoformansis the major cause of fungal meningitis in HIV infected patients. Several studies have highlighted the important contribution of Ca2+signalling and homeostasis to the virulence ofC. neoformans. Here, we identify the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) and demonstrate its role in Ca2+homeostasis, bud emergence, cell cycle progression and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signalling cascade. Our work provides evidence of a link between Ca2+homeostasis and cell cycle progression inC. neoformans.

The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1β level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.

Excessive microglial cell activation in the brain can lead to the production of various neurotoxic factors (e.g., pro-inflammatory cytokines, nitric oxide) which can, in turn, initiate neurodegenerative processes. Recent research has been reported that mitochondrial dynamics regulate the inflammatory response of lipopolysaccharide (LPS). Isoliquiritigenin (ISL) is a compound found in Glycyrrhizae radix with anti-inflammatory and antioxidant properties. In this study, we investigated the function of ISL on the LPS-induced pro-inflammatory response in BV-2 microglial cells. We showed that ISL reduced the LPS-induced increase in pro-inflammatory mediators (e.g., nitric oxide and pro-inflammatory cytokines) via the inhibition of ERK/p38/NF-κB activation and the generation of reactive oxygen species (ROS). Furthermore, ISL inhibited the excessive mitochondrial fission induced by LPS, regulating mitochondrial ROS generation and pro-inflammatory response by suppressing the calcium/calcineurin pathway to dephosphorylate Drp1 at the serine 637 residue. Interestingly, the ISL pretreatment reduced the number of apoptotic cells and levels of cleaved caspase3/PARP, compared to LPS-treated cells. Our findings suggested that ISL ameliorated the pro-inflammatory response of microglia by inhibiting dephosphorylation of Drp1 (Ser637)-dependent mitochondrial fission. This study provides the first evidence for the effects of ISL against LPS-induced inflammatory response related and its link to mitochondrial fission and the calcium/calcineurin pathway. Consequently, we also identified the protective effects of ISL against LPS-induced microglial apoptosis, highlighting the pharmacological role of ISL in microglial inflammation-mediated neurodegeneration.

Calcineurin is a critical enzyme in fungal pathogenesis and antifungal drug tolerance and, therefore, an attractive antifungal target. Current clinically-accessible calcineurin inhibitors, such as FK506, are immunosuppressive to humans, so exploiting calcineurin inhibition as an antifungal strategy necessitates fungal-specificity in order to avoid inhibiting the human pathway. Harnessing fungal calcineurin-inhibitor crystal structures, we recently developed a less immunosuppressive FK506 analog, APX879, with broad-spectrum antifungal activity and demonstrable efficacy in a murine model of invasive fungal infection. Our overarching goal is to better understand, at a molecular level, the interaction determinants of the human and fungal FK506-binding proteins (FKBP12) required for calcineurin inhibition in order to guide the design of fungal-selective, non-immunosuppressive FK506 analogs. To this end, we characterized high-resolution structures of theM. circinelloidesFKBP12 bound to FK506, and of theA. fumigatus, M. circinelloidesand human FKBP12 proteins bound to the FK506 analog, APX879, which exhibits enhanced selectivity for fungal pathogens. Combining structural, genetic and biophysical methodologies with molecular dynamics simulations, we identify critical variations in these structurally similar FKBP12-ligand complexes that will guide the rational design of inhibitors with enhanced fungal-selectivity.Significance statementInvasive fungal infections are a leading cause of death in the immunocompromised patient population. The rise in drug resistance to current antifungals highlights the urgent need to develop more efficacious and highly selective agents. Numerous investigations of major fungal pathogens have confirmed the critical role of the calcineurin pathway for fungal virulence, making it an attractive target for antifungal development. Although FK506 inhibits calcineurin, it is immunosuppressive in humans and cannot be used as an antifungal. By combining structural, genetic, biophysical, andin silicomethodologies, we pinpoint regions of FK506 and a less immunosuppressive analog, APX879, that could be altered to enhance fungal selectivity. This work represents a significant advancement toward realizing calcineurin as a viable target for antifungal drug discovery.

As the prevalence of systemic fungal infections caused by Candida albicans gradually increases, it is necessary to explore potential and effective antifungals. Carvacrol is reported to be lethally toxic to C. albicans, involving several potential mechanisms. However, the form and specific mechanism of cell death caused by this compound has not been delineated. In this study, we found that carvacrol could significantly decrease C. albicans survival rates, consistent with previous researches. Further examination proved that carvacrol treatment caused cell membrane permeability and depolarization. To elucidate the association between cell death and apoptosis, DNA fragmentation and metacaspase activation were determined; as expected, these two apoptosis-related markers were clearly observed. Moreover, total and mitochondrial reactive oxygen species (ROS) levels were elevated, and both mitochondrial transmembrane potential and morphology were disrupted. Additionally, cytosolic and mitochondrial calcium levels were also increased by carvacrol. Calcineurin inhibition experiments revealed cyclosporine A (CsA) addition notably rescued cell growth and inhibited metacaspase activation, indicating that carvacrol triggered C. albicans apoptosis through inducing calcineurin activation. Carvacrol was demonstrated to both have low toxicity and be effective in alleviating systemic infections with C. albicans, which might be via its antifungal and immunomodulation activities. This study suggests that carvacrol has excellent potential as a natural protective compound against C. albicans infections.

Background/Aims: Angiotensin II type I receptor agonistic autoantibody (AT1-AA) is closely related to pre-eclampsia, which is characterized by proteinuria and hypertension. AT1-AA has been shown to enhance the effect of AngII in pre-eclampsia, such as production of endothelin-1, activation of ROS, and vasoconstriction, which are considered to be associated with hypertension; however, whether or not AT1-AA participates in podocyte damage leading to the generation of proteinuria has not been reported. In this study we investigated the role of pre-eclamptic serum AT1-AA on podocytes and the mechanism underlying the generation of proteinuria. Methods: The levels of AT1-AA isolated from pre-eclamptic sera were determined by an enzyme-linked immunosorbent assay. Human podocytes were cultured in vitro and treated with various concentrations of AT1-AA. Whether or not an ERK1/2 inhibitor and TRPC6 siRNA inhibit the effect of AT1-AA on podocytes was determined. Western blot was used to detect the expression of podocyte-specific proteins (nephrin, synaptopodin, and podocin) and the phosphorylation of ERK1/2 and TRPC6. The arrangement of F-actin was observed by immunofluorescence. A Calcineurin Cellular Activity Assay Kit was used to detect calcineurin activity. Changes in the intracellular Ca2+ concentration was determined by confocal laser. Results: AT1-AA induced a decrease in podocyte-specific protein expression and calcineurin activity and increased expression of p-ERK1/2 and TRPC6 protein and the intracellular Ca2+ concentration. Immunofluorescence revealed rearrangement of F-actin. PD98059, an inhibitor of ERK1/2, and TRPC6 siRNA attenuated the decreased expression of podocyte-specific proteins and decreased intracellular Ca2+ concentration. The expression of TRPC6 was reduced following the addition of ERK1/2 inhibitor. Conclusion: AT1-AA induced podocyte damage in a dose-dependent manner. The underlying mechanism might involve activation of the TRPC6 –calcium/calcineurin pathway. This study provides new details regarding podocyte injury and the mechanism underlying the generation of proteinuria in pre-eclampsia.

Target of rapamycin (TOR) regulates cellular homeostasis by coordinating cellular growth pathways in response to different environmental signals. Rapamycin, an allosteric TOR complex 1 (TORC1) inhibitor, has proven to be invaluable for elucidating various aspects of the TOR signalling pathway; however, its applications are limited due to its inability to completely suppress TORC2. In the present study, we examined the effects of a newly discovered potent TOR inhibitor, Torin2, which inhibits both TORC1 and TORC2, on Saccharomyces cerevisiae growth. Genome-scale expression profiling of Torin2 treated yeast cells showed an expression profile similar to that of other TOR inhibitors such as rapamycin and caffeine. Distinct inhibition of cell growth by Torin2 treatment is indicated by the fact that a smaller number of transcripts are altered, compared to the changes after rapamycin and caffeine treatments. Our results revealed that Torin2 leads to increased expression of the calcineurin pathway genes favouring a synergistic therapeutic response of Torin2 in combination with calcineurin inhibitors. Further, Torin2 causes defective bud site selection during asymmetric cell division, indicating a role of TOR signalling in regulation of the budding pattern. Torin2 treated yeast cells exhibit increased expression of metalloreductases which affects iron homeostasis leading to iron toxicity. Notably, the enhanced expression of TOR1 and TOR2 rescue the Torin2 augmented iron toxicity of yeast cell. This study has revealed novel conduits and our results suggest that using Torin2 will enable the dissection of TORC2 mediated functions of the TOR signalling pathway.

Honokiol, a bioactive compound isolated from the cone and bark of Magnolia officinalis, has been shown to have various activities including inhibition of the growth of Candida albicans. We investigated the roles of the Hsp90-calcineurin pathway in the antifungal activity of honokiol. The pharmacologic tool was employed to evaluate the effects of Hsp90 and calcineurin in the antifungal activity of honokiol. We also evaluated the protective effects of the calcineurin inhibitor cyclosporin A (CsA) on honokiol-induced mitochondrial dysfunction by the fluorescence staining method. The Hsp90 inhibitor potentiated the antifungal activity of honokiol. A C. albicans strain with the calcineurin gene deleted displayed enhanced sensitivity to honokiol. However, co-treatment with calcineurin inhibitor CsA attenuated the cytotoxic activity of honokiol due to the protective effect on mitochondria. Our results provide insight into the action mechanism of honokiol.

Calcineurin modulates environmental stress survival and virulence of the human fungal pathogen Cryptococcus neoformans. Previously, we identified 44 putative calcineurin substrates, and proposed that the calcineurin pathway is branched to regulate targets including Crz1, Pbp1, and Puf4 in C. neoformans. In this study, we characterized Had1, which is one of the putative calcineurin substrates belonging to the ubiquitously conserved haloacid dehalogenase β-phosphoglucomutase protein superfamily. Growth of the had1∆ mutant was found to be compromised at 38° or higher. In addition, the had1∆ mutant exhibited increased sensitivity to cell wall perturbing agents, including Congo Red and Calcofluor White, and to an endoplasmic reticulum stress inducer dithiothreitol. Virulence studies revealed that the had1 mutation results in attenuated virulence compared to the wild-type strain in a murine inhalation infection model. Genetic epistasis analysis revealed that Had1 and the zinc finger transcription factor Crz1 play roles in parallel pathways that orchestrate stress survival and fungal virulence. Overall, our results demonstrate that Had1 is a key regulator of thermotolerance, cell wall integrity, and virulence of C. neoformans.

The Ca2+/calmodulin-dependent protein phosphatase calcineurin orchestrates sexual reproduction, stress responses, and virulence via branched downstream pathways in the opportunistic human fungal pathogen Cryptococcus neoformans. The calcineurin-binding protein Cbp1, the calcineurin temperature suppressor Cts1, the calcineurin-responsive zinc finger transcription factor Crz1, and the calcineurin targets Pbp1, Tif3, and Puf4, all function downstream of calcineurin to orchestrate distinct cellular processes. To elucidate how the calcineurin pathway regulatory network governs unisexual reproduction, stress responses, and virulence, we have analyzed the self-filamentous C. deneoformans strain, XL280α, and generated double mutants of these calcineurin downstream genes. We demonstrated that calcineurin governs unisexual reproduction at different sexual developmental stages, in which the initiation of the yeast–hyphal morphological transition is independent of Crz1, whereas the sporulation process is dependent on Crz1. Calcineurin-dependent unisexual reproduction is independent of the pheromone response pathway. Crz1 synergistically interacts with different calcineurin downstream targets in responding to ER, high-calcium, and cell wall stresses. We observed a widespread synergy suggesting that these proteins function in complex branched pathways downstream of calcineurin with some functional redundancy, which may allow efficient signaling network rewiring within the pathway for prompt adaptation to changing environments. Finally, we showed that deletion of PBP1 or TIF3 in the cna1∆ mutant background conferred a modest level of growth tolerance at 37°, but that the cna1∆ pbp1∆ and cna1∆ tif3∆ double mutants were both avirulent, suggesting that calcineurin may control virulence via mechanisms beyond thermotolerance.

Aim of research: determination of the diagnostic value of serum concentration of biomarkers of calcineurin signal pathway (calcineurin, protein S100, interleukin-2) in children with the bicuspid aortic valve at absence of heart failure signs and influence of revealed changes on the early remodeling and hypertrophy of myocardium.Materials and methods: 38 children with the bicuspid aortic valve (SAV), diagnosed by doppleroechocardiography, and 28 children of the control group underwent the measuring of concentrations of calcineurin, protein S100, interleukin-2 and general calcium by the immune-enzyme method with the further estimation of the influence of these changes on morphofunctional hearth parameters by the method of correlation analysis.Results: children with SAV demonstrated the reliable thickening of the back wall of the left ventricle and interventricular septum at the correspondent increase of the blood velocity and pressure gradient on the aortic valve. Serum concentrations of calcineurin and calcium in children with SAV were reliably decreased, and protein S100 correspondingly increased comparing with the control group. At that the SAV group, as opposite to the control one, demonstrated reliable negative correlations between the calcineurin level and right ventricle sizes, protein S100 and left and right atria sizes interleukin-2 and left ventricle systolic size at the reliable positive correlation between levels of calcineurin and general calcium.Conclusions: Children with SAV demonstrated the moderately expressed left ventricle myocardium hypertrophy, decrease of calcineurin serum concentrations that have a negative influence on the geometry of right heart parts and serum calcium level at the correspondent increase of protein S100. The revealed changes allowed to establish the predicative role of calcineurin in the development of early morphofunctional changes of myocardium in children with SAV

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Chitin is an essential structural polysaccharide component of the cell walls of fungi. Because chitin is not found in human cells it also represents an attractive target for antifungal therapy. Recent reports have suggested that Candida cells can resist killing by the antifungal echinocandin both in vitro and in vivo by up-regulating chitin synthesis and thereby sustaining cell wall integrity (Lee et al., 2012). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of echinocandins often have supra-high rather than high chitin levels, and therefore having too much chitin in the cell wall may be detrimental for viability. Furthermore, mutants in the Ca2+/calcineurin pathway are associated with appearance of viable super-high chitin cells following cell wall stress, suggesting suggest that this pathway may negatively regulate other chitin stimulation pathways. We, therefore, propose that C. albicans has evolved a mechanism to maintain their chitin content high enough to protect cells against cell wall damage, but not so high that will negatively affect cell viability. In our model, the Ca2+/calcineurin pathway acts as a buffering system or attenuator in maintaining viable high chitin cells by coordinating both chitin upregulation and modulation of the cell wall integrity pathway, through negative regulation of the MAPK Mkc1 pathway. In summary, we have uncovered a novel mechanism used by C. albicans to survive cell wall stress imposed by echinocandin treatment.

The calcineurin pathway is an important signaling cascade for growth, sexual development, stress response, and pathogenicity in fungi. In this study, we investigated the function of CrzA, a key transcription factor of the calcineurin pathway, in an aflatoxin-producing fungus Aspergillus flavus (A. flavus). To examine the role of the crzA gene, crzA deletion mutant strains in A. flavus were constructed and their phenotypes, including fungal growth, spore formation, and sclerotial formation, were examined. Absence of crzA results in decreased colony growth, the number of conidia, and sclerocia production. The crzA-deficient mutant strains were more susceptible to osmotic pressure and cell wall stress than control or complemented strains. Moreover, deletion of crzA results in a reduction in aflatoxin production. Taken together, these results demonstrate that CrzA is important for differentiation and mycotoxin production in A. flavus.

Jagaricin is a lipopeptide produced by the bacterial mushroom pathogen Janthinobacterium agaricidamnosum, the causative agent of mushroom soft rot disease. Apart from causing lesions in mushrooms, jagaricin is a potent antifungal active against human-pathogenic fungi. We show that jagaricin acts by impairing membrane integrity, resulting in a rapid flux of ions, including Ca(2+), into susceptible target cells. Accordingly, the calcineurin pathway is required for jagaricin tolerance in the fungal pathogen Candida albicans. Transcriptional profiling of pathogenic yeasts further revealed that jagaricin triggers cell wall strengthening, general shutdown of membrane potential-driven transport, and the upregulation of lipid transporters, linking cell envelope integrity to jagaricin action and resistance. Whereas jagaricin shows hemolytic effects, it exhibited either no or low plant toxicity at concentrations at which the growth of prevalent phytopathogenic fungi is inhibited. Therefore, jagaricin may have potential for agricultural applications. The action of jagaricin as a membrane-disrupting antifungal is promising but would require modifications for use in humans.

Chitin is an essential structural polysaccharide component of the cell walls and septa of fungi. Recent reports have suggested that Candida cells can resist killing by echinocandins by up-regulation of chitin synthesis thereby sustaining cell wall integrity both in vitro and in vivo (Lee et al. 2012). This increase in chitin content seen in C. albicans cells that are less susceptible to caspofungin is coordinated simultaneously by the PKC, Ca2+/calcineurin and HOG pathways (Munro et al. 2007, Walker et al. 2008). However, when echinocandins are removed, the chitin content quickly returns to basal levels, suggesting that elevated chitin cell wall content represents a fitness cost. We show here that those cells that die in the presence of caspofungin often have supra-normal chitin levels rather than low chitin levels, and therefore that having too much chitin in the cell wall may be detrimental for viability. Chitin content may therefore need to be clamped at levels that enable cells to survive cell wall stresses but are not so high that they negatively affect cell viability.

Luciferase reporter gene systems based on the NFAT-response element (RE) have been used to monitor intracellular Ca2+ elevation. However, Ca2+ mobilization agent (e.g., ionomycin) alone is not adequate to activate the currently often employed reporter gene that contains the NFAT-RE found in the IL2 promoter. In addition to activation of NFAT through the Ca2+-calmodulin/calcineurin pathway, activation of AP-1 as a partner transcription factor is essential for the IL2-based NFAT-RE system. Here, we describe a detailed method for the recently developed new reporter gene system containing the NFAT-RE from the IL8 promoter. This system enables us to monitor endpoint effects of Ca2+-mobilizing agonists independent of AP-1 activation.

In order to discover plant-derived signaling pathway inhibitors with antifungal properties, a two-component screening system utilizing the calcineurin and Hog1 mitogen-activated protein kinase pathways responsible for the virulence networks of Cryptococcus neoformans was employed, owing to the counter-regulatory actions of these pathways. Of the 1,000 plant extracts tested, two bioactive compounds from Miliusa sinensis were found to act specifically on the calcineurin pathway of C. neoformans. These compounds, identified as pashanone and 5-hydroxy-6,7-dimethoxyflavanone, exhibited potent antifungal activities against various human pathogenic fungi with minimum inhibitory concentration values ranging from 4.0 to >128 μg/ml.

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity.

Transient receptor potential vanilloid 2 (TRPV2) acts as a Ca(2+)-permeable non-selective cation channel that has been reported to be sensitive to temperature, mechanical force, and some chemicals. We recently showed that TRPV2 is critical for maintenance of the thermogenic function of brown adipose tissue in mice. However, the involvement of TRPV2 in the differentiation of brown adipocytes remains unexplored. We found that the expression of TRPV2 was dramatically increased during the differentiation of brown adipocytes. Non-selective TRPV2 agonists (2-aminoethoxydiphenyl borate and lysophosphatidylcholine) inhibited the differentiation of brown adipocytes in a dose-dependent manner during the early stage of differentiation of brown adipocytes. The inhibition was rescued by a TRPV2-selective antagonist, SKF96365 (SKF). Mechanical force, which activates TRPV2, also inhibited the differentiation of brown adipocytes in a strength-dependent manner, and the effect was reversed by SKF. In addition, the inhibition of adipocyte differentiation by either TRPV2 ligand or mechanical stimulation was significantly smaller in the cells from TRPV2KO mice. Moreover, calcineurin inhibitors, cyclosporine A and FK506, partially reversed TRPV2 activation-induced inhibition of brown adipocyte differentiation. Thus, we conclude that TRPV2 might be involved in the modulation of brown adipocyte differentiation partially via a calcineurin pathway.

Candida albicans CNB1 plays a role in the response in vitro and in vivo to stress generated by PB-WUT-01, namely 1,3-dimethyl-7-(2-((1-(3-(perbromo-2H-benzo[d][1,2,3]triazol-2-yl)propyl)-1H-1,2,3-triazol-4-yl)methoxy)propyl)-1H-purine-2,6(3H,7H)-dione. The antifungal mechanism involved the calcineurin pathway-regulated genes SAP9-10. Galleria mellonella treated with PB-WUT-01 (at 0.64 µg/mg) showed limited candidiasis and remained within the highest survival rates. The molecular mode of action of PB-WUT-01 was rationalized by in silico docking studies toward both human and C. albicans calcineurin A (CNA) and calcineurin B (CNB) complexes, respectively. PB-WUT-01 acting as a calcineurin inhibitor in the C. albicans cells enhances the cells’ susceptibility. Therefore it could be a suitable alternative treatment in patients with candidiasis.

FK506 (tacrolimus) is an FDA-approved immunosuppressant indicated for the prevention of allograft rejections in patients undergoing organ transplants. In mammals, FK506 inhibits the calcineurin-nuclear factor of activated T cells (NFAT) pathway to prevent T-cell proliferation by forming a ternary complex with its binding protein, FKBP12, and calcineurin. FK506 also exerts antifungal activity by inhibiting calcineurin, which is essential for the virulence of human-pathogenic fungi. Nevertheless, FK506 cannot be used directly as an antifungal drug due to its immunosuppressive action. In this study, we analyzed the cytotoxicity, immunosuppressive activity, and antifungal activity of four FK506 analogs, 31-O-demethyl-FK506, 9-deoxo-FK506, 9-deoxo-31-O-demethyl-FK506, and 9-deoxo-prolyl-FK506, in comparison with that of FK506. The four FK506 analogs generally possessed lower cytotoxicity and immunosuppressive activity than FK506. The FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against Cryptococcus neoformans and Candida albicans, which are two major invasive pathogenic yeasts, due to the inhibition of the calcineurin pathway. Furthermore, the FK506 analogs, except for 9-deoxo-prolyl-FK506, had strong antifungal activity against the invasive filamentous fungus Aspergillus fumigatus. Notably, 9-deoxo-31-O-demethyl-FK506 and 31-O-demethyl-FK506 exhibited robust synergistic antifungal activity with fluconazole, similar to FK506. Considering the antifungal efficacy, cytotoxicity, immunosuppressive activity, and synergistic effect with commercial antifungal drugs, we selected 9-deoxo-31-O-demethyl-FK506 for further evaluation of its in vivo antifungal efficacy in a murine model of systemic cryptococcosis. Although 9-deoxo-31-O-demethyl-FK506 alone was not sufficient to treat the cryptococcal infection, when it was used in combination with fluconazole, it significantly extended the survival of C. neoformans-infected mice, confirming the synergistic in vivo antifungal efficacy between these two agents.

There are many different pathogenic stimuli that are able to activate the immune system, ranging from microbes that include bacteria, viruses, fungi, and parasites to host-derived triggers such as autoantigens that can induce autoimmunity as well as neoantigens involved in tumorigenesis. One of the key interactions shaping immunity toward these triggers involves the encounter of antigen-processing and -presenting cells such as macrophages and dendritic cells with T cells, resulting in immune responses that are highly selective for the antigenic trigger. Research over the past few years has implicated members of the coronin protein family, in particular coronin 1, in responses against several pathogenic triggers. While coronin 1 was initially described as a host factor allowing the intracellular survival of the pathogen; Mycobacterium tuberculosis; , subsequent work showed it to be a crucial factor for naïve T cell homeostasis. The activity of coronin 1 in allowing the intracellular survival of pathogenic mycobacteria is relatively well characterized, involving the activation of the Ca; 2+; /calcineurin pathway, while coronin 1's role in modulating naïve T cell homeostasis remains more enigmatic. In this mini review, we discuss the knowledge on the role for coronin 1 in immune cell functioning and provide a number of potential scenarios; via; which coronin 1 may be able to regulate naïve T cell homeostasis.

Iron is an essential element for neuronal as well as cellular functions. However, Iron overload has been known to cause neuronal toxicity through mitochondrial fission, dysregulation of Ca2+, endoplasmic reticulum (ER) stress, and reactive oxygen species (ROS) production. Nevertheless, the precise mechanisms of iron-induced oxidative stress and mitochondria- and ER-related iron toxicity in neuronal cells are not fully understood. In this study, we demonstrated that iron overload induces ROS production earlier in the ER than in the mitochondria, and peroxiredoxin 5 (Prx5), which is a kind of antioxidant induced by iron overload, prevents iron overload-induced mitochondrial fragmentation mediated by contact with ER and translocation of Drp1, by inhibiting ROS production and calcium/calcineurin pathway in HT-22 mouse hippocampal neuronal cells. Moreover, Prx5 also prevented iron overload-induced ER-stress and cleavage of caspase-3, which consequently attenuated neuronal cell death. Therefore, we suggested that iron overload induces oxidative stress in the ER earlier than in the mitochondria, thereby increasing ER stress and calcium levels, and consequently causing mitochondrial fragmentation and neuronal cell death. So we thought that this study is essential for understanding iron toxicity in neurons, and Prx5 may serve as a new therapeutic target to prevent iron overload-induced diseases and neurodegenerative disorders.

International audience; Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1μM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.