TNNC1 . Here, we report the case of two siblings with severe, early onset DCM who were found to have compound heterozygous variants in TNNC1: p.Asp145Glu (D145E) and p.Asp132Asn (D132N), which were inherited from the parents. We began our investigation with CRISPR/Cas9 knockout of TNNC1 in Xenopus tropicalis, which resulted in a cardiac phenotype in tadpoles consistent with DCM. Despite multiple maneuvers, we were unable to rescue the tadpole hearts with either human cTnC wild-type or patient variants to investigate the cardiomyopathy phenotype in vivo. We therefore utilized porcine permeabilized cardiac muscle preparations (CMPs) reconstituted with either wild-type or patient variant forms of cTnC to examine effects of the patient variants on contractile function. Incorporation of 50% WT/50% D145E into CMPs increased Ca2+ sensitivity of isometric force, consistent with prior studies. In contrast, incorporation of 50% WT/50% D132N, which had not been previously reported, decreased Ca2+ sensitivity of isometric force. CMPs reconstituted 50–50% with both variants mirrored WT in regard to myofilament Ca2+ responsiveness. Sinusoidal stiffness (SS) (0.2% peak-to-peak) and the kinetics of tension redevelopment (kTR) at saturating Ca2+ were similar to WT for all preparations. Modeling of Ca2+-dependence of kTR support the observation from Ca2+ responsiveness of steady-state isometric force, that the effects on each mutant (50% WT/50% mutant) were greater than the combination of the two mutants (50% D132N/50% D145E). Further studies are needed to ascertain the mechanism(s) of these variants.Familial dilated cardiomyopathy (DCM), clinically characterized by enlargement and dysfunction of one or both ventricles of the heart, can be caused by variants in sarcomeric genes including  Pediatric cardiomyopathies, or diseases of the heart muscle in young patients, have an annual incidence of 1.1–1.5 per 100,000 and are the most common indication for heart transplantation in children . DilatedTNNC1 gene and is abundantly expressed in cardiomyocytes, functions as a myofilament Ca2+ sensor and plays a critical role in regulating contraction (2+ and/or Mg2+) at two EF-hand motifs – referred to as sites III and IV based on their location in the primary sequence – and has important structural and modulatory roles in the sarcomere , troponin T (TnT), and troponin C (TnC) . Cardiactraction . The tertraction . The C-darcomere . The N-d systole .TNNC1. As a result of our attempts to understand the molecular basis of these pediatric DCM cases, we provide in vivo experimental evidence confirming that TNNC1 is a crucial gene for heart function, and further provide in vitro experimental evidence that altered contractile mechanics – unexpectedly – cannot always explain the pathogenicity of TNNC1 variants.Missense variants in cTnC have been associated with both dilated and hypertrophic cardiomyopathies , and knoXenopus were maintained and cared for in an aquatics facility in accordance with Yale University Institutional Animal Care and Use Committee protocols.The human subjects research in this study was approved by the Institutional Review Board of Yale University School of Medicine. Genomic DNA was isolated from either venous blood or saliva samples. Whole exome sequencing was performed by using IDT xGen capture kit followed by Illumina DNA sequencing (HiSeq 4000). Paired end sequence reads were converted to FASTQ format and were aligned to the reference human genome (hg19). SNVs and indels were called using a GATK pipeline and annotated using AnnoVar.Xenopus tropicalis tadpoles was used as previously described (tnnc1 were designed to generate knockdowns.CRISPR/Cas9-mediated genome editing in escribed . BrieflyCRISPR 1 oligo (targets exon 4):CTAGCtaatacgactcactataGGTTCTTGGTCATGATGGTCgttt tagagctagaaTAGCAAGand CRISPR 2 oligo (targets exon 5):CTAGCtaatacgactcactataGGAGGAACTCATGCGAGATGgtt ttagagctagaaTAGCAAG.The sgRNAs were synthesized using the EnGen sgRNA synthesis kit (NEB), and underwent subsequent purification and concentration using the RNA Clean & Concentrator-5 kit (Zymo). Individual sgRNAs were injected at 400 pg/embryo with 1.6 ng of Cas9 protein into single celled embryos according to standard methods . UninjecWild-type (WT) and mutant (either D132N or D145E) human cTnC proteins were cloned, expressed, and purified as previously described . Site-di2+ solutions ranging from pCa 8.0 (relaxing) to 4.0 (activating) were calculated utilizing a pCa Calculator , varying [Ca2+], pH 7.0. Anion of choice for the pCa solutions was propionate . Solutions were prepared at room temperature (20–21°C).Different Calculator . All sol–8 M free [Ca2+]) containing non-ionic detergent Triton X-100 (1% v:v). After incubating for 4 h at 4°C, skinned cardiac muscle preparations (CMPs) were transferred to 51% glycerol-relaxing pCa 8.0 solution (v:v), stored at −20°C, and utilized within 1 month. Strips of muscle, 0.8–1.0 mm in length and 0.15–0.25 mm in diameter, were mounted using aluminum foil T-clips to a force transducer on one end and a motor on the other end. CMPs were immersed in pCa 8.0 relaxation solution and the initial length (L0) was set at 2.1 μm sarcomere length measured by HeNe laser diffraction. Initial maximal Ca2+-activated tension was measured at pCa 4.0 (10–4 M free [Ca2+]).Left ventricular papillary muscles were harvested from porcine hearts obtained from a local abattoir and dissected into small muscle bundles . Dissect2+-activated tension was measured by immersing the CMPs in pCa 4. CMPs exhibiting residual force >40% (n = 2) were excluded from analysis. CMPs with residual force ranging from 8.4 to 39.4% were reconstituted by incubation for ∼5 min total (five incubations of ∼1 min each) at 21°C with one of four conditions: drops of pCa 8.0 solution containing 1.0 mg mL–1 of either 100% WT cTnC, 50% WT/50% D132N cTnC mix, 50% WT/50% D145E cTnC mix or 50% D132N/50% D145E cTnC mix. Tension recovery was evaluated by comparing the reconstituted maximal tension at pCa 4.0 to the initial maximal Ca2+-activated tension measured prior to cTnC extraction (P/P0) and ranged from 81% to 123%.Native (endogenous) cTnC was extracted by incubating CMPs in 5 mM CDTA solution (pH 8.4) for ∼1.5 h at 21°C as previously described . To eval2+-dependence of steady-state, isometric force was measured by incubating reconstituted CMPs in a series of Ca2+ solutions ranging from pCa 8.0 to 4.0 at 21°C. Normalized [Ca2+]-force data for each CMP were fit using non-linear least squares regression to a 2-parameter Hill equation to obtain parameter estimates for pCa50 and nHill, as described (pCa-Force: Caescribed .kTR): After force reached steady-state in each pCa solution, measurement of the rate of tension redevelopment was obtained by shortening CMPs by 20% L0, followed by rapid, 25% re-stretch, then release back to L0 (kTR) was obtained from each tension recovery time course as described previously  were computed in MatLab as previously described  as that measured experimentally for the same condition.Kinetics of Tension Redevelopment  was obtained by oscillating CMP length ∼0.2% Leviously .post hoc Student-Newman–Keuls test. Data are shown as mean ± S.E.Statistical analyses including non-linear regression were performed using SigmaPlot v.12.0. Data were tested for significant statistical differences using one-way ANOVA with Z score of 0.8) and a low decreased M-mode left ventricular ejection fraction (LVEF) of 48%. Given the family history, she was admitted to the hospital briefly with a diagnosis of DCM for initiation of medical therapy with captopril, furosemide and aspirin. She was followed closely as an outpatient, with electrocardiogram having signs of left ventricular enlargement and serial echocardiograms demonstrating gradually worsening left ventricular function, necessitating a steady escalation of her medical management (Z score of 8.55) and M-mode LVEF of 33%. She was being managed with enalapril, carvedilol, digoxin, furosemide and aspirin, and had been listed for cardiac transplantation due to her severely depressed heart function. She suffered a cardiac arrest while awaiting a suitable donor and expired at 14 months of age.The proband was a female delivered full term following an uncomplicated pregnancy that included two normal fetal echocardiograms. She had initially been well without symptoms referable to the cardiovascular system. She demonstrated no feeding intolerance, premature fatigue, irritability, pallor, cyanosis, increased work of breathing, tachypnea, excessive diaphoresis, or lethargy. She had a good appetite and breast fed without difficulty. Screening echocardiography was done at 18 days of age due to an older sibling with neonatal cardiomyopathy of presumed idiopathic etiology. This showed borderline enlargement of the left ventricle with a left ventricular end diastolic diameter (LVEDD) of 2.18 cm . Subsequent analysis of the family  revealed that these variants were inherited in trans  single gene variants across the entire exome that were shared by both affected siblings, and thus no other rare or pathogenic variants were found in other known cardiomyopathy genes in either of the siblings. The proband was sequenced to a mean depth of 102 independent reads per targeted base and her affected brother and their unaffected parents were sequenced to a mean depth of 44–49X. Greater than 10X coverage was achieved for 98% of the proband’s exome and 94% for the rest of family members’ exomes. Greater than 20X coverage was achieved for 96% of the proband’s exome and 88% for her other family members.Due to the strong family history, the proband underwent genetic testing with whole exome sequencing, which identified two variants of uncertain significance, D132N  and D145E , in the in trans . Both vain trans . Both ofin trans . In the in vivo system, we began by using CRISPR/Cas9-mediated gene editing to knockout TNNC1 in Xenopus. Loss of the TNNC1 gene did not prevent the early stages of development, and resulted in a dramatic cardiac phenotype in tadpoles consistent with DCM  or mixtures of cTnCs . The range of average of residual tension post-extraction was 18% ∼ 35%, with individual values ranging from 8.4 to 39.4%, indicating that the majority of native cTnC had been extracted from the myofilaments (2+ sensitivity of force generation (pCa50 = 5.32 ± 0.03), with a rightward shift of 0.11 pCa units compared with CMPs reconstituted with 100% WT cTnC (pCa50 = 5.43 ± 0.02) (2+ sensitivity of tension (pCa50 = 5.58 ± 0.04), with a leftward shift of 0.15 pCa units (2+ sensitivity of tension upon reconstitution of CMPs with a mixture of 50% D132N and 50% D145E (D132N/D145E) (nHill) is an indicator of cooperativity of thin filament activation. Compared to WT (nHill = 1.70 ± 0.20), only the WT/D132N mixture exhibited a significant change – an increase – in the apparent cooperativity (nHill = 2.60 ± 0.45) (CMPs reconstituted with a mixture of 50% WT and 50% D132N variant (WT/D132N) displayed significantly reduced myofilament Ca ± 0.02) . Consist ± 0.02) , we founCa units . InteresN/D145E) . AlthougN/D145E) . The Hil ± 0.45) .2+ sensitivity of tension for the 50%:50% combination of the two variant cTnCs (D132N/D145E) compared with 100% WT (kTR). SS is used as a metric of the overall number of cross-bridges, whereas kTR is used a metric of the kinetics of cross-bridge cycling at maximal Ca2+-activation and also informs about thin filament regulatory unit dynamics at submaximal Ca2+-activation. We found no significant difference in maximum SS values when comparing CMPs reconstituted with the variants  to those reconstituted with 100% WT  to those reconstituted with 100% WT (f+g) is approximately constant for all four conditions. To explore the possibility that the variants may have altered cross-bridge attachment (f) and detachment (g) rates even though the sum remained constant, we used a 3-state model of muscle regulation to evaluate the Ca2+-dependence of kTR  appear to be less curvilinear than for WT  are all lumped parameters that primarily inform about cross-bridge cycling (f and g) and thin filament regulatory unit dynamics including Ca2+ binding to and dissociation from cTnC (kON and kOFF). Computational modeling indicates that kOFF is, as expected, generally faster as Ca2+ sensitivity decreases , but the magnitudes of the changes were less; in other words, the combination of the two variants appeared to partially ameliorate the effects of the individual variants, as was also found for Ca2+ sensitivity of steady state isometric force  would have adverse consequences for Ca2+ regulation of contractility. However, in contrast to what might be expected from the family pedigree, our functional data indicate that these two variant cTnC proteins when present as a mixture of one variant with WT (as in the mother and father) significantly altered Ca2+ regulation of steady-state isometric force in opposite directions, while the combination of the two variants (as in the affected siblings) did not.Given the central role of cTnC in cardiomyocyte contraction, it is little surprise that a growing number of variants in the in silico predictors of variant effects – including SIFT, PolyPhen2 and CADD – predict that each of these variants is likely to be detrimental, they are both classified as variants of uncertain significance in ClinVar. The D132N variant has not been reported in the Genome Aggregation Database (gnomAD). The population frequency of the D145E variant is reported as 0.0001228 in gnomAD, and the Atlas of Cardiac Genetic Variations classifies this uncommon variant as “unlikely to be pathogenic.” This classification of D145E is consistent with individuals such as the proband’s mother, who is heterozygous and asymptomatic. The available evidence, therefore, suggests that D145E could possibly be a risk factor for cardiomyopathy in some cases, but does not appear to be disease-causing when present in the heterozygous state. The two affected siblings presented here, in contrast to the parents, have D145E in trans with the D132N variant. When viewed in this light, the D145E population frequency is well within an expected range for a recessive disease.Although multiple TNNC1 alleles, we performed knockout experiments in Xenopus, revealing a dramatic DCM phenotype in tadpoles  and it was the only variant found in a screen of fifteen HCM-susceptibility genes . In contTNNC1 can affect skeletal muscle function because cTnC is also expressed in slow skeletal muscle. The D145E mutant was reported to increase slow skeletal muscle ATPase activity at low and high calcium concentrations in reconstituted myofibrils  coordinating residues (+Z position) in site IV. While the regulatory site for Ca2+ binding is site II in the N-domain of cTnC, our prior results indicate that there is communication between the C- and N-domains  had the opposite effect of decreasing Ca2+ sensitivity  values, the latter generally reflecting cooperative processes associated with isometric force generation. While incorporation of the two mutants appeared to normalize opposite effects of the two variants on Ca2+ dependence of isometric force, what is difficult to reconcile is the severe DCM pathology associated with the presence of the two mutations.Both variants are located in the C-domain of cTnC . The mis-domains  such tha-domains . The ressitivity  even thositivity . In genesitivity , and tha2+ sensitivity commonly found in HCM (increased) and DCM (decreased) is generally well-accepted  or the parents (WT/D132N or WT/D145E), along with the control condition (100% WT). Previous studies showed that D145E cTnC dissociates more slowly from the thin filament compared to WT cTnC indicating that D145E has a higher binding affinity for the thin filament  is considered a common feature of DCM and HCM . In the sitivity . Studiestization . AlthougnHill for D132N/D145E (kTR (kTR data (kTR relations (kTR relation, and alters contractile kinetics (kTR in f and g in Alternative possibilities exist because the two mutations are not present in the same protein. In the affected siblings, the two variant proteins are expected to be distributed randomly in thin filaments, and differences in function might affect communication between adjacent regulatory units when they contain different cTnCs. The lack of change in 2N/D145E  suggests45E (kTR . Both thkTR data  and the elations  suggest We and others have previously suggested that troponin subunits may participate in cellular functions that extend beyond contractile regulation in the sarcomere . In factTNNC1 in siblings with early onset familial DCM. We provide functional evidence that both variants separately lead to abnormal myofilament Ca2+ sensitivity in which the variants D132N and D145E are, respectively, associated with reduced or increased myofilament Ca2+ sensitivity relative to WT, while these effects compensated for no change relative to WT when the two variants were combined. Analyses of activation-dependence of kTR suggest that both variants have similar effects on contractile kinetics, although there was no significant change in maximal kTR, and that the combination of the two variants again partially ameliorated the effects of the individual variants. None of the observed changes in mechanics, however, can explain the clinical outcomes in this family. Further studies are needed to understand precisely how these variants combine to result in a severe DCM phenotype.In summary, we report the association of compound heterozygous variants in SL is part owner of Qiyas Higher Health, a startup company unrelated to this work.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The studies involving human participants were reviewed and approved by the Institutional Review Board of Yale University School of Medicine. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. The animal study was reviewed and approved by the Yale University Institutional Animal Care and Use Committee.JP and SL conceptualized the study and oversaw project administration. ML-V, JJ, WJ, EM, JT, MS-M, LJ, EH, DP-M, MK, and ED contributed to the data curation. PC contributed to computational data analysis and modeling. ML-V, JJ, JT, EM, PC, SL, and JP wrote the manuscript. All authors reviewed and approved the final version of the manuscript.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.


Calcium is one of the most important signaling factors in mammalian cells. Specific temporal and spatial calcium signals underlie fundamental processes such as cell growth, development, circadian rhythms, neurotransmission, hormonal actions and apoptosis. In order to translate calcium signals into cellular processes a vast number of proteins bind this ion with affinities from the nanomolar to millimolar range. Using classical biochemical methods an impressing number of calcium binding proteins (CBPs) have been discovered since the late 1960s, some of which are expressed ubiquitously, others are more restricted to specific cell types. In the nervous system expression patterns of different CBPs have been used to discern different neuronal cell populations, especially before advanced methods like single-cell transcriptomics and activity recording were available to define neuronal identity. However, understanding CBPs and their interacting proteins is still of central interest. The post-genomic era has coined the term “calciomics,” to describe a whole new research field, that engages in the identification and characterization of CBPs and their interactome. Secretagogin is a CBP, that was discovered 20 years ago in the pancreas. Consecutively it was found also in other organs including the nervous system, with characteristic expression patterns mostly forming cell clusters. Its regional expression and subcellular location together with the identification of protein interaction partners implicated, that secretagogin has a central role in hormone secretion. Meanwhile, with the help of modern proteomics a large number of actual and putative interacting proteins has been identified, that allow to anticipate a much more complex role of secretagogin in developing and adult neuronal cells. Here, we review recent findings that appear like puzzle stones of a greater picture. In order to fulfill these tasks resting cells actively expel calcium ions from the cytosol with the help of Ca2+ transport ATPase (PMCA) and Na+/Ca2+ exchanger (NCX) that are localized in the plasma membrane and transport Ca2+ against a massive concentration gradient into the extracellular space  or the mitochondrial Ca2+ uniporter. Activation of Ca2+-dependent signaling pathways can be initiated by membrane depolarization or extracellular signaling molecules that activate voltage- or ligand-activated calcium channels in the plasma membrane or via intracellular messengers that cause the release of Ca2+ from intracellular stores, mainly via the 1, 4, 5- triphosphate receptor (IP3R) or the ryanodine receptor (RyR) from the ER or SR  was discovered in an “expression dictates function” manner. Wagner et al.  generate2+ with an overall half maximal affinity of approximately 25 μM, a relatively low affinity compared to calbindin [with a (Ca2+) 0.5 of approx. 1 μM] or calretinin [with a (Ca2+) 0.5 of approx. 1.5 μM; Rogstam et al., Secretagogin is a hexa EF-hand protein that binds Ca2+ binding, but not upon Mg2+ binding, suggesting that SCGN belongs to the “sensor” family of Ca2+ -binding proteins  channels, that, when activated, induce nuclear translocation of the Sp1 transcription factor to initiate secretagogin transcription. Sp1 has previously been shown to also mediate upregulation of the CBP calmodulin in response to an insulin stimulus  in LC-MS/MS. SNAP-25 is one of the components of the soluble N-ethylmaleimide-sensitive-factor attachment receptor  complex, which is the main complex for membrane fusion events  Tau was initially demonstrated by GST-pull downs from pancreatic cell lines and later confirmed from rat brains  and used micropunches from that area in co-immunoprecipitations. Similarily, Romanov et al. , in the CA1-CA3 of the hippocampus, and outer cortex around the posterolateral cortical amygdaloid area. This specific expression of SCGN anticipates an important role in the mechanisms of olfaction, vision, activity of the hypothalamus, and memory formation in hippocampus.As mentioned before, it is apparent, that expression of secretagogin frequently occurs in cell clusters, shown for rat brain by Maj et al. , implica+ clusters were often found in direct contact with GM130 clusters, a marker of the cis-Golgi network. Adding to the fact, ultracentrifugation experiments using sucrose density centrifugation of rat hippocampal tissue extracts revealed that SCGN immunoreactivity was detectable in the membrane-fraction as well as in the cytosolic fraction  in the inner nuclear layer with neurites extending into the inner plexiform layer. The expression of SCGN shows significant overlap with calbindin and calretinin, but not parvalbumin expression.A detailed characterization of SCGN-positive cells in zebrafish retina has been provided by Dudczig et al. . SCGN stSecretagogin has been demonstrated to label several subtypes of cone bipolar cell in the mouse, rat, and rabbit retina  neurons in the dorsal horn of mouse and human that coexpress calcitonin gene-related peptide (CGRP) and in rat DRGs not CGRP. SCGN-positive neurons were further found in the mouse, rat and human dorsal horn implicating a role of these neurons in processing of sensory information including pain  from the anterior pituitary gland and ACTH then activates the adrenal glands to release steroid hormones like cortisol, the primary stress hormone. Romanov et al.  cells on one hand are directly innervated by CRH+ cells  release into the cerebrospinal fluid. In the locus coeruleus, CNTF binds to its cognate Trk receptor on NE+ neurons initiating a signaling cascade of ERK1 activation (a secretagogin-interactor) and subsequent tyrosine hydroxylase (TH) phosphorylation thereby stimulating its enzymatic activity. Alpár et al.  coordinates vigilance, alertness and behavioral responses to acute stress facilitated by noradrenergic afferents originating in the locus coeruleus. These norepinephrinergic , major depression, and serious metabolic consequences including hypoglycemia.The crucial roles of secretagogin in cellular function as defined by today already put it into an important position in the context of pathological developments.Clinical data have suggested links between Type II diabetes (T2D) and AD, which some researchers have proposed to call a form of “Type III diabetes.” Animal models have shown that T2D promotes the formation of amyloid-β plaques, tau phosphorylation and neurofibrillary lesions, however in humans this evidence is scarce or ambigous. What has been shown, is that brain insulin resistance with an impaired signaling response to insulin is also a pathomechanism in AD  protect neurons from detrimental Ca2+ overload as it occurs for instance in ischemic conditions . Future research will prove, if secretagogin is engaged in the release of further hormones, or eventually even neuropeptides or other neurotransmitters. With this aim in mind, single-cell transcriptomics can be a suitable methodology for expression profiling of SCGN+ cells. However secretagogin’s protein interaction network, as known by today, is vast. Therefore it can be assumed, that in those selected cell types, where it is expressed, it might function as a kind of Ca2+-dependent switch or moderator for more cellular processes including cytoskeletal rearrangements and modulation of cellular stress. In order to delineate individual mechanisms, further functional investigations with known interactors will have to be undertaken.Secretagogin’s role as a CaAll authors were involved in the conception of the article, MM and VT wrote the final version of the manuscript.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.


We demonstrate that Ca2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP‐PG 722 is mediated by a Na+/Ca2+ exchanger critical for virulence. Using the fluorescent Ca2+ probe Fura 2‐AM, we demonstrate that Ca2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca2+ entry is a Na+/Ca2+ exchanger. In silico analysis of the Psav DAPP‐PG 722 genome revealed the presence of a single gene coding for a Na+/Ca2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P.syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity  and virulence (ptz) genes was reduced in this Psav‐cneA mutant. Complementation of the Psav‐cneA mutation restored both Ca2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.In a number of compatible plant‐bacterium interactions, a rise in apoplastic Ca The cytosolic Ca2+ levels are influenced by the difference in its intracellular‐to‐extracellular concentration   , the causal agent of olive knot disease.Although the molecular mechanisms that cause the cytosolic fluctuations of CaPsav survives as an epiphyte in the phyllosphere penetrating its host through wounds  that the plant perceives through specific receptors and via which it activates plant immunity have been extensively studied in the last decades  whether an increase in external Ca2+ resulted in an increase in the cytosolic Ca2+ concentrations in Psav. We find that the cytosolic Ca2+ concentrations rapidly increase in response to external Ca2+ concentration in the medium  influences Ca2+ entry. However, addition of IAA or L‐tryptophan to the incubation buffer did not significantly alter Ca2+ entry in Psav DAPP‐PG 722 cells , which belongs to the ChaA antiporter superfamily  that encompasses the PRK10599, caca2 and Na+/Ca2+ exchanger protein domains  dicitrate transport system. A comparative phylogenetic analysis of the nucleotide sequences of the cneA gene was performed using the Geneious resource  of housekeeping genes  have a different position in the cneA gene tree than in the MLSA tree  infiltrated in the non‐host tobacco, (ii) inoculated on 1‐year‐old wounded olive plants. When infiltrated in tobacco leaves, the Psav‐cneA mutant was unable to induce an HR . Moreover, we found that the Psav‐cneA mutant strain was unable to proliferate in olive plants in comparison with the Psav wild type  or a mini‐Tn7 transposon encoding cneA from its own promoter and inserted in the chromosome of the mutant strain (Psav‐cneA mutant [miniTn7::cneA]). Ca2+ entry into the complemented strains was restored to more than 60%, in the absence  was not significantly different to that of Psav wild type  in olive plants was comparable to the Psav wild type   was detected in the Psav‐cneA mutant strain 48 h after incubation at static conditions, while no formation was detected in the wild‐type strain under these conditions  were not able to swim as the Psav wild type under the conditions tested, and they only recovered partially the capacity to produce EPSs (Table In order to determine if the Ss Table . Other pSs Table  and the Psav infection and in particular when the bacterium reaches the apoplast (intercellular spaces and xylem), the abundant presence of Ca2+  and virulence (ptz) genes again confirming that Ca2+ is an important host signal that is perceived by the bacterium. We find that the Ca2+ influx reaches its maximum levels when the energy supply is limiting. In fact, the presence of glucose, fructose, sucrose or ATP inhibited calcium entry entirely.The importance of Can et al. , who chaPsav virulence was largely linked to the bacterial secretion of the phytohormones IAA and cytokinins at the site of infection, which stimulates olive cell activity to produce new tissue and gives rise to knot development  probably due to the suppression of type III secretion system. Here, in fact, it was noted that β‐galactosidase activity associated to hrpL and hrpA promoters was statistically reduced in the Psav‐cneA mutant. Wei et al. , the expression was driven by the lac promoter). However, it should be emphasized that complementation of the Psav‐cneA mutation restored both Ca2+ entry and pathogenicity in olive plants.Our results obtained with the deletion mutant were confirmed by plasmid and chromosomal mutant complementation, except for restoration of the HR. AHLs production and biofilm formation were also not recovered. This, may be due to the different expression levels of et al., Escherichia coli was grown at 37 °C in LB broth. Chromobacteriumviolaceum strain CVO26 , agarose gel electrophoresis, DNA fragment purification, ligation with T4 ligase, end filling using the Klenow enzyme and k et al. . Plasmid2+ levels were determined using a fluorimetric method, which employed the fluorescent probe Fura 2‐AM . Approximately 5 ×  106 cells of Psav DAPP‐PG 722 grown at 27 ± 1°C for 16 h in LB broth to a stationary phase, were suspended in 0.12 M Tris (pH 7.8) and 2 mM EGTA. At 200 s after incubation at 25 °C, 2 mM CaCl2 was added to stop the EGTA effect as reported by Grynkiewicz et al.  supplemented with 2 mM Fura 2‐AM (dissolved in DMSO) or in HBSS buffer supplemented with 2 mM Fura 2‐AM and different carbon sources  or ATP 50 µM. The fluorescence intensities of Fura 2‐AM  were monitored with a spectrofluorophotometer . The cytosolic Ca2+ concentration was calculated following the formula reported by Grynkiewicz et al.  was created as follows. An internal 305 bp fragment of the cneA gene was amplified from Psav DAPP‐PG 722 genomic DNA using the primers cneA For and cneA Rev .A genomic null mutant of the Naev Table , generatnt Table  after tr Psav‐cneA mutant with a plasmid encoding the cneA gene was performed as follows. The complete sequence of the cneA open reading frame (ORF) with its ribosome binding site was amplified from Psav DAPP‐PG 722 genomic DNA using primers cneA compl 1 and cneA compl 2 . The amplified fragment was purified from an agarose gel using the EuroGOLD Gel Extraction Kit  following the instructions of the manufacturer. After A‐tailing , the fragments were cloned in pGEM‐T Easy vector  and sequenced at Macrogen Europe. Having verified the correctness of the sequence, the cneA ORF was excised from pGEM‐T Easy Vector using XhoI and SpeI and cloned in the corresponding sites of the plasmid pBBR MCS‐5. The resulting plasmid   and the primers PromAP_Fw and AP_Rev  (Table 7 transposon into the correct site was verified using the primers GlmS_savastanoi (hybridizing at the 3′ of the glmS gene) and the Tn7Rev primer (hybridizing at the Tn7R end of the integrated plasmid) . For each bacterial strains, the relationship between the number of cells (log10 transformed) and the hpi was investigated by means of a second‐order polynomial model. Likelihood ratio test was used to assess the differences between wild type and Psav‐cneA mutant strains under R statistical environment  plants. To prepare the inoculum, the strains were grown in NA at 27 °C for 24 h, resuspended in sterile deionized water and spectrophotometrically adjusted to 108 CFU/mL. About 10 µL of the bacterial suspensions or water (control) was infiltrated into the mesophyll of tobacco leaves using a needleless syringe. The appearance of the HR was scored at 24 hpi.The HR assay was carried out in et al.  grown in KB broth, were diluted to OD600nm = 0.1 and loaded in a 96‐well plate . Plates were incubated under static or shaking conditions and biofilm formation was quantified by measurement of the A595 after 24 h and 48 h after crystal violet staining  and Psav‐cneA mutant (miniTn7:: cneA)  was placed in wounds (five per plant) made in the bark of olive plants using a sterile scalpel as previously described  plants inoculated with the strains 2+ entry promotes the expression of pathogenicity  and virulence (ptz) genes of Psav, transcriptional fusions of their promoters were constructed with LacZ reporter gene. For amplification of the iaaM and ptz promoters, the regions upstream of the iaaM and ptz ORFs  were amplified by PCR using primers iaaM For, iaaM Rev, ptz For and ptz Rev . The cultures were harvested by centrifugation, washed twice with 10 mM MgCl2 and the cells were transferred to Hrp medium  were used as negative controls. To determine the activity associated exclusively to the promoter fusions to lacZ, the background activity detected in the control strains was subtracted from those obtained for each of their corresponding transformants.To verify whether Ca Miller,  and modi Miller, . Psav DAConflict of interest: The authors have declared that no conflict of interest exists.Research Involving Human Participants and/or Animals: This article does not contain any studies with human participants or animals (vertebrates) performed by any of the authors.Informed consent: Informed consent was obtained from all individual participants included in the study.Fig. S1 Maximum likelihood tree based on the nucleotide sequence of the cneA gene showing the phylogenetic relation within the P. syringae complex. Phylogroup (PG) designations are indicated on the appropriate branches. Numbers at branching points are bootstrap percentages based on 1000 replications. Psy = Pseudomonas syringae; Psav = Pseudomonas savastanoi; Pca = Pseudomonas cannabina and P = Pseudomonas.Click here for additional data file.Fig. S2In vitro growth on KB medium of Pseudomonas savastanoi pv. savastanoi (Psav) DAPP PG 722 (wild type [wt]) and the calcium exchanger Psav mutant (Psav cneA mutant). Number of cells (mean ± SE) and fitted polynomial models of wt  and Psav cneA mutant . Standard error bars are not visible in the plot as their values are smaller than the dimensions of the closed squares and open circles.Click here for additional data file.


In the present study, we compared the strategies for cold acclimation between stenothermic tilapia and eurythermic goldfish. Whole-body calcium content was more prominently diminished in tilapia than in goldfish after long-term cold exposure. This difference can be attributed to alterations in the transportation parameters for Ca2+ influx, i.e., maximum velocity (maxV) and binding affinity (1/mK). There was also a significant difference in the regulation of Ca2+ efflux between the two fishes. Transcript levels for Ca2+ related transporters, including the Na+/Ca2+ exchanger and epithelial Ca2+ channel, were similarly regulated in both fishes. However, upregulation of plasma membrane Ca2+ATPase expression was more pronounced in goldfish than in tilapia. In addition, enhanced Na+/K+-ATPase abundance, which provides the major driving force for ion absorption, was only detected in tilapia, while upregulated Na+/K+-ATPase activity was only detected in goldfish. Based on the results of the present study, we have found that goldfish and tilapia differentially regulate gill epithelial plasma membrane Ca2+-ATPase (PMCA) expression and Na+/K+-ATPase activity in response to cold environments. These regulatory differences are potentially linked to more effective regulation of Ca2+ influx kinetics and better maintenance of whole body calcium content in goldfish than in tilapia.The body temperatures of teleost species fluctuate following changes in the aquatic environment. As such, decreased water temperature lowers the rates of biochemical reactions and affects many physiological processes, including active transport-dependent ion absorption. Previous studies have focused on the impacts of low temperature on the plasma ion concentrations or membrane transporters in fishes. However, very few  Despite this relatively low availability from the environment, constitutive increase of whole-body calcium content is an important factor during development that affects survival  on the apical membrane (+/Ca2+ exchanger (NCX)  is thought to be mediated by paracellular transport through the gill epithelium  , and enemembrane . In contithelium , which i10 = 2–3) than does permeability (Q10 = 1–2) between fish cells (2+ levels were previously reported to be decreased in both carp (Cyprinus carpio) (Carassius auratus)   and in gauratus) . However of carp  and in h of carp . Other a2+ regulation, resulting in growth inhibition or even death of fish . The tilapia, a tropical species, lives exclusively in warm temperature waters, while the goldfish is a cyprinid that has the ability to survive in a wider temperature range. Based on this key distinction, we proposed that these two species may exhibit different compensatory mechanisms for whole-body Ca2+ regulation during acclimatization to a hypothermic environment. Whole-body Ca2+ influx, efflux and net flux rates, as well as kinetic constants (mK and maxV) for Ca2+ influx were examined in these two species after acute and chronic exposures to low temperature. In order to assess potential mechanisms by which Ca2+ flux was modulated by low temperature, we probed the mRNA expression levels of the major Ca2+ transporters, ECaC, NCX, and PMCA, on the gill in both fishes after cold acclimation. Furthermore, we measured the activity, quantity and sensitivity of Na+/K+-ATPase, one of the major active transporters involved in maintaining epithelial cell ionic gradients, including those that drive Ca2+ transport , weighing 5–10 g, were obtained from a laboratory stock, and goldfish (C. auratus), weighing 5–12 g, were obtained from local aquariums. All fish were reared in aerated local tap water [(Ca2+) = 0.2 mM] at 27–29°C with a 12L:12D photoperiod. The fish were fed a daily diet of commercial pellets . All animal experiments and handling procedures were overseen by the Institutional Animal Care and Use Committee (Protocol No. 104078) at National Cheng Kung University, Taiwan. The 3R policy  for animal handling was followed, according to guidance from the Ministry of Science and Technology, Taiwan.Juvenile tilapia  to measure calcium and sodium concentrations. Calcium and sodium standard solutions  were used to create standard curves for the measurements.At the end of the 2-week acclimation periods, fish were euthanized with an overdose of anesthetic . Body weights were measured, and then fish were dried and digested with 13.1 N HNO2+ flux rates were determined according to previous methods  into aerated tap water [(Ca2+) = 0.2 mM], such that the specific activity of the media was 510,000–540,000 cpm μmole-1. The tracer media (100 ml) in flux chambers was gently aerated without filter equipment, and the water quality (pH and ion concentration) showed no significant change during the period of incubation. Preliminary experiments indicated that the radioisotope adheres nonspecifically to the surfaces of flux chamber and the fish, but this non-specific redistribution of material was saturated within the first 10 min of incubation. In addition, the plot of accumulated radioisotope in fish against time was linear over the first 6 h, and the calculated influxes at each period within the first 6 h were constant. Therefore, water samples  were collected 10 min and 6 h 10 min after incubation in order to exclude the effects of non-specific adsorption. Counting solution  was added to water samples from the 45Ca2+ media, and then radioactivity was counted with a β-counter . The Ca2+ influx  was determined by the formula:Ca methods  with somi and [R]f (cpm mL-1) refer to initial (10 min) and final (6 h 10 min) concentrations of radioactivity in the tracer media, Vi and Vf (mL) refer to volumes of the initial and final tracer media, [Ca2+]i and [Ca2+]f were the initial and final concentrations of Ca2+ in the tracer media, t (6 h) is incubation time, and W is body weight of fish in grams.where [R]2+ net flux  was determined by the formula:Water samples  were collected 10 min and 6 h 10 min after the beginning of the incubation. The Caeff, nmole g-1 h-1) were determined by the equation:Efflux rates  at room temperature in order to remove extra Ca2+ ions from the body surface. Flux chambers were filled with tracer media (20 ml) that was prepared by adding CaSO4 to artificial Ca2+-free solution with final concentrations of Ca2+ ranging from 0.01 to 5 mM. 45Ca2+-labeled CaCl2 stock solutions were also added to the prepared Ca2+ solutions so that the same Ca2+/45Ca2+ ratio was maintained in each solution. Flux experiments and calculation of Ca2+ influxes at different external Ca2+ levels were performed for 2 h. Eadie–Hofstee plots were used to calculate the values of mK (Michaelis constant) and maxV (maximum velocity) . These values were applied to the Michaelis–Menten equation:The protocol for measuring Caeriments  with som2+] is the concentration of Ca2+ in the tracer media. The influx kinetics experiments were repeated three times.where [Ca+/K+-ATPase activity was measured as previously described  with or without 0.5 mM ouabain for 30 min at 37°C. The reactions were stopped by 8.5% trichloroacetic and were centrifuged 4000 × g for 10 min at 4°C. Supernatant (500 μl) was mixed with 1000 μl colorization buffer  for 30 min at 20°C. The concentration of inorganic phosphate was determined by spectrophotometry (Hitachi). Protein concentration was determined by the Bradford assay, and BSA was used as a standard. An inorganic phosphate standard curve was prepared with Na2HPO4 in concentrations ranging from 0 to 80 μg/ml. The Na+/K+-ATPase activity was calculated as the concentration differences of inorganic phosphate inhibited by ouabain.Gill Naescribed . Fish gi+/K+-ATPase  or β-actin  primary antibodies and HRP-conjugated goat anti-mouse  or HRP-conjugated goat anti-rabbit  secondary antibodies were used for western blotting analysis as previously described (+/K+-ATPase sensitivity was measured according to an earlier report (50) against Na+/K+-ATPase activity.The Naescribed . Immunobr report , where iNCX1b , ECaC , PMCA (2+ ATPaseplasma membrane Ca), and internal controls of ribosomal protein L7 (RPL7) for tilapia or 18S ribosomal RNA (18S rRNA) for goldfish are shown in Supplementary Table S1. Experiments were repeated three times, and groups were compared by Student’s t-test.Quantitative  RT-PCR was performed and analyzed as described in a previous study . Gill cDIn situ hybridization was performed following a previous study  mixtures. The rehydrated slides were incubated in the pre-hybridization buffer for 2 h and hybridized with RNA probes at 70°C overnight. The hybridized slides were washed with mixture of hybridization buffer and 20X SSC, followed by 0.2X SSC at 70°C. Next, the gill slides were washed with mixture of 0.2X SSC and PBST at room temperature. The washed slides were transferred into blocking buffer containing 2% normal goat serum and 2 mg/ml BSA for 2 h at room temperature. The samples were incubated with alkaline phosphatase-conjugated anti-DIG antibody  overnight at 4°C. The tissues were washed with PBST six times for 15 min each. The washed samples were then transferred into alkaline Tris buffer, containing 0.2 M Tris HCl, pH 9.5, 0.1 M MgCl2, 0.2 M NaCl and 0.2% Tween 20. Tissues were labeled with a mixture of 225 μg/ml NBT (Nitro Blue Tetrazolium) and 175 μg/ml BCIP (5-Bromo 4-Chloro 3-indolyl Phosphate). The labeling reaction was stopped with stop solution, containing 1 mM EDTA, 0.1% Tween 20 in 1X PBS, pH 5.5. After in situ hybridization, the gill slides were washed with PBST three times for 15 min each; then slides were incubated with 3% BSA in PBST for 2 h at room temperature. Slides were transferred into Na+/K+-ATPase  antibody and incubated overnight at 4°C. The gill sections were washed with PBST six times for 10 min each, and then incubated with Alexa 488-conjugated goat anti-mouse antibody  for 2 h at room temperature. The stained gill slides were mounted with VECTASHIELD antifade mounting medium. Images were captured with a Zeiss LSM 780 confocal microscope and the colocalization of NKA and PMCA signaling was determined using the profile function in Zen2010 software (Zeiss). The Na+/K+-ATPase, PMCA or double-stained cell numbers per sections were counted and recorded. The cell number was recorded from one middle section per gill filament, three gill filaments per fish and three fish from each experimental group. Values are expressed as mean ± S.D. Differences were considered significant for p ≤ 0.05, n = 3, as calculated by Student’s t-test. Fluorescence intensities of Na+/K+-ATPase immunostaining and DAPI were measured using the histology function in Zen2010 software (Zeiss). The Alexa 488 intensity was normalized to DAPI intensity in the same area of the gill section.us study . Brieflypost hoc Tukey’s multiple comparison was used to compare the Ca2+ influx, net flux and efflux kinetics among groups in the same species. Na+/K+-ATPase expression, activity, Ca2+ transporter expression and cell numbers were compared by Student’s t-test. P < 0.05 was considered statistically significant.All values are shown as mean ± S.D. Data were first analyzed with Shapiro–Wilk’s test for normality and Levene’s test for equality of variance before further analysis. One-way ANOVA with n = 8 per group) were directly transferred to 25°C and 15°C tracer media, after which the Ca2+ flux rates were measured.Both tilapia and goldfish were acclimated in tap water at 25 ± 1°C for 2 weeks. After acclimation, fish (n = 8) was used to measure Ca2+ influx, efflux and net flux at the acclimation temperature. The second group of fish (n = 7 for tilapia and n = 9 for goldfish) was digested and used for the whole-body Ca2+ concentration determinations. The third group of acclimated fish (n = 4) was used to determine the Ca2+ influx kinetics at 25°C directly.Tilapia and goldfish were randomly divided into three groups per species and the temperature was gradually decreased by 2°C per day from 25°C to 15°C, after which the fish were acclimated for 2 weeks with the filter equipment. During the acclimation period, the fish from both temperature groups were not fed. During the cold acclimation, tilapia exhibited decreased motility with 10–20% mortality, but goldfish did not. At the end of acclimation, the first group of each species  with recirculated dechlorinated local tap water for 3 days in order to exclude the handling effects . However, the calcium reduction was less severe in goldfish (8.7% reduction compared with the 25°C control) than in tilapia (17.2%). The same was true for the reduction in sodium content (11.6% in goldfish and 25.2% in tilapia). This result indicated that goldfish can more effectively maintain whole-body calcium and sodium contents in cold environments.After long-term cold acclimation, whole-body calcium and sodium contents were reduced significantly both in goldfish and tilapia . However, the reduction in tilapia  was more extreme than in goldfish . This result indicated that the Q10 between 25°C and 15°C for Ca2+ influx was higher in tilapia than goldfish. After long-term acclimation, goldfish showed a complete recovery of Ca2+ influx. However, no significant compensation for reduced Ca2+ influx was detected in tilapia after long-term cold acclimation.Upon acute exposure to low temperature (15°C), Ca2+ efflux rates compared with the 25°C control group . In contrast, significant increases in Ca2+ efflux were detected in tilapia after both acute and long-term cold exposure .During acute and long-term cold exposure, goldfish did not show any significant change in Ca2+ influx, goldfish maintained a positive but very low level of Ca2+ net flux upon acute exposure and a complete recovery after long-term acclimation . However, the decrease of Ca2+ net flux upon acute cold exposure in tilapia did not significantly recover after long-term exposure .Similar to Ca2+ influx rates, determined at different Ca2+ concentrations, displayed typical Michaelis–Menten kinetics in both goldfish  and tilapia  after acclimation at two different temperatures. As shown in Table 2, 2 weeks of hypothermic acclimation resulted in a decreased mK (Michaelis constant) and an increased maxV (maximum velocity) for Ca2+ influx in goldfish. However, neither the mK nor the maxV were changed after the same treatment in tilapia. These results indicated that binding affinity and maximum velocity for Ca2+ transport are both increased after cold acclimation in goldfish but not in tilapia.Ca+/K+-ATPase is one of the most important molecules in the control of active ion absorption in freshwater fish . However, Na+/K+-ATPase activity was enhanced in cold acclimated goldfish . Interestingly, both cold acclimated tilapia  and goldfish  showed increased activity when measurements were made at 25°C and 35°C. However, only cold acclimated goldfish showed increased activity with a 15°C reaction temperature. In order to test whether Na+/K+-ATPase has similar ion binding affinity after cold acclimation, Na+/K+-ATPase sensitivity, as determined by IC50 of ouabain was measured (Table 3). The goldfish showed a significant decrease in ouabain IC50 after cold acclimation.Nater fish . Changeslimation . TherefoNCX1b, ECaC, and PMCA were determined after cold acclimation in tilapia and goldfish. The results showed that the expression levels of NCX1b but not ECaC are increased both in tilapia and goldfish after cold acclimation . Interestingly, the expression of PMCA was increased significantly only in goldfish , suggesting that increase of PMCA mRNA may be an especially important factor for successful cold acclimation.The calcium related transporters, NCX1b, ECaC, and PMCA are located on the gill and are thought to constitute the major driving force for calcium absorption in freshwater fish . Increas+/K+-ATPase immunostaining. The fluorescence intensity was increased in tilapia after cold acclimation , suggesting that the increase of Na+/K+-ATPase abundance detected by western blotting in tilapia may due to increased protein density per cell instead of cell number. In addition, the transcription of PMCA, which is the major Ca2+ active transporter, was increased significantly in goldfish but not in tilapia after cold acclimation. Previous studies have shown that PMCA and ECaC-expressing cells are not completely colocalized in zebrafish embryos  but not in tilapia after cold acclimation.Surface area and cell numbers of gill ionocytes were previously reported to be increased after cold acclimation in goldfish . Therefo embryos . Therefo2+ balance in eurythermic and stenothermic teleosts. The major findings are as follows. (1) Upon acute cold exposure, stenothermic tilapia showed a higher Q10 (3.1) for whole-body Ca2+ influx than goldfish (1.7). (2) After long-term cold acclimation, goldfish recovered Ca2+ net flux due to compensatory increases in influx. However, recovery of influx was not detected in tilapia. (3) After long-term cold acclimation, goldfish showed compensatory increases in maxV and decreases in mK for Ca2+ influx, while no compensation was observed after cold acclimation in tilapia. (4) This compensatory response in goldfish may be related to the observed upregulation of Na+/K+-ATPase activity and PMCA expression, as well as the increased number of PMCA-expressing cells.This study is the first to compare the effects of hypothermic exposure on the whole-body Ca10 of Ca2+ influx and lack of recovery for net uptake in tilapia indicated that the Ca2+ active transport mechanism is more sensitive to low environmental temperature in tilapia than in goldfish. Acute hypothermic effects on enzyme activities have been well-studied in various cells or tissues. Homeotherms and poikilotherms were compared on the basis of their mitochondrial succinate oxidase activities in rat and trout livers  and subtropical tilapia (Tristramella simonis simonis) intestinal alkaline phosphatase  was more sensitive than eurythermic shiner (Notropis cornutus) upon acute cold exposure. Therefore, our current data agree with the previous literature, suggesting that animals living within a narrower range of temperatures are generally more sensitive to temperature fluctuations.Upon acute hypothermic exposure, the larger Qt livers , or in at livers . Stenothsphatase . Based osphatase . In addi2+ influx from the environment, and this process relies on energy-consuming transcellular transport mechanisms  was detected in goldfish after long-term cold acclimation. Several mechanisms have been proposed to compensate for reduced enzyme activity during cold acclimation. These include upregulation of enzyme density, post-translational modifications or induced synthesis of kinetically distinct protein variants . However, this observation should only account for the increase of maxV of Ca2+ influx but not the affinity. Although there is still no direct evidence showing that different Ca2+ transporter isoforms participate in cold acclimation, a few lines of evidence have suggested it may be an efficient mechanism  and goldfish, respectively. However, the isoforms of PMCA in other Teleostei species are known to be highly homologous (more than 70% identity of nucleotide sequences). Since tilapia (O. mossambicus) and goldfish are not commonly used model species, the sequences of PMCA isoforms are not available. Therefore, our data cannot exclude the possibility that upregulation of one or several PMCA variants may be used as a strategy for goldfish acclimation. Furthermore, identification and enzymatic characterization of the specific isoform of PMCA that is regulated in response to cold exposure in goldfish still needs further exploration.The gill has been shown to be the major organ responsible for whole-body Cachanisms . In the variants . We demoechanism . First, echanism . Second,re curve  and vari2+/Ca2+ ATPase  in order to maintain membrane fluidity (or viscosity) , and thi+ ATPase  and incrion rate  can be dion rate . The hom2+ efflux was observed from tilapia than from goldfish upon acute and long-term cold exposure. 2+ influx, but it is also the major site of efflux in freshwater tilapia. However, the mechanism for Ca2+ excretion from the branchial is still not fully described , but not changes in sensitivity. These observations suggested that tilapia acclimate to the cold temperature by simply increasing expression of the same protein isoform used at warm temperature . However, this isoform may not perform well in cold temperatures, which could explain why increased activity cannot be detected at a cold reaction temperatures . In contrast to tilapia, Na+/K+-ATPase quantity in goldfish does not change when detected by western blotting, but shows an increase in cell numbers and activity after cold acclimation . The inability to detect differences by western blotting may be due tissue dilution effects. If the total cell numbers are altered after cold acclimation (gill remodeling), the overall tissue increase in Na+/K+-ATPase abundance may not be sufficient to observe by immunoblotting. It was previously suggested by Flik et al. .Upon cold temperature exposure, upregulation of Na in carp  and gold in carp , indicatk et al.  that car2+ influx maxV. Together, our data and previous studies suggest that effective cold adaptation may require changes in basic protein folding and activities, instead of simple increases of protein amounts.The increase of PMCA in goldfish was tightly associated with upregulation of CaSupplementary Information Files.All data generated or analyzed during this study are included in this published article and its F-IL and P-PH designed the study. T-YH, C-YWu, H-CT, Y-PC, W-FC, T-CL, C-YWa, J-RL, and F-IL performed the study and analyzed the results. F-IL and P-PH drafted the manuscript. All authors reviewed and approved the final manuscript.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.


However, a direct link between these two hypotheses can be drawn. Mitochondria actively participate to global Ca2+ signaling, and increases of [Ca2+] inside organelle matrix are known to sustain energy production to modulate apoptosis and remodel cytosolic Ca2+ waves. Most importantly, while mitochondrial Ca2+ overload has been proposed as the no-return signal, triggering apoptotic or necrotic neuronal death, until now direct evidences supporting this hypothesis, especially in vivo, are limited. Here, we took advantage of the identification of the mitochondrial Ca2+ uniporter (MCU) and tested whether mitochondrial Ca2+ signaling controls neuronal cell fate. We overexpressed MCU both in vitro, in mouse primary cortical neurons, and in vivo, through stereotaxic injection of MCU-coding adenoviral particles in the brain cortex. We first measured mitochondrial Ca2+ uptake using quantitative genetically encoded Ca2+ probes, and we observed that the overexpression of MCU causes a dramatic increase of mitochondrial Ca2+ uptake both at resting and after membrane depolarization. MCU-mediated mitochondrial Ca2+ overload causes alteration of organelle morphology and dysregulation of global Ca2+ homeostasis. Most importantly, MCU overexpression in vivo is sufficient to trigger gliosis and neuronal loss. Overall, we demonstrated that mitochondrial Ca2+ overload is per se sufficient to cause neuronal cell death both in vitro and in vivo, thus highlighting a potential key step in neurodegeneration.Neurodegenerative diseases are a large and heterogeneous group of disorders characterized by selective and progressive death of specific neuronal subtypes. In most of the cases, the pathophysiology is still poorly understood, although a number of hypotheses have been proposed. Among these, dysregulation of Ca Changes in cytosolic Ca2+ concentration ([Ca2+]) are controlled by either influx from the extracellular space (triggered by the opening of voltage- or ligand-gated Ca2+ selective channels) or release from the intracellular store such as the endoplasmic reticulum (through the IP3 or ryanodine receptors). The spatiotemporal patterns of cytosolic Ca2+ signals are strongly shaped by both proteins that bind and transport Ca2+ as well as by intracellular organelles, especially mitochondria. Thanks to their highly negative membrane potential, mitochondria can rapidly accumulate large amount of Ca2+ through a highly selective, ruthenium red-sensitive channel named mitochondrial Ca2+ uniporter (MCU) [2+ sponges and buffer either local or global increases in cytosolic [Ca2+]. Inside the organelle matrix, Ca2+ plays a pleiotropic role, since mitochondrial Ca2+ is a priming signal that can control many organelle features, including morphology, energy supply, ROS production, and cell death  measurements, mouse primary cortical neurons at 5 DIV were cotransfected with either GCamp6f or 2mtGCamp6m probes  causing depolarization-induced Ca2+ influx through voltage-sensitive Ca2+ channels. Upon stimulation, there was a sharp increase in cpVenus fluorescence and a decrease in CFP fluorescence intensity in both control and MCU-overexpressing primary cortical neurons due to the increased FRET efficiency . Confocal microscopy analysis of RFP fluorescence revealed a consistent difference between control and MCU-overexpressing neurons. In mock-transfected neurons, mitochondria appeared elongated and well distributed in the whole cell, in the soma, dendrites, and axons. Conversely, MCU overexpression induced a clear alteration of the overall organelle morphology, with numerous rod-like and fragmented mitochondria, mostly absent at the level of dendrites. Increase in the number of objects per cell was detected, as well as a decrease in their volume and surface  or a MCU-EGFP (synMCU-GFP) constructs. After infection, the percentage of GFP-positive cells at three different time points  was determined. Neurons infected with the synGFP construct did not show any loss over this period. In contrast, neurons infected with the synMCU-GFP adenoviral particles were progressively lost over time Figures . This suTo further confirm our findings, the TUNEL assay was employed. Mouse primary cortical neurons were infected as in the previous experiments, and apoptotic cells were labeled with a specific fluorescent marker of fragmented DNA. The percentage of TUNEL-positive cells in synGFP-infected neurons was low and constant over time. Conversely, the percentage of dead neurons progressively increased over time in MCU-overexpressing cultures Figures .2+ overload can induce opening of the mitochondrial permeability transition pore, leading to loss of inner mitochondrial membrane potential cyt. MCU overexpression mimics the effects of the excitotoxic stimulus treatment, precisely glutamate, on both mitochondrial morphology and neuronal cell death . These differences can however be explained by considering that mitochondrial Ca2+ is a pleiotropic signal that can be differentially decoded according to the global characteristics of cell-specific signaling toolkit. On the one hand, Ca2+-dependent stimulation of oxidative metabolism appears to be critical for cell survival  (2+ cycling across the IMM is occurring in these cells. As a consequence, the increased flux through the TCA cycle determined by elevated mitochondrial [Ca2+] is likely to be undermined by the energy waste caused by this futile cycle. A similar scenario has been recently proposed in cells derived from patients lacking MICU1, i.e., another condition that chronically increases matrix [Ca2+]  , thus inx [Ca2+] . Tissuesll death .2+ levels can be a key mechanism underlying neurodegeneration, thus uncovering a new putative target with enormous therapeutic potential.Overall, these data clearly indicate that exagerated organelle Ca2+ overload is per se sufficient to cause neuronal cell death both in vitro and in vivo, thus highlighting a potential key step in neurodegeneration. This evidence opens the possibility to clinical intervention through the regulation of intracellular Ca2+ signaling and, in particular, through the modulation of mitochondrial Ca2+ uptake.In conclusion, we demonstrated that mitochondrial Ca


Acute leukemia (AL) is a family of blood cancers that arises from the malignant transformation of hematopoietic cells of lymphoid (acute lymphoblastic leukemia (ALL)) or myeloid (acute myeloid leukemia (AML)) origins. While ALL occurs mostly in children, AML is mostly a disease of older adults, although it also peaks in infants (children aged 0-1 years). Treatment of AL remains challenging across age, with standard therapeutic treatments allowing 5-year overall survival rates of about 70% and 90% in children and 30–40% in adults, for AML and ALL, respectively. The major cause of treatment failure is relapse, and the prognosis for relapsed AL is extremely poor. In recent years, many efforts to improve the prognosis for these patients resulted in profound changes in supportive care and minimal residual disease monitoring. In addition, significant achievements have been made in our understanding of the genetic basis of both lymphoid and myeloid leukemias, which in turn allowed a better risk-adapted patients' stratification and the identification of novel biomarkers suitable for innovative targeted therapy-based approaches. We are pleased to introduce this special issue that contains original research as well as review articles covering several aspects of AL biology and clinical management.Two interesting manuscripts in this special issue focus on the complexity of AML biology, which has implications for therapeutic intervention. L. Handschuh provided a comprehensive review of the molecular alterations of AML and their use for AML subtype classification, risk stratification, outcome prediction, and novel therapeutic approaches. In addition, novel findings identified by applying next-generation sequencing approaches were detailed. They include novel chimeric or alternative spliced transcripts, as well as small RNAs, piwi-interacting RNAs, small nucleolar RNAs, long noncoding RNAs, and circular RNAs. All these functional transcripts dramatically impair expression and increase the genetic heterogeneity of AML. The second review by P. Bernasconi and O. Borsani focused on AML stem cell-niche and the possible interventions that might affect the cross talk between leukemic cells and bone marrow microenvironment, aimed to restore the normal niche ecology and to sensitize AML cells to chemotherapy, thus overcoming niche-mediated drug resistance.WT1 mutations, FLT3-ITD, and NUP98-NSD1, was re-evaluated in a contemporary pediatric AML trial. Analysis of a large cohort of pediatric AML patients treated in Germany with AML-BFM protocols between 2004 and 2017 confirmed that co-occurrence of these three alterations defines a subgroup of AML patients with dismal outcome. The original research article by S. Bruno et al. reported the identification of two novel alterations in DNMT3A gene in two adult AML patients, a single nucleotide variant (p.Trp795Ter) and a 35 nucleotides insertion (p. Thr862_Glu863fsins), both resulting in a premature STOP codon. The authors demonstrated that both these mutations affect DNMT3A protein expression and DNMT3A-mediated DNA methylation. Finally, the research article by A. Vitkevičienė et al. suggested the addition of 3-deazaneplanocin A (HMT inhibitor) and belinostat (HDAC inhibitor) to the conventional therapy (idarubicin and retinoic acid) of acute promyelocytic leukemia (APL) patients, based on enhanced efficacy observed in their in vitro study.Three further manuscripts investigating genetic alterations or innovative therapeutic approaches in AML are included in this special issue. In the research article by N. Niktoreh et al., the prognostic relevance of co-occurrence of The manuscript of C. Grobbelaar and A. M. Ford described the roles of different miRNAs in pediatric ALL, particularly focusing on their role for ALL classification, risk stratification, prediction of therapy response, and potential therapeutic targets. The authors also reported an interesting section about the recently identified gut miRNAs that likely participate in shaping the gut microbiota and might be useful as potential noninvasive diagnostic and prognostic biomarkers, or even as tools for prevention of certain subtypes of childhood ALL.Involvement of central nervous system (CNS) by leukemia is a relatively common event and it is still a major clinical issue. To target malignant cells in the CNS, intrathecal (IT) chemotherapy is commonly used. Intrathecal therapy associates with acute and long-term neurotoxicity, as it is well documented in pediatric patients but data about long-term toxicity of IT in adults are lacking. D. M. Byrnes et al. investigated the effects of IT in adult patients, including ALL patients, who were treated over a two-year period. The results of this retrospective study demonstrated a high frequency of neurological complications secondary to IT therapy, suggesting that less toxic forms of therapy may be warranted.Finally, E. Ledesma-Martínez et al. reviewed the biological effects of caseins, the main milk proteins, and peptides released during their digestion, focusing in particular on their interaction with the immune system, normal hematopoietic and leukemic cells. Although few researches have explored the role of caseins as antileukemic agents, what emerges is the influence of dietary intake of foods on cancer development or predisposition, sustaining the importance to pay attention on patients' nutrition during leukemia therapy.We hope that the articles included in this special issue will provide the opportunity to deepen some aspects of acute leukemias and will stimulate future researches in the field.


According to stopped-flow experiments performed in vitro, YTnC had 4-fold faster calcium-dissociation kinetics than NTnC. In HeLa cells, YTnC exhibited a 3.3-fold lower brightness and 4.9-fold increased response to calcium transients than NTnC. The spontaneous activity of neuronal cultures induced a 3.6-fold larger ΔF/F response of YTnC than previously shown for NTnC. On patched neurons, YTnC had a 2.6-fold lower ΔF/F than GCaMP6s. YTnC successfully visualized calcium transients in neurons in the cortex of anesthetized mice and the hippocampus of awake mice using single- and two-photon microscopy. Moreover, YTnC outperformed GCaMP6s in the mitochondria and endoplasmic reticulum of cultured HeLa and neuronal cells.The NTnC genetically encoded calcium indicator has an advantageous design because of its smaller size, GFP-like N- and C-terminal ends and two-fold reduced number of calcium binding sites compared with widely used indicators from the GCaMP family. However, NTnC has an inverted and modest calcium response and a low temporal resolution. By replacing the mNeonGreen fluorescent part in NTnC with EYFP, we engineered an NTnC-like indicator, referred to as YTnC, that had a positive and substantially improved calcium response and faster kinetics. YTnC had a 3-fold higher calcium response and 13.6-fold lower brightness than NTnC  As we previously described, FP-based calcium indicators may be classified into three major types by their molecular design  based on green fluorescent protein-like (GFP-like) fluorescent proteins (FPs) are broadly utilized for the 2+)-binding domain from the toadfish Opsanus tau, utilized in the Twitch family of FRET indicators3, and the single fluorescent domain from the cpFP-based indicators. Thus, NTnC-like GECIs simultaneously possess the reduced number of Ca2+-binding sites  and the smaller molecular size of the indicator . The reduced number of Ca2+-binding sites in NTnC-like GECIs is thought to provide a more linear response to Ca2+ transients, thus facilitating the quantification of changes in the Ca2+ concentration4.The first two types of GECIs, which include the fluorescence resonance energy transfer (FRET)-based and circularly permutated FP (cpFP)-based indicators, do not have the optimal design compared with the third type, the NTnC-like type of GECIs. The latter combines the advantages of the minimal troponin C (TnC) calcium  and M13-peptide pair of these GECIs with intracellular environments. The CaM-based GCaMP6 indicator interferes with L-type calcium channels (Cav1)/CaM-mediated excitation-transcription coupling in neurons and disrupts Ca2+ dynamics and gene expression6. Expression of GCaMP6 causes aberrant cortical activity in transgenic mouse lines7. The truncated version of TnC in NTnC-like GECIs has a smaller chance of having interacting partners in neurons8. Therefore, the NTnC-like type of GECIs is preferable among other types because it may be potentially inert to the neuronal environment and intermolecular interactions.The NTnC-like design has additional advantages compared with other designs. For example, GCaMP6s and G-GECO1.2, representatives of the second type GECIs with green fluorescence, demonstrated reduced fluorescence recovered after photobleaching- (FRAP-) mobility in mammalian cells at both low and high CaO. tau (tsTnC) as the Ca2+-binding moiety, inserted in the mNeonGreen FP2  as the fluorescent moiety9, we assumed that the replacement of the mNeonGreen fluorescent part in NTnC with EYFP may result in an indicator with an enhanced fluorescence response and faster kinetics. The previously reported NTnC indicator has an inverted phenotype, and GECIs with a positive fluorescence response to calcium are not currently available for the NTnC-like design, although GECIs with a positive phenotype have two major advantages over GECIs with an inverted phenotype. First, GECIs that have a positive Ca2+ response potentially enable three dimensional (3D) calcium imaging of the large-scale neuronal circuitry with high precision because neurons remain inactive most of the time10. Second, GECIs that have a positive phenotype should undergo less photobleaching during prolonged calcium imaging because their photobleaching rate in the Ca2+-unbound state is significantly reduced and they are not fully saturated with Ca2+ even during neuronal calcium activity 11).As another green fluorescent GECI with the insertion of the Ca2+-binding sites, which is similar to the NTnC GECI but with the positive fluorescence response to Ca2+ and substantially improved fluorescence contrast and kinetics. This indicator, named YTnC, has TnC as the Ca2+-binding domain, which is inserted in the EYFP protein. We validated the main characteristics of the YTnC indicator in vitro. YTnC demonstrated an 11- and 3-fold higher fluorescence ΔF/F in vitro in the absence and presence of 1 mM Mg2+ ions, respectively, compared with the respective characteristics of NTnC in vitro. However, it had a 13.6- and 3.6-fold lower brightness and photostability, respectively, compared with NTnC in vitro. According to stopped-flow fluorimetry data, the Ca2+-dissociation kinetics for YTnC were 4-fold faster than for NTnC and 2-fold slower than for GCaMP6f. However, the kinetics of the Ca2+-association in the case of YTnC was up to 8-fold faster than that for GCaMP6f. During Ca2+ transients in HeLa mammalian cells, YTnC demonstrated a 4.9-fold higher ΔF/F than identified for the NTnC indicator. Using YTnC, we visualized Ca2+ dynamics during spontaneous activity in primary neuronal cultures with a 3.6-fold improvement in the ΔF/F values over NTnC. We also assessed the YTnC characteristics in neurons using patch clamp recordings and determined that YTnC robustly and linearly responded to the stimuli in the range of 2–100 APs; however, it had lower ΔF/F values than GCaMP6s. We successfully applied YTnC for the in vivo visualization of Ca2+ neuronal activity in the V1 cortical area of anesthetized mice and the hippocampus of freely moving mice using two-photon microscopy and an nVista miniscope, respectively.Herein, we developed a single-fluorophore-based GECI that contains two Ca12. We selected the EYFP protein as the fluorescent moiety for the new GECI because it was successfully used in the Camgaroo1 GECI and demonstrated high fluorescence contrast upon interaction with Ca2+ ions9. As a Ca2+-binding motif, we used the point mutated derivative of tsTnC excised from the NTnC GECI. We started with the generation of three libraries with the insertion of the TnC part between residues 144 and 146 (L1), 144 and 145 (L2), or 147 and 149 (L3) of EYFP (according to EGFP enumeration) and randomized both the 3- and 2-amino-acid-long linkers between the fluorescent EYFP and TnC components  to select clones with the highest fluorescence ratio. Second, the selected clones were analyzed in a bacterial suspension in a 96-well plate format. After the second step of screening, we confirmed that all three libraries have clones with a positive fluorescence response to elevation of the calcium concentration. In the L1 library, there were two clones with the maximal fluorescence contrast of 2.8-fold. This response was superior to the response observed for NTnC, thus demonstrating that our design strategy was valid.To enhance the contrast and kinetics of NTnC GECI, we swapped its mNeonGreen fluorescent part with the EYFP protein and performed several rounds of further optimization using directed molecular evolution in a bacterial systemand 149 L of EYFP 2+-dependent changes in green fluorescence, according to the previously described protocol. After 5 rounds of random mutagenesis and selection, we chose a variant with the best performance in terms of contrast and brightness, named YTnC , which had 19 mutations relative to the original template  to another form (with absorbance at 495 nm) and by an increase of the quantum yield of the 495 nm-absorbing form. The 413 nm- and 495 nm-absorbing forms likely originate from different protonation states of the GFP chromophore. At pH 11, at alkaline conditions that favored the deprotonation of the GFP chromophore, YTnCsat had one absorbance peak at 495 nm  and protonated (form B) forms of the GFP-like chromophore, similar to that observed for GFP14. Thus, the high fluorescence contrast of the YTnC indicator is mainly a result of the effective transition from the B form to the A form upon Ca2+ binding compared to the NTnC indicator for which the inverse A to B transition was incomplete2.To estimate different factors that may contribute to the value of the YTnC contrast, we investigated the spectral transitions of YTnC upon calcium binding. The binding of YTnC to Ca nm Fig. . Thus, t nm Fig. . At pH 516, it was important to characterize the pH stability of the YTnC indicator to ensure its proper utilization in neurons. YTnC exhibited a shift in pKa values from two transitions with pKa 5.2 and 8.2 in the Ca2+-free state to 6.3 in the Ca2+-saturated state 17. Thus, sensitivity to pH was most likely inherited by YTnC and NTnC GECIs from their FP progenitors, and pH variations may contribute to the YTnC Ca2+ response to a greater extent than in the Ca2+ response of the NTnC indicator.Given that the pH in neurons may vary up to 0.3 unitsate Fig. , Table 12+ ions. It is known that the free calcium concentration may vary in the range of 50–100 nM to 250–10000 nM in neuronal cytoplasm19. According to equilibrium binding titration experiments, YTnC demonstrated a Kd value of 223 ± 10 nM, which was 2.7-fold larger and 1.7-fold lower than the respective constants for NTnC and GCaMP6f , the YTnC photobleaching rate in the Ca2+-bound state was 15- and 3.6-fold faster than in the case of EGFP  to 1.3-fold (at 1300 nM free Ca2+) faster kinetics of Ca2+ binding than GCaMP6f GECI. In the similar free Ca2+ concentration range, YTnC had slightly slower calcium binding kinetics than NTnC (from 1.09–fold slower at 300 nM free Ca2+ to 1.3–fold slower at 1100 nM free Ca2+). Fitting the dependence of the observed Ca2+-association rate constants on the Ca2+ concentrations to the equation kobs = kon × [Ca2+]n + koff enabled us to estimate the Hill coefficient and the dissociation constant. Both the Kdkin and Hill coefficient values for the YTnC, NTnC and GCaMP6f indicators were rather similar to those determined from the equilibrium studies  transients of intracellular calcium; thus, it is important to validate the temporal resolution of GECIs at physiological calcium concentrations. Therefore, we investigated the Caasm Fig. . The assasm Fig. . Two expted Fig. ; therefo2+ transients in HeLa Kyoto cells. After the addition of 2 mM CaCl2/2.5 µM ionomycin in Dulbecco’s phosphate-buffered saline (DPBS) with 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.40, the YTnC indicator demonstrated an average ΔF/F value of 198 ± 42% and a normalized ΔF/F value of 73 ± 33% relative to the red R-GECO1 in approximately 2 min. These values are 2.8 and 4.9-fold larger than the average and normalized ΔF/F values of 72 ± 5 and 15 ± 1% for NTnC, respectively, and 1.4-fold less than the response of R-GECO1  before and after the addition of 2.5 µM ionomycin  dimmer than the EGFP and NTnC proteins under identical expression and imaging conditions, respectively, and a ΔF/F response of 271 ± 33%. Taking into account that the brightness of the purified YTnCsat protein was not affected by the presence of 1 mM Mg2+, the observed 2-fold decrease in YTnCsat brightness in HeLa cells as a result of the DPBS to DMEM exchange may be explained by the influence of other factors but not the presence of Mg2+ ions. Thus, under the conditions most identical to in vivo, the YTnC indicator in fusion with P2A-mCherry has a 1.6- and 3.3-fold lower brightness than EGFP and NTnC proteins, respectively.To measure the brightness of YTnC under conditions that closely match in Table . In thesin vivo experiments.Overall, the results suggested that in terms of brightness, ΔF/F response, kinetics and calcium sensitivity, YTnC was appropriate for its further application in To validate the functionality of the YTnC indicator in neurons, we compared its response during spontaneous activity in dissociated neuronal cultures with those of the NTnC, GCaMP6s and R-GECO1 GECIs. Accordingly, we co-transduced neuronal cultures with recombinant AAV (rAAV) particles carrying YTnC, NTnC or GCaMP6s green indicators together with the reference R-GECO1 red calcium indicator under the control of the CAG promoter. The spontaneous activity of neurons in two- to three-week-old cultures was accompanied by an increase in the green fluorescence of the YTnC and GCaMP6s indicators and a decrease in the green fluorescence of the NTnC indicator with normalized ΔF/F values of 82 ± 12, 91 ± 115 and 23 ± 4%, respectively, relative to R-GECO1 Fig. . Thus, Yin vitro, we estimated its resistance to photobleaching during confocal microscope imaging. The imaging of neurons that expressed YTnC during 33 min under a 60x oil objective lens using 80% power of a 488 nm laser (17.3 µW/cm2 before objective lens) resulted in a 24 ± 5% loss of green fluorescence  less than those for GCaMP6s  of the V1 visual cortex of anesthetized head-fixed mice during the presentation of drifting grating as a visual stimulus. The YTnC and control GCaMP6s indicators were delivered to L2/3 of the primary visual cortex (V1) of the mice using rAAV particles. In the 6–7 weeks post infection, we identified cytoplasmic expression with additional nuclear expression of YTnC in neuronal bodies (15–25 μm in diameter) according to the green fluorescence , respectively Fig. . The non24 were 1.4-fold lower or 3.5-fold higher than for YTnC. The fraction of specifically evoked neurons expressing GCaMP6s in this study was also 6.3-fold lower than the published data24. This discrepancy may be explained by the significant variation of the fractions of specifically responding cells depending on the imaging region in the visual cortex  and the different types of anesthesia . YTnC showed a 1.9-fold lower (p < 0.0001) averaged ΔF/F response of 0.62 ± 0.23 compared with the ΔF/F value of 1.17 ± 0.53 for the GCaMP6s indicator. The decreased response of YTnC during stimulus-evoked neuronal activity correlates with its 2.6-fold lower ΔF/F values than GCaMP6s on patched cultured neurons , respectively  to YTnC, GCaMP6f, and GCaMP6s, respectively  higher for the Mito, ER and Lyn fusions, respectively, and 2.7-fold (p = 0.0113) higher for the IMS fusion (Supplementary Table sat in the cytoplasm was 4.7-fold (p < 0.01) dimmer than the brightness of GCaMP6ssat. The brightness of YTnCsat N-terminally fused to the Mito, IMS, and ER signals was 3.1-, 3.4-, and 2.6-fold (p < 0.01) higher than the brightness of NES-YTnCsat, respectively. The YTnC indicator in fusion with H2B and Lyn signals had a brightness similar to NES-YTnCsat. The brightness of GCaMP6ssat N-terminally fused to the Mito, IMS, ER, and Lyn signals was 7.1-, 3.8-, 40-, and 9.3-fold (p < 0.01) lower than the brightness of non-fused GCaMP6ssat, respectively. GCaMP6ssat in fusion with H2B had a similar brightness to non-fused GCaMP6ssat. Thus, YTnC in N-terminal fusions shows proper localization and preserves the high brightness in contrast to GCaMP6s, which demonstrated a higher percent of cytoplasmic mislocalizations and lost its brightness by 3.8–40-fold in most N-terminal fusions examined.According to the confocal imaging of the HeLa cells that transiently expressed different fusions, YTnC demonstrated good localization for N-terminal fusions that nicely targeted it to the mitochondrial lumen (Mito), mitochondrial intermembrane space (IMS), endoplasmic reticulum (ER) Fig. , nuclei We also compared the ΔF/F responses of the YTnC and GCaMP6s indicators in N-terminal fusions in HeLa cells to the calcium increase induced by a 2.5 μM ionomycin addition. YTnC outperformed GCaMP6s in this parameter by 3.4- and 2.5-fold (p < 0.01) for the Mito and IMS fusions, respectively  larger or similar normalized (relative to R-GECO1) ΔF/F responses compared with the respective fusions for GCaMP6s  higher for the Mito and IMS fusions, respectively  reduced, respectively. In fusions with β-actin and α-tubulin, the brightness of GCaMP6ssat was also 3.3- and 10.3-fold (p < 0.01) reduced, respectively. C-terminal fusions with β-actin and α-tubulin also reduced the ΔF/F response of YTnC by 1.8- and 2.9-fold (p < 0.01), respectively. The GCaMP6s indicator in fusion with β-actin and α-tubulin demonstrated the same and 2.4-fold (p < 0.01) lower ΔF/F response than the non-fused GCaMP6s. Thus, in C-terminal fusions with β-actin, GCaMP6s outperforms YTnC in terms of the proper localization, brightness and ΔF/F response; however, in fusion with α-tubulin, both indicators demonstrated improper localization, and GCaMP6s-α-tubulin showed a higher brightness and ΔF/F response than with YTnC-α-tubulin.C-terminal fusions of YTnC and GCaMP6s with β-actin and α-tubulin were aimed to target indicators to cytoskeletal microtubules , the myosin-binding domain of melanophilin (MBD or MLPH motif), the ankyrin-binding domain from voltage-gated Na+ channel 1.6 (AIS or Nav1.6 motif) and the N-terminal end from glutamate receptor kainate type2 KA2 protein (KA2 motif)  and a faster maturation rate than NTnC in vitro. The limited brightness of YTnC as a purified protein in vitro correlated with its relative brightness in mammalian cells. Moreover, in HeLa cells, the YTnC indicator in fusion with P2A-mCherry had a 1.6- and 3.3-fold lower brightness than EGFP and NTnC in the same fusion, respectively  of YTnC-infected mice with a fixed head. Using the YTnC indicator, we identified spontaneous and drifting grating-evoked neuronal activity in the soma of neurons located in the mouse visual cortex  infected with rAAV particles carrying AAV-CAG-NES-YTnC or AAV-CAG-GCaMP6s, respectively, and through an installed cranial window as described in ref.2 with modifications (Supplementary Methods).Surgeries for V1 2+in vivo imaging with the nVista HD miniature microscope were accomplished on twelve adult male C57BL/6 mice , aged 20 weeks at the beginning of the experiments, and transduced with rAAV particles carrying AAV-CAG-NES-YTnC, AAV-CAG-GCaMP6f, or AAV-CAG-GCaMP6s as described in ref.30 with modifications (Supplementary Methods).Animals and surgeries for imaging with an nVista HD miniature microscope and CaAfter the experiments, the mice were anesthetized and transcardially perfused with 30 mL of phosphate buffered saline (PBS) and 30 mL of 4% paraformaldehyde in PBS (pH 7.4). The extracted brains were further post-fixed in 4% paraformaldehyde in PBS overnight at 4 °C and stored in PBS with 0.1% sodium azide at 4 °C until sectioning. The brains were sagittally or coronally sectioned at a thickness of 50 µm with a vibratome (Leica). After rinses with PBS, the sections were incubated for blocking and permeabilization in PBS with 2% Triton X-100 and 5% goat serum at room temperature for 2 h. After rinses with washing solution (PBS with 0.2% Triton X-100), the sections were incubated at 4 °C overnight in antibody solution (PBS with 0.2% Triton X-100 and 5% goat serum) that contained primary antibodies: chicken anti-GFP , mouse anti-NeuN , rabbit anti-glial fibrillary acidic protein  and rabbit anti-Iba1 . The primary antibody reaction was extended for 2 h at room temperature, and the sections were rinsed with washing solution. The sections were subsequently incubated for 2 h at room temperature with Alexa Fluor (AF) fluorescent dye-conjugated goat secondary antibody AF-488, AF-568, or AF-647 . After rinses with washing solution, the sections, mounted on poly-L-lysine-coated slide glasses, were cover-slipped over fluorescence mounting media (Dako) for confocal microscopy.The mosaic Z-stack images were collected with the spinning disk confocal microscope Andor Revolution WD  and analyzed using Imaris  software. Representative images were imported into ADOBE PHOTOSHOP 13.0 (Adobe Systems) and were minimally processed to adjust the brightness, contrast, and background.+) and microglia (Iba+) were counted. The mean number of cells per frame was obtained by successively averaging the number of cells in frames, sections, and animals.To compare the cytotoxicity effects and distribution of the YTnC and GCaMP6f indicators in neurons, rAAV viral particles carrying AAV-CAG-NES-YTnC or AAV-CAG-NES-GCaMP6f were injected as previously described into the left and right hemispheres of the same mouse. The animals were perfused four weeks after the injections. The sagittal sections were immunochemically stained as previously described and captured under a 60x objective lens with the scanning confocal microscope Olympus Fluoview10i . The quantification was produced in Imaris  software on three sections for each hemisphere and animal into two 210 × 210 μm frames using a blind method. The total numbers of astrocytes  calculated for the one-tailed hypothesis.approved by the National Research Center “Kurchatov Institute” Committee on Animal Care  and were in accordance with the Russian Federation Order Requirements N 267 МЗ and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Nineteen and five C57BL/6 mice were used in this study, ages ~2-4 months and P0-P2 old, respectively. Mice were used without regard to gender.All methods for animal care and all experimental protocols were Supplementary InformationSupplementary Video 1Supplementary Video 2Supplementary Video 3