APOE) gene exists in three isoforms in humans: APOE2, APOE3 and APOE4. APOE4 causes structural and functional alterations in normal brains, and is the strongest genetic risk factor of the sporadic form of Alzheimer’s disease (LOAD). Research on APOE4 has mainly focused on the neuronal damage caused by defective cholesterol transport and exacerbated amyloid-β and Tau pathology. The impact of APOE4 on non-neuronal cell functions has been overlooked. Astrocytes, the main producers of ApoE in the healthy brain, are building blocks of neural circuits, and Ca2+ signaling is the basis of their excitability. Because APOE4 modifies membrane-lipid composition, and lipids regulate Ca2+ channels, we determined whether APOE4 dysregulates Ca2+signaling in astrocytes.The apolipoprotein E (2+ signals were recorded in astrocytes in hippocampal slices from APOE3 and APOE4 gene targeted replacement male and female mice using Ca2+ imaging. Mechanistic analyses were performed in immortalized astrocytes. Ca2+ fluxes were examined with pharmacological tools and Ca2+ probes. APOE3 and APOE4 expression was manipulated with GFP-APOE vectors and APOE siRNA. Lipidomics of lysosomal and whole-membranes were also performed.Ca2+responses in APOE4 versus APOE3 astrocytes in male, but not female, mice. The immortalized astrocytes modeled the male response, and showed that Ca2+ hyperactivity associated with APOE4 is caused by dysregulation of Ca2+ handling in lysosomal-enriched acidic stores, and is reversed by the expression of APOE3, but not of APOE4, pointing to loss of function due to APOE4 malfunction. Moreover, immortalized APOE4 astrocytes are refractory to control of Ca2+ fluxes by extracellular lipids, and present distinct lipid composition in lysosomal and plasma membranes.We found potentiation of ATP-elicited CaAPOE4 versus APOE3 astrocytes present: increased Ca2+ excitability due to lysosome dysregulation, altered membrane lipidomes and intracellular cholesterol distribution, and impaired modulation of Ca2+ responses upon changes in extracellular lipids. Ca2+ hyperactivity associated with APOE4 is found in astrocytes from male, but not female, targeted replacement mice. The study suggests that, independently of Aβ and Tau pathologies, altered astrocyte excitability might contribute to neural-circuit hyperactivity depending on APOE allele, sex and lipids, and supports lysosome-targeted therapies to rescue APOE4 phenotypes in LOAD.Immortalized  APOE exists in three isoforms: APOE2, 3—the most common allele—and 4. Although the three isoforms differ only in the amino acids in positions 112 and 158 at the N-terminal domain of the protein : NaCl 124, KCl 2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and glucose 10, gassed with 95% O2/5% CO2 (pH 7.3–7.4). Slices were loaded with sulforhodamine 101  for 20 min at 34 °C for astrocyte labeling [2, Hamamatsu, Japan). Cells were illuminated for 100–200 ms at 490 nm using LED system (CoolLED pE-100), and images from stratum radiatum astrocytes were acquired every 1 s. The LED system and the camera were controlled and synchronized by the NIS-Elements software  that was also used for epifluorescence measurements. Astrocyte Ca2+ levels were recorded from the astrocyte cell body, and Ca2+ variations were quantified as changes in the fluorescence signal (F) over the baseline (F0) ((F-F0)/F0). Two protocols of Ca2+ monitoring were used. Firstly, spontaneous events were studied by recording Ca2+ events for 120 s. Secondly, ATP-induced responses were studied by recording Ca2+ baseline for 30 s, followed by local application of ATP  to activate purinergic receptors for 30 s, and post ATP period for 60 s. Local application of ATP was delivered by pressure pulses through a micropipette  in the presence of tetrodotoxin . Matlab software  custom-written plugin was used for computation of fluorescence values of each region of interest (ROI).Animals were sacrificed; the brain was rapidly removed and placed in ice-cold modified artificial cerebrospinal fluid medium (ACSF). To reduce swelling and damage in superficial layers, N-methyl-D-glucamine (NMDG)-ACSF was used during brain sectioning, NMDG being a substitute for sodium ions in a wide range of adult ages and applications . NMDG-AClabeling –48, and 2+ measurement was performed using 2 μM of fura-2/AM, a ratiometric fluorescence indicator whose fluorescence ratio (R340/380) is proportional to Ca2+ concentration. Imaging of Ca2+ signaling in organelles was performed using genetically encoded Ca2+ indicators (GECI). The plasmids for endoplasmic reticulum (pCMV G-CEPIA1er) and mitochondrial (pCMV CEPIA3mt) were a gift from Masamitsu Iono [0). Ratiometric fluorescence values were obtained using the MetaFluor software and selecting individual cells (ROI). Two to four coverslips with 15 to 25 cells per coverslip were analyzed for each condition. Data values were further analyzed with GraphPad Prism 6.Intracellular Catsu Iono . FluoresAPOE plasmids  or negative control 1 μg/mL siRNA (Thermo Fisher Scientific).Cells were transfected with plasmids using Lipofectamine 2000 (Thermo Fisher Scientific) while transfection with siRNA to achieve silencing was accomplished with Lipofectamine RNAiMAX (Thermo Fisher Scientific). Briefly, lipofectamine and DNA or siRNA were added to medium without FBS and antibiotics. This solution was kept at room temperature for 20 min and administered to the cells. After 5 h, the medium was replaced by growth medium. The quantities of lipofectamine and DNA or siRNA were: 9 μL of lipofectamine and 2.5 μg/mL GECI plasmids; 12 μL of lipofectamine and 4.5 μg/mL of ribed in ), and 4.Lysosomal pH was measured using the ratiometric dye LysoSensor Yellow/Blue DND-160 (Thermo Fisher Scientific) as described  with minCells were fixed with 4% paraformaldehyde for 15 min and permeabilized by adding 0.1% of Triton buffer. Then, 5% of normal goat serum (NGS) was used to block the unspecific unions. Primary antibodies  and rat monoclonal anti-Lamp1  were incubated overnight, followed by one-hour incubation with the secondary antibody Cy3-donkey anti-rat IgG  and Alexa fluor 488 goat anti-mouse IgG . Coverslips were mounted on a slide with Fluoromount G. Images were acquired with confocal laser scanning microscopy ZEISS LSM 700. Analysis of lysosome localization was carried out using the ImarisCell tool of IMARIS software (Bitplane), which permits manual selection of the nucleus and the membrane of each cell and computes the distance (μm) from each Lamp1-positive vesicle to the nucleus center. Lamp1-positive vesicles larger than 0.5 μm were considered. The frequency distribution of vesicles from the nucleus center to the cellular membrane was represented.Filipin III (Sigma-Aldrich) was used to stain cholesterol. Cells were fixed with 4% paraformaldehyde for 15 min and incubated with 25 μg/mL Filipin for 30 min at room temperature in the dark. Images were acquired with a CCD ORCA-EG monochromatic camera (Hamamatsu) and the Eclipse TE-2000E (Nikon) epifluorescence microscope, using a 20x objective.Lysosomes from astrocytes were isolated as described . Briefly3:MeOH in a proportion of 1 to 2 with 0.1% formic acid to promote the ionization of molecules. Then, samples were vortexed, centrifuged at 4000 rpm, and analyzed with mass spectrometry using the QSTAR XL hybrid system . The sample was injected at a flux of 15 μL/min through the infusion integrated pump. Spectra were acquired during positive ionization in a range of m/z from 50 to 1100 uma. The ionization parameters were: 3300 V of voltage ion spray, 60 V of decluttering potential, and 250 V of focusing potential. Nitrogen was used as a collision gas for the spectra acquisition. Markerview™ and SIMCA-P™ software were used to reduce the results into a two-dimensional matrix of peak spectra and intensity of peaks, and for the statistical analysis. Inter-genotype comparisons were carried out with the multivariate analysis Partial Least Squares-discriminant analysis (PLS-DA). Next, the Variable Importance in Projection (VIP) was used to establish which metabolites had more impact in the segregation of samples according to APOE3 and APOE4 genotypes. ANOVA with a Tukey correction post-test was applied to the group of metabolites with a VIP > 1 to assess, again, inter-genotype differences with the identified metabolites. We then proceeded to identify metabolites with VIP > 1 comparing their accurate masses with those available in metabolomics databases  [APOE4 vs APOE3 astrocytes were calculated and a multi-t statistical test with corrected probability  was applied.The extraction of metabolites for the untargeted lipidomic assay was carried out by adding 200 μL CHClPIDMAPS) . Finally0D1  and monoclonal anti β-actin , were incubated overnight. The next day, the secondary antibody was incubated for 1 h . Finally, the membrane was developed using the chemiluminescence kit of BioRad according to the manufacturer’s protocol, and membrane chemiluminescence was detected with a Chemidoc MP Image System (BioRad). Image lab software (BioRad) was used for the quantification of bands.Cells for protein extraction were lysed with RIPA buffer and extracts were sonicated and centrifuged. Protein extracts were quantified with BCA kit  according to the manufacturer’s protocol. 20 μg protein of samples was loaded in 12% polyacrylamide gels electrophoresis (PAGE). Electrophoresis was conducted at a constant amperage (30 mA) for approximately 2 h followed by the protein transference to a PVDF membrane at constant voltage (100 mV) for 1.5 h. 5% non-fat milk was used to block unspecific unions, and the primary antibodies mouse monoclonal anti-V-ATPase subunit VCells were collected adding Trizol Reagent. Then, 0.2 mL chloroform was added to the extracts, and these were centrifuged at 11,500 rpm for 15 min at 4 °C allowing the formation of three phases. The RNA-containing phase (superior phase) was isolated by pipetting carefully, and 0.2 mL of isopropanol was added to precipitate of RNA, which was then washed with cold ethanol 75%. RNA-sample concentration was determined with a Nanodrop 200 spectrophotometer (Thermo Fisher Scientific). 2 μg of RNA was reverse transcripted to cDNA using 1 μM of oligo DT, 1 μM of hexamers, 0.5 mM dNTPs, 0.45 mM DTT, 10 U RNAse out, RT buffer, and 200 U of retrotranscriptase. The PCR program was divided into four steps: 65 °C for 10 min, 25 °C for 10 min, 42 °C for 1 h, and 72 °C for 10 min.ΛCq of each gene analyzed. Expression data were normalized with housekeeping genes (Gapdh and/or18s), using their geometric mean calculated according to the geNorm algorithm [APOE (Hs00171168_m1), Trpml (Mm00522550_m1), Gapdh (Mm9999915_g1), and 18S (Mm03928990) were purchased from Thermo Fisher Scientific. Primers for SYBR green were designed according to the sequence of the gene: Tpc1 , Tpc2 , and Gapdh .Gene expression was carried out with quantitative real-time PCR (qPCR) using Taqman and SYBR green technology. Fluorescence was detected with the 7500 Fast Real-Time PCR System. qPCR cycles were the following: a holding stage of 50 °C (2 min), 10 min at 95 °C and 40 cycles of 95 °C (15 s), and 60 °C (1 min). A similar protocol was used for SYBR assay but with an extra stage for the melting curve . Data analysis was performed using Cq value, and the average of the gene efficiency provided by LinReg PCR software, following the formula 1 + eficiencelgorithm . TaqMan p-value < 0.05 was considered significant , p-value < 0.01 (**), p-value < 0.001 (***) and p-value < 0.0001(****).Each determination was carried out with cells from at least three different passages. A parametric unpaired T-test was used for the comparison of a given variable in two different conditions or cell types, whereas one-way or two-way analysis of variance (ANOVA), with a Tukey’s or Dunn’s post hoc test, was used when comparing more than two conditions. The software employed was GraphPad Prism 6, and data are represented as mean ± SEM (standard error of the mean). A APOE4 alters astrocyte excitability, we recorded Ca2+ in hippocampal slices of 9–12-week-old male and female mice in which the endogenous mouse APOE gene had been replaced with human APOE3 or APOE4 genes. Recordings were made in ACSF medium supplemented with 10% FBS, in order to keep lipoprotein and lipid concentration as close as possible to physiological conditions. Slices were incubated with the Ca2+ indicator fluo-4/AM and the astrocytic marker sulforhodamine (SR101)  to basal levels in APOE3 astrocytes, but remained significantly increased over its own basal levels, and with respect to APOE3 cells, in APOE4 cells , by at least 2-fold, in astrocytes from APOE3 females as compared to APOE3 males , the fluorescence ratio being 0.28 ± 0.01 and 0.41 ± 0.03, respectively . Taken together, the data support immortalized APOE3 and APOE4 astrocytes as a model to study the mechanisms underlying the regulation of Ca2+ responses by APOE alleles in males.To gain insight into the mechanism by which expression of different or APOE4  since thAPOE4 astrocytes, we examined Ca2+ fluxes among the principal intracellular Ca2+ sources with organelle-specific probes and pharmacological manipulations. First, we investigated the mitochondrial Ca2+ uptake that characteristically buffers increases in cytosolic Ca2+. Since APOE4 has been described as harming mitochondria in neurons [APOE4 astrocytes could result in deficient Ca2+ uptake, and hence in increased intracellular Ca2+ responses. However, expression of the mitochondrial Ca2+ indicator CEPIA3mt , and transient receptor potential mucolipin (Trpml) [2+ uptake by acidic stores is accomplished through an indirect mechanism whereby V-ATPase pumps H+ into the vesicles, and then H+ are exchanged with Ca2+ through the Ca2+/H+ exchanger (CAX) [2+ release from acidic stores in APOE4 astrocytes might be due to two phenomena: greater expression or activation of NAADP receptors, or greater Ca2+ stored in these organelles, due to increased activity of V-ATPase and/or CAX. First, we analyzed the expression of NAADP receptors in astrocytes with real time qPCR. Expression of Trpml and Tpc1 channels is the same in APOE3 and APOE4 cells, while expression of Tpc2 channel is lower in APOE4 cells  . By conter (CAX)  , followed by its proteolysis by cathepsin C, such that the stored lysosomal Ca2+ is released into the cytoplasm. Then, the area under the curve (AUC) of the cytosolic Ca2+ increase after addition of GPN to the astrocytes was used to calculate the total amount of stored calcium in these organelles. The results showed greater AUC in APOE4 cells , whereas there was no effect on non-stimulated intracellular Ca2+ levels in APOE3 (0.44 ± 0.08 for cells treated with bafilomycin A1 compared to 0.39 ± 0.02 of cells treated with vehicle). This suggests that the aforementioned lower basal cytoplasmic Ca2+ concentration in APOE4 cells is due to greater V-ATPase-mediated Ca2+ uptake into acidic stores, consistent with the greater intralysosomal Ca2+ levels revealed by the GPN experiments. Accordingly, bafilomycin A1 reduced the ATP-induced Ca2+ release in APOE4 astrocytes, confirming the dependence of these Ca2+ responses on acidic stores  loss-of-function due to decreased contents [APOE3 and APOE4 astrocytes by quantifying mRNA levels with qPCR. As shown in Fig. APOE expression is lower in APOE4 than in APOE3 astrocytes. Therefore, it is plausible that lower expression of APOE4 accounts for Ca2+ signaling alterations. To test this possibility, we modulated the quantity of APOE3 and APOE4 with the rationale that, if APOE expression matters, decreasing ApoE in APOE3 cells would increase Ca2+ responses, whereas increasing ApoE in APOE4 cells would decrease Ca2+ responses. Upon decreasing APOE expression in APOE3 astrocytes with an APOE siRNA  malRNA Fig. b, ATP-inlls Fig. c. We alsion Fig. d. The GFner Fig. e, suggesGFP Fig. f, again 2+ responses between genotypes, we studied whether ApoE was localized in the acidic stores. Because the quality of staining of such organelles with the fluorophore lysotracker was not optimal for confocal studies (data not shown), we resorted to using Lamp1, a marker for lysosomes, autophagosomes, and different vesicles of the endolysosomal pathway, including late-endosomes [APOE3 astrocytes, we used ApoE and Lamp-1 immunostaining  Krebs medium (KH), 2) DMEM supplemented with lipoproteins-deficient FBS and 3) DMEM with B27, a supplement without lipoproteins and restricted composition of lipids: linoleic acid, linolenic acid, progesterone, and corticosteroids. In the absence of lipids, non-stimulated basal Ca2+ levels were significantly lower in immortalized APOE4 vs APOE3 astrocytes, as detected in the presence of lipids. Thus, in KH, basal levels were 0.29 ± 0.02 in APOE3 vs 0.23 ± 0.03 in APOE4 ; in DMEM supplemented with lipoprotein-deficient FBS, 0.39 ± 0.07 in APOE3 vs 0.29 ± 0.06 in APOE4 ; and in DMEM with B27, 0.30 ± 0.02 in APOE3 vs 0.22 ± 0.01 in APOE4 . By contrast, in all three conditions, stimulation of purinergic responses elicited Ca2+ responses of similar magnitude in APOE3 and APOE4 astrocytes  and high in the presence of low lipid concentration, or no lipids. Consistent with the greater ATP-triggered Ca2+ responses, the responses also lasted longer in APOE3 astrocytes in the absence of lipids: the response was 36.0 ± 4.0% of the maximum peak signal after 20 s in KH, 51.6 ± 4.3% in DMEM supplemented with lipoproteins-deficient FBS and 34.6 ± 3.0% in DMEM with B27, as compared to the previously reported 27.7 ± 1.5% in DMEM with FBS (p < 0.05). Thus, the results point to a potentiation of long-lasting amplification Ca2+ pathways, perhaps by Ca2+-induced Ca2+ release from the ER or store-operated Ca2+ entry (SOCE)—that is, Ca2+ influx across the plasma membrane in response to depletion of intracellular Ca2+ stores. Still, ATP responses were significantly shorter  in lipid-depleted APOE3 astrocytes as compared to APOE4 astrocytes , indicating that some differences in Ca2+ fluxes remain between APOE3 and APOE4 astrocytes.Thus far, all our Ca2+ response in APOE3 astrocytes is a fast-onset process, as it was observed just a few min after lipoprotein removal. It is not transient, because it persisted 12 h after replacement of DMEM supplemented with FBS for DMEM supplemented with B27  was abrogated by Ned-19, the inhibitor of Ca2+ release from acidic stores  ATP-induced Ca2+ responses in APOE3 astrocytes kept in KH without lipoproteins, but not in APOE3 astrocytes kept in KH with FBS  of these 35 metabolites revealed significant differences due to the APOE phenotype with a p-value < 0.05, confirming, again, that expression of APOE4 alters the lipidome of lysosomes. We then proceeded to identification of the particular metabolites and calculation of their fold change (FC) in APOE4 vs APOE3 astrocytes. We could identify 19 lipids: 10 phosphatidylcholines , 5 phosphatidylethanolamine , 2 lysophosphatidylcholine (2 decreased), 1 lysophosphatidylethanolamine (decreased), and 1 carnitine (increased) , such that lipids with VIP > 1 were the ones with greater weight on the group change. In lysosomal membranes, there were 35 lipids with VIP > 1 in the ed) Fig. d. HoweveAPOE4 vs APOE3 astrocytes. ANOVA analysis with a Tukey correction post-test of the intensities of these 41 metabolites confirmed significant differences due to the APOE phenotype with a p-value < 0.01, in agreement with the previous PLS-DA analysis. Twety-one lipids were identified according to their m/z, and their FC in APOE4 vs APOE3 calculated .Overall, this is the first demonstration that the expression of APOE3 and APOE4 we found that APOE4, in comparison to APOE3, increases receptor-induced Ca2+ responses due to increased release of Ca2+ from acidic organelles, which integrate lysosomes and related organelles. Further, APOE4-expressing astrocytes present distinct lipid profiles and are refractory to Ca2+-signaling regulation by lipids  rodents [Whatever the case, our study supports that immortalized astrocytes from human unctions  lends crn humans , 8, althng APOE4 , 96, andOE4 mice , 89, mos rodents .APOE4 phenotype observed in the aforementioned studies, as well as in our study, occurs in the absence of LOAD pathology, although it may render brains more vulnerable to age-dependent ailments. Thus, a wealth of data suggests that APOE4 exacerbates the impairment of Aβ processing and clearance caused by ApoE, leading to increased accumulation, and hence aggregation, of Aβ in the brain [2+ signaling play a role in LOAD, too. The Ca2+ hypothesis in LOAD contends that aberrant Ca2+ responses in neurons associated with Aβ, Tau, and the glutamatergic system underlie cognitive impairment [2+ signaling is profoundly dysregulated in astrocytes in animal models of AD [APOE4 may exacerbate the dysregulation of Ca2+ signaling in male astrocytes in LOAD owing to lysosomal dysfunction caused by lipid dyshomeostasis. Dysregulation of astrocyte excitability in LOAD may, in turn, contribute to neural-circuit hyperactivity independently of Aβ and Tau pathologies [We emphasize that the he brain . In addihe brain . Howeverhe brain , lipid hhe brain , and Ca2pairment . In addils of AD , 101, mals of AD . Our reshologies , supportAPOE4-targeted therapeutics is whether the mutated domain renders ApoE4 toxic, or less efficient, than ApoE3 and ApoE2. Alternatively, since ApoE is lower in cerebrospinal fluid (CSF) of APOE4 individuals [APOE3 and APOE4 as compared with APOE2 targeted replacement mice [APOE4 with APOE3 or APOE2. It seems unlikely that the effects of APOE4 on lysosomal-related Ca2+ fluxes are due to decreased expression of APOE4, because APOE4 over-expression did not revert Ca2+ responses to the levels observed in APOE3 cells. It is also unlikely that the APOE4 phenotypes are caused by direct interaction between ApoE4 and V-ATPase and/or NAADP receptors in the acidic organelles, as shown for APOD, another brain apolipoprotein that participates in vesicle-mediated astrocyte-to-neuron communication [APOE3, as shown here, and perhaps APOE2 [APOE allele modifies lipid contents and distribution in astrocyte membranes remain to be explored. Alternatively, because APOE4 exacerbates ApoE-mediated Aβ aggregation [APOE as a lipid carrier between cells, and plausibly inside cells, chronic removal of ApoE may have secondary effects. All in all, development of APOE4-specific therapeutics is in order. To this end, better understanding of the basic biology of APOE, and of the particulars of APOE4 action and sex bias independently of Aβ and Tau, are necessary. Overall, our findings that APOE4 per se disrupts the lipid-based regulation of Ca+ 2 fluxes in astrocytes due to lysosomal dysregulation supports the notion that astrocyte dysfunction contributes to APOE4 pathology in neurodegeneration.An outstanding question in ividuals , and in ent mice , is it tnication  and prevnication , given tps APOE2 . Whetherps APOE2  would reregation  and Tau regation , ApoE reregation . HoweverAPOE4 malfunction augments Ca2+-based excitability in astrocytes due to dysregulation of calcium fluxes in and out the lysosome, associated with lipid dyshomeostasis, and that the phenomenon might be male-associated. One implication of the study is that it supports the use of therapies aimed to restoring lysosomal dysfunction in astrocytes, including targeted overexpression of APOE3 or APOE2, in order to halt the accelerated progression of LOAD in male APOE4 carriers. Since Ca2+ signaling is central to the regulation of neural circuits by astrocytes, another implication of the study is that the therapeutic correction of astrocyte excitability might reverse the neural-circuit hyperactivity observed in APOE4-harboring humans and mice in the absence of Aβ and Tau pathologies. Finally, clarifying the sex bias in the efficacy of APOE4 targeted therapeutics is a must, for the mechanisms underlying APOE4 pathology might differ in males and females.Taking together ex vivo and in vitro data, we conclude that


The endoplasmatic reticulum plays a significant role in Ca2+ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca2+ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca2+ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca2+-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca2+ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca2+ uptake by pathogenic protists.Ca Thereincrease .2+ signaling inside the cell, reaching micromolar values of Ca2+. Ca2+ accumulation into the mitochondria stimulates ATP production by modulating enzymes of the tricarboxylic acid cycle (TCA cycle) such as pyruvate dehydrogenase, 2-oxoglutarate, and isocitrate-dehydrogenases. Additionally, a high concentration of Ca2+ inside the cell may trigger cell death by resulting in an excessive mitochondrial Ca2+ uptake and the release of apoptotic factors [Apart from their role in energy metabolism, mitochondria also participate in the Ca factors .2+ into and out of the mitochondria is tightly regulated by channels and transporters located in the outer and inner mitochondrial membrane. The increase in cytoplasmatic (Ca2+) occurs through two mechanisms in eukaryotes: the exit of Ca2+ from intracellular stores to the cytoplasm, and the entry of Ca2+ through the cell membrane [2+ permeable channels, such as voltage Ca2+ channels (VGCC) and P2X ionotropic receptors, participate in the increase of Ca2+ influx through the plasma membrane in excitable cells. In non-excitable cells, the mobilization of intracellular stores guarantees Ca2+ dependent cell signaling. In addition to membrane channels, ATP-dependent pumps and Na+/Ca2+ exchangers promote the cell’s Ca2+ efflux, maintaining the low intracellular concentration.The transport of Camembrane . Ca2+ pe2+, K+, Cl−, Na+) through the plasma membrane’s depolarization regulate essential activities that generate physiological responses, such as muscle contraction, neurotransmitter secretion, learning, and memory mechanisms. Voltage-operated Ca2+ channels are examples of proteins present on the plasma membrane’s surface that regulate ion concentration in the cytoplasm, generating signaling and cellular response [2+).In excitable cells, changes in ion gradients  ion channels are membrane proteins found in several tissues and cell types, permeable to mono- or divalent cations, and are involved in cellular responses such as the perception of stimuli  and ion homeostasis ,12,13. Tesponses .2+ in animal cells is the endoplasmic reticulum (ER). Two ion channels present in the ER membrane are responsible for the release of Ca2+ ions from the organelle to the cytosol: the inositol 1,4,5-triphosphate receptor (IP3R) and the ryanodine receptor (RyR). IP3R activation occurs through a signaling pathway mediated by second messengers that involve the activation of G protein-coupled receptors (GPCRs) [2+ from the ER, and increasing cytoplasmatic (Ca2+). Three types of IP3R are described in vertebrates, IP3R types 1–3, and differences in splicing and phosphorylation sites. In affinity for IP3 and associated molecules, they promote unique responses attributed to each different destination of the activated signaling pathways [One of the most extensive stocks of Ca (GPCRs) ,15. Briepathways .2+ to the cytosol is RyR. This receptor can be activated indirectly by stimulating the voltage modulated channel type-L Cav1.1–1.2, and by Ca2+, Mg2+ ions, protein kinase A (PKA), calmodulin (CaM), Ca2+ dependent protein kinase/calmodulin (CaMK), FK506 binding proteins, calsequestrin (CSQ), triadin, and junction [Drosophila melanogaster, Caenorhabditis elegans, and Homarus americanus.Another channel that regulates the output of Cajunction . In vert2+) in the ER lumen stimulates a process of interaction between Stromal interaction molecules (STIM), anchored in the ER membrane, and the Calcium release-activated calcium channel protein (ORAI) Ca2+ permeable channels present in the cell’s plasma membrane. This process, called store-operated calcium entry (SOCE), first described by JW Putney Jr (1986) [2+ stocks. Due to the high concentration of Ca2+ in the ER, the N-terminal domain of the STIM protein, located in the ER membrane, is linked to these ions. After the depletion of Ca2+ stores mediated by IP3 and consequently uncoupling of Ca2+ from the STIM protein, the latter dimerizes and translocates to the junction region of the ER membrane with the plasma membrane, allowing the interaction with the ORAI channels. This channel opens, allowing the passage of Ca2+ into the cell’s cytoplasm [The decrease in (Car 1986) , has bee86 , has 2+ is sequestered into the ER for the maintenance of intracellular Ca2+ stocks through an ATP-dependent protein called SERCA-ATPase, present in all eukaryotic cells [2+, regulating the termination of signaling pathways and preventing cell intoxication by an excess of Ca2+.Cytoplasmatic Caic cells . This pu2+ oscillations. Two-pore channels (TPCs) found in the membrane of endo-lysosomes of animals and plants allow the passage of Ca2+ ions to the cell cytoplasm when activated [Trypanosoma brucei [2+ and are rich in orthophosphate, pyrophosphate, and polyphosphate, being conserved from bacteria to humans [2+ permeable channels’ presence participates in the cytosolic Ca2+ oscillations, and ATP-dependent exchanger pumps transport Ca2+ back into the organelle, contributing to homeostasis.Acidic organelles, such as endo-lysosomes and acidocalcisomes, also contribute to cellular Cactivated ,22,23. Aa brucei  that maio humans ,26,27. T2+. Ca2+ permeable channels, such as IP3R, RyR, and TRPs are present in the organelle membrane and contribute to the increase in the cytoplasmatic (Ca2+). On the other hand, the presence of SERCA and SPCAs (secretory-pathway ATPases) guarantees the sequestration of Ca2+ into the GA [Golgi Apparatus (GA) is also considered an organelle that participates in oscillations and homeostasis of Cao the GA ,29.2+ can bind to different molecules and regulate different cell signaling pathways. A wide range of proteins present in other tissues has a specific motif for Ca2+ binding: EF-hand. Calmodulin (CaM) is an abundant and conserved protein among eukaryotes. It has the EF-hand domains and, when bound to Ca2+, regulates the activation of calmodulin kinase (CaMK) [Once free in the cell’s cytoplasm, Cae (CaMK) ,31, whice (CaMK) .Trypanosoma cruzi life cycle begins when a vector insect bites and releases metacyclic trypomastigotes in its feces. These forms enter the injury site and invade nearby cells, and then differentiate into intracellular amastigotes. This form can divide by binary fission and differentiate into trypomastigotes, which are released into the bloodstream. Trypomastigotes in the bloodstream can invade multiple cells, resulting in new intracellular amastigotes, or they can remain in the extracellular medium to be ingested by the insect vector. Ingested trypomastigotes differentiate into epimastigotes in the midgut of the insect vector, multiply, and differentiate into metacyclic trypomastigotes in the hindgut, ready to be released and infect a new host and continue the cycle. In parallel, the Plasmodium falciparum cycle is more complex: it begins with the bite of an infected anopheles mosquito, which injects sporozoites into the host. Once in the bloodstream, sporozoites migrate to the liver and invade hepatocytes, where they can remain inactive or replicate asexually, forming a large number of merozoites in the host cell. The release of merozoites into the bloodstream marks the beginning of the erythrocytic stages. Merozoites invade erythrocytes and develop within the parasitophorous vacuole, undergoing various biochemical and morphological transformations, which can be identified by three stages called ring, trophozoite, and schizont. The erythrocyte rupture by schizonts releases new merozoites, continuing the intra-erythrocytic cycle. During the cycle, a small percentage of parasites differentiate in female or male gametocytes, capable of infecting the vector mosquito during blood ingestion. In the mosquito’s intestine, gametocytes mature in macrogametocyte  and exflagellated microgametocyte , which is followed by fertilization and zygote formation. The zygote migrates to the intestinal epithelium, where it develops into an oocyst. The rupture of the oocyst releases sporozoites, which migrate to the salivary gland and are injected into the human bloodstream during the mosquito’s feeding, completing the cycle.The life cycle of parasitic protozoa is complex, involving multiple hosts, several cell types, different tissues, and microenvironments. This great diversity implies a finely regulated cell signaling mechanism that allows the parasites to adapt to different stimuli. For example, the 2+ signaling is essential for cell mobility, invasion, and egress of the host cell and cell differentiation in some protozoa. Among them, the ciliates (Paramecium spp.), Trypanosomatids , and apicomplexan Plasmodium spp., Toxoplasma spp., Cryptosporidium spp., Babesia spp.) [Paramecium and Apicomplexa, little is known about the mechanisms that regulate calcium-mediated concentration and signaling in these organisms [Intracellular Caia spp.) ,34. Due rganisms ,36.Paramecium spp. is a model organism that has been widely studied on the mechanisms of intracellular (Ca2+) regulation. A range of Ca2+ permeable channels have already been described in these ciliates. These include IP3R and RyR homologs present in different cell compartments, ATP-dependent channels (SERCA and PMCA), voltage-operated, and mechanosensitive channels. Ca2+ binding proteins, such as calmodulins, calcineurins, and protein kinases, have also been identified [The free-living ciliated protozoan entified ,38,39,40Trypanosoma and Leishmania. Several Ca2+ permeable channels have been described in Trypanosoma spp. and Leishmania spp. among them: Ca2+-ATPase channels SERCA homologs are present in the ER; Ca2+-ATPases (PMCA) present in the plasma membrane and acidocalcisomes; voltage-operated channels present in the plasma membrane; IP3R homolog channels present in acidocalcisomes; TRP channels present in acid organelles; and Ca2+ influx and efflux channels present in the mitochondria. Ca2+ binding proteins, such as CaM, CaM-like proteins, calcireticulin, and Ca2+ binding proteins present in the flagellum membrane were also identified [Trypanosomatids are a large group of protozoa that include two genera of great importance for human health: entified ,42,43.Plasmodium, Toxoplasma, and Cryptosporidium organisms, some Ca2+-ATPases proteins are known, such as SERCA in the ER, Ca2+ transporters in the GA membrane, Ca2+/H+ exchanger pumps, and components that regulate upstream signaling via Ca2+ [Toxoplasma gondii, TPC and TRPs homologous Ca2+ channels were identified, but none of the three organisms shows a canonic mitochondrial calcium uniporter (MCU)-type channel [Plasmodium and Toxoplasma indicate that the mobilization of Ca2+ from intracellular stocks occurs via the PLC-IP3 pathway [P. falciparum parasites display four GPCR-like proteins [2+ and PLC signaling [2+ binding proteins in apicomplexan parasites, such as calmodulins and most notably calcium-dependent protein kinases (CDPKs) [In via Ca2+ ,44,45,46 channel ,47. Phar pathway ,48 and b pathway , respectproteins  and one ignaling . However (CDPKs) ,36,44,52Plasmodium, ER is one of the main intracellular stores of Ca2+, in addition to the mitochondria and an acidocalcisome [P. falciparum (PfGCaMP3) that expresses the genetically encoded Ca2+ indicator GCaMP3. The authors showed the dynamics of Ca2+ release and influx elicited by inhibitors of the SERCA pumps, cyclopiazonic acid (CPA), and Thapsigargin (Thg) [2+-ATPase (PfATP6) transporter was identified in the P. falciparum genome [In alcisome ,53,54. Palcisome  recentlyin (Thg) . Only onm genome .2+) are essential for the hepatic stage of the malaria parasite, thus activating motility, regulating the secretion of adhesion and invasion proteins sporozoites. Carey et al. (2014) demonstrated that sporozoites treated with a Ca2+ chelating agent, PLC inhibitor, or IP3R inhibitor harmed motility and adhesin secretion, highlighting the importance of the PLC-IP3 pathway during the parasite’s liver cycle [P. berghei sporozoites’ invasion in HepG2 cells and the development throughout the liver cycle [Oscillations in the cytoplasmatic  [P. falciparum merozoites from a high (K+) (140 mM) to a low (K+) (5 mM) buffer results in cytoplasmatic (Ca2+) rise. Moreover, a shift in K+ concentration leads to an increase in the expression of the microneme (secretory organelles of parasitic apicomplexans) proteins EBA175 (erythrocyte-binding antigen-175) and AMA-1  on the surface of the parasites [2+ A23187 ionophore and inhibited after treatment with BAPTA-AM. Even in the absence of extracellular Ca2+, the change in (K+) induced the same effect, but treatment with PLC inhibitor U73122 abolished the response. The authors proposed that the contact of merozoites with the extracellular environment induces changes in cytoplasmatic (Ca2+) via PLC-IP3 and secretion of apical proteins responsible for interaction with the host cell.The egress process in . (2010)  demonstrarasites . These ePlasmodium protein kinase G (PKG) is involved in parasite motility and invasion during the hepatic phase through the activation of CDPK4 (Ca2+ dependent protein kinase). Using P. berghei parasites mutants for PKG or that carried deletions in the gene encoding CDPK4, Govindasamy et al. (2016) [2+ dependent signaling process [. (2016)  demonstr. (2016) . Protein process .Plasmodium parasites encounter low temperatures, pH differences, and xanthurenic acid. The last is capable of inducing an increase in cytoplasmatic (Ca2+) via PLC-IP3 in gametocytes, and the formation of cGMP, regulating Ca2+ oscillations through the production of IP3 [P. falciparum. On the other hand, both in the presence of xanthurenic acid and high cGMP concentrations, treatment with BAPTA-AM inhibits the maturation of the male gametocytes [P. berghei gametocytes resistant to PKG inhibitor treatment showed inhibition of Ca2+ oscillation in the presence of xanthurenic acid [In the Anopheles mosquito’s intestine, n of IP3 . Using gn of IP3  demonstretocytes . In anotnic acid .P. falciparum phosphoproteome revealed that PfCDPK1 may be downstream to GMPc/PKG signaling cascade [P. falciparum parasites showed deficiency during intra-erythrocytic development, with low growth compared to wild-type parasites, and changes in the expression of AP2-G and GDV1 genes in gametocytes [ cascade ,64 and a cascade ,66. A reetocytes . FurtherP. berghei, the cpdk3 gene’s deletion results in ookinetes with impaired motility and access to the mosquito’s intestinal epithelium, which decreases the transmissibility of the parasites [In arasites ,69. SporP. berghei and in P. falciparum, after the infection of mosquitoes, CPDK4 participates in the regulation of the parasites’ cell cycle, in the replication of genetic material, as well as in the gametogenesis process [P. falciparum, the induction of PfCDPK5 deficiency in schizonts results in merozoites unable to egress, even with the apical complex protein secretion [Like CDPK1 and CDPK3, CDKP4 plays an important role during the infection of parasites in mosquitoes. Through the use of inhibitors and the generation of genetically modified parasites, it was possible to determine that, both in  process ,71. In Pecretion ,73.2+ homeostasis. After the release of Ca2+ through the IP3R and RyR channels present in the ER membrane, these ions can be quickly incorporated by the mitochondria through regions of interaction between the two organelles observed in fungi and different mammalian cells, and where membranes associated with the mitochondria (MAM) interact with the ER network [2+ oscillations, for example, during ER stress and control over the generation of reactive oxygen species (ROS) [Considered the energetic matrix of the cell, the mitochondria are also fundamental in cellular Ca network ,75. In aes (ROS) ,77.2+ signaling, in apoptosis, and in aerobic respiration , who used comparative in silico analysis of conserved sequences to determine the sequence corresponding to the MCU [Mitochondrial calcium uniporter (MCU) is an essential protein for the transport of calcium across the mitochondrial membrane and has a fundamental role in the regulation of Caiew, see ). The fima cruzi . Similar the MCU .2+ [2+ into the space between membranes, being fundamental for the decrease of cytoplasmatic (Ca2+), and on the other hand, also transports the Ca2+ back to the cytoplasm [2+ ions in the intermembrane space are transported to the mitochondrial matrix via the mitochondrial calcium uniporter (MCU), present in the inner mitochondrial membrane [+/Ca2+ exchange pumps found in the inner membrane allow the Ca2+ to go from the mitochondrial matrix to the intermembrane space [Several proteins located at the inner and outer mitochondrial membrane play a central role in regulating the absorption and release of Ca2+ . The volytoplasm . Free Camembrane . Na+/Ca2ne space .2+, the uptake of this ion by the mitochondria is also fundamental for the ATP synthesis, activating Ca2+-dependent enzymes, oxidative phosphorylation, metabolic carriers, and reactive oxygen species (ROS). Ca2+ overload of the mitochondrial matrix compromises the functioning of this subcellular compartment. This event results in a decrease in ATP production, an increase in ROS concentration, and induction of the cell death process [P. chabaudi and P. falciparum’s mitochondria can sequester cytoplasmatic Ca2+ during the increase in the concentration of this ion within the cells. This increase may be due to an ER discharge caused by Thg and CPA, or stimulation with an agonist that results in a signaling event and culminates in the release of Ca2+ [2+ into the mitochondria is membrane potential-dependent since the pretreatment with electron transport chain uncoupler Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) prevents this process. The action of melatonin on the mitochondrial dynamics of P. falciparum has also been reported. This hormone can activate the expression of mitochondrial fission-related genes in a stage-specific manner [2+/H+ exchange protein  may be responsible for the mitochondrial Ca2+ efflux in P. falciparum [In addition to participating in the reduction of the cytosolic concentration of Ca process ,83,84. Llciparum .2+ to a buffer containing digitonin permeated P. berghei trophozoites causes stimulation of mitochondrial respiration, proportionally to Ca2+ concentration in the medium, altering stage 4 of cellular respiration. Oximetry experiments revealed a decrease in mitochondrial membrane potential, proportional to Ca2+ concentration added to the buffer, which is compatible with Ca2+ influx into the matrix. The addition of succinate to a medium containing digitonin permeabilized trophozoites and 3.5 µM Ca2+ led to a drop in this concentration to 0.6 µM. The subsequent addition of FCCP caused a massive increase in free Ca2+ in the medium, indicating an efficient mitochondrial calcium transport mechanism is operative [The addition of Caperative .2+/H+ antiporter (PfCHA) has been described as a mitochondrial transport of divalent ions responsible for exchanging H+ for Ca2+ or Mn2+, with kinetics supporting the role of mitochondria as a dynamic Ca2+ stock. Although a homologous protein in humans could not be found, PfCHA has the function of transporting the excess of these ions back to the cytoplasm, helping to maintain mitochondrial concentrations. The low affinity for Ca2+ (Tm of 2.2 mM) supports the hypothesis that this transporter would only act in conditions where the mitochondria is loaded with Ca2+ [Caith Ca2+ .2+ in trypanosomes is limited when compared to mammalian cells because Ca2+ regulated dehydrogenases are not present or have not been well studied so far. Pyruvate dehydrogenase E1, which is sensitive to Ca2+, had its gene identified in T. cruzi and has phosphorylation sites to regulate the activity, but there is no evidence that this enzyme works in the same way as in mammals [2+ [2+ in animals, in trypanosomes [2+, are present in trypanosomes but do not have the EF-hand domain for binding to Ca2+ and are probably insensitive to this ion [Knowledge of the role of mitochondrial Ca mammals . Mitochomals [2+ . There ianosomes . Moreovethis ion .2+ influx mechanism, although some proteins are absent [2+. It directly reflects in the metabolism, resulting in abnormal ATP concentrations, growth defects, and autophagy. These effects become more prominent when oxidative phosphorylation is essential, such as in low-glucose media found in the insect vector. On the other hand, MCU overexpression results in a pro-apoptotic state with Ca2+ overloaded mitochondria [Similar to mammals, Trypanosomatids have a well-characterized MCU-mediated Cae absent ,92. Intechondria . Other schondria ,94.Plasmodium, trypanosome mitochondria are also capable of acting as a Ca2+ buffer. The uptake of cytoplasmic Ca2+ into the mitochondria can occur in both molar and micromolar concentrations, depending on the membrane potential [2+ concentration, such as plasma membrane, acidocalcisomes, or endoplasmic reticulum [2+ space-temporal fluctuations resulting from various cellular processes [2+ is its involvement with apoptosis. This process in trypanosomes is well studied, although some important effector and regulatory molecules have not yet been described, such as caspases and TNF receptors [T. brucei, the production of reactive oxygen species blocks the mitochondrial transport of Ca2+, which results in its accumulation in the nucleus and causes cell death [T. cruzi, calcium overload-related apoptosis is dependent on the formation of superoxide ions [As observed in otential . This pheticulum . As IP3Reticulum . Despiterocesses ,99. Anoteceptors ,101. In ll death . In T. cide ions .Leishmania infantum promastigotes. Using concentrations between 25 and 50 nM, significant inhibition of complexes I, III, and IV of the electron transport chain was noted, and eventually, the death of the parasite [2+ homeostasis by altering the functioning of the mitochondrial permeability transition pore, controlling the capacity of mitochondrial Ca2+ retention and release, which can also be associated with cell death [Elmahallawy et al. (2014) investigated the harmful effects of melatonin in parasite . In addill death . Apicomplexa organisms have peculiar and unusual mitochondria, with primitive characteristics that place the organisms of this group as strong candidates to the first existing eukaryotes. Among these characteristics, the presence of a single mitochondria during most of the life cycle, a small and highly fragmented mitochondrial genome, simple protein import machinery, and low activity of the electron transport chain stand out, all of this in organisms with a need to adapt to several different microenvironments. In addition to the obvious public health need to study these organisms in order to identify new therapeutic targets, the study of parasites’ biology can help to understand molecular aspects still unknown in more complex organisms, such as mammals.


Arabidopsisthaliana  is grown in zero or full phosphate conditions, then roots or root tips are mechanically stimulated. Plants also are grown vertically on a solid medium so their root skewing angle  can be determined as an output of mechanical stimulation. Phosphate starvation results in significantly impaired cytosolic free calcium elevation in both root tips and whole excised roots. Phosphate-starved roots sustain a significantly lower root skewing angle than phosphate-replete roots. These results suggest that phosphate starvation causes a dampening of the root mechano-signalling system that could have consequences for growth in hardened, compacted soils.The root tip responds to mechanical stimulation with a transient increase in cytosolic free calcium as a possible second messenger. Although the root tip will grow through a heterogeneous soil nutrient supply, little is known of the consequence of nutrient deprivation for such signalling. Here, the effect of inorganic phosphate deprivation on the root’s mechano-stimulated cytosolic free calcium increase is investigated.  Candidates include MCA1  would be competent to elevate [Ca2+]cyt [2+]cyt elevation and sensitivity varies along the root [2+]cyt signal lies downstream of the PM kinase FERONIA (FER), can be decoded by Calmodulin-like (CML) proteins (CML12 and CML24), and has been linked to mechanically-induced changes in root system architecture [2+]cyt to response at the whole root level is far from being resolved.Mechanical stimulation can alter root growth and development ,2,3,4,5.esponses ,7. Plasmtivity1, , OSCA1 , MSLcrease1, ) and DEKcrease1, ,13). AltCa2+]cyt ,15. Regathe root ,17. Thisitecture . Thus fa2+]cyt signalling but, recently, it was shown that inorganic phosphate (Pi) deprivation can alter the patterns of abiotic stress-induced [Ca2+]cyt elevation of Arabidopsis thaliana roots [2+]cyt elevation. This could potentially be important as agricultural soils are commonly both compacted and Pi-deficient and use of Pi-fertiliser is not sustainable [2+]cyt touch response of Arabidopsis thaliana roots is examined using cytosolic (apo)aequorin as a luminescent [Ca2+]cyt reporter. Additionally, the ability of roots to skew as an output of mechanical stimulation and [Ca2+]cyt signalling is tested [Arabidopsis mutants for FER and CML24 have an enhanced skewing angle [2+]cyt touch response, the [Ca2+]cyt signal might negatively regulate skewing. Here, Pi deprivation impairs the root’s [Ca2+]cyt touch response but also results in a lower skewing angle, in contrast to fer [2+]cyt response acts as a positive regulator of skewing.As the root tip grows through the soil, it will experience both mechanical stimulation and a heterogeneous nutrient supply. Few studies have examined whether nutrient supply can influence cyt elevation caused by mechanical stimulation of Pi-replete and Pi-starved Col-0 individual root tips expressing cytosolic (apo)aequorin was first determined  and it is held that mechanical stimulation of the root apex is a contributory factor, with [Ca2+]cyt signalling implicated as a negative regulator cyt elevation) and at the whole root level (root skewing). The mechanistic basis of a dampened [Ca2+]cyt touch response of Pi-starved roots could have a variety of origins. These might include the effects of wall stiffening that occurs during deprivation, or PM lipid remodelling [2+]cyt response during Pi starvation. Downregulation of mechano-sensitive PM Ca2+ influx pathways  also might occur. However, in previous studies on Pi starvation of Arabidopsis roots, no putative Ca2+ channel subunits were found to be downregulated at the protein level cyt response could be the effect of an upregulated PM Ca2+ efflux transporter. Early work with tomatoes reported a strong upregulation of LCA1  encoding a root Ca2+-ATPase, under Pi starvation cyt response of fer was determined for epidermal cells cyt touch response measured with aequorin can be highly variable [Arabidopsis Pi-starved root tips reported either a significant diminution of the [Ca2+]cyt touch response or no effect [Arabidopsis had a significantly lower [Ca2+]cyt touch response than Pi-replete samples in terms of peak elevation and total [Ca2+]cyt mobilised aequorin was as described in cyt were estimated according to Knight et al. [2+]cyt mobilised was estimated as “Area Under the Curve” , pH 5.6 with MES , after After 8 days of vertical growth, the position of the root tip of each seedling was marked on the growth plate. Plants closest to the edge of the growth plate were not considered for analysis. Since plants on both full Pi as well as Pi-deficient conditions developed similarly up to day 7 (possibly due to seed reserves), marking the root tips on day 8 was considered as a point from where plant development diverged depending on the nutrient condition. After 11 days of growth, plates were scanned using an Epson scanner  with 300 dpi resolution. The software, ImageJ, was used to quantify the root skewing angle, resulting from the root deviating away from vertical. The direction of root skewing was recorded, where growth to the right of vertical was considered as positive skewing and growth to the left as negative skewing, when viewed from the back of the plate. Root length was measured with the ImageJ plugin NeuronJ. During the test of growth at a 45° incline, plants were grown vertically for 6 days then root tip position was marked on the plate. Half the plates were grown vertically, and half the plates were placed at a 45° angle. The final scans were taken after a further 3 days.www.r-project.org, version 3.5.1) in an R studio environment. The ‘MESS’  package was used to calculate Area Under the Curve (AUC) in aequorin studies. A Welch two sample t-test was used to test for statistically significant differences, using a significance threshold of p < 0.05.Statistical analyses were performed using the open-source software R (


Two microbe-associated molecular patterns (MAMPs), bacterial flg22 and fungal chitin, and one damage-associated molecular pattern (DAMP), AtPep1, were used to study the differential Ca2+ signatures in Arabidopsis leaves. The results revealed that flg22, chitin, and AtPep1 induced distinct changes in Ca2+ dynamics in both the cytosol and nucleus. In addition, Flg22 and chitin upregulated the expression of salicylic acid-related genes, ICS1 and EDS1, whereas AtPep1 upregulated the expression of jasmonic acid-related genes, JAZ1 and PDF1.2, in addition to ICS1 and EDS1. These data demonstrated that distinct Ca2+ signatures caused by different molecular patterns in leaf cells lead to specific downstream events. Furthermore, these changes in the expression of defense-related genes were disrupted in a knockout mutant of the AtSR1/CAMTA3 gene, encoding a calmodulin-binding transcription factor, in which a calmodulin-binding domain on AtSR1 was required for deciphering the Ca2+ signatures into downstream transcription events. These observations extend our knowledge regarding unique and intrinsic roles for Ca2+ signaling in launching and fine-tuning plant immune response, which are mediated by the AtSR1/CAMTA3 transcription factor.Plants encrypt the perception of different pathogenic stimuli into specific intracellular calcium (Ca The eviously ; the equ4/mL Arabidopsis protoplasts cells were isolated from three-week-old WT or three mutant plants  grown at 22 °C, and the cells were transfected with 10 µg of AEQ-red fluorescent protein (RFP) or AEQ-NLS plasmids with the PEG-mediated transfection method, as described previously [2. The following morning, the protoplasts were harvested at 100× g with centrifuge and washed with fresh Ca2+-containing WI buffer once; then, the cells were centrifuged and resuspended with the same WI buffer with a 5 µM final concentration of coelenterazine. Then, 80 µL Arabidopsis protoplasts cells were put into wells of black 96-well microplates  and incubated for 1 h in the dark before starting the Ca2+ measurements. The MAMPs- or DAMPs-triggered Ca2+ signals were tested as described above. To rule out variations in transfection efficiencies, RFP signals were measured as an internal control.Here, 5 × 10eviously . Samples3 were dissolved in Ca2+ imaging solution  as stock solution. For immune-stimulus treatment, the working solutions were freshly prepared with stock solution by diluting 1:1000 in Ca2+ imaging solution. Ca2+ imaging was performed as described above.Stock solutions of 1 mM flg22, 1 mM chitin, 1 mM AtPep1, and 1 M LaClArabidopsis plants were transferred into a single well of a 24-well plate and incubated in half-strength MS liquid medium (pH 5.8) overnight in a growth chamber. Control and treated leaf discs were collected and flash frozen in liquid nitrogen (N2). The frozen tissues were ground to powder in 1.5 mL microfuge. Total RNA was prepared using TRIzol Reagent (Invitrogen) followed by DNase-I (Roche) treatment. Two micrograms of total RNA were used to synthesize cDNA with random primer and oligo (dT) primer using an advantage RT-for-PCR kit . The cDNA was diluted five times and 1 μL/reaction (10 μL) was used as a template. Real-time PCR was performed using an Eppendorf single-color real-time PCR detection system with SYBR Green Supermix (Bio-Rad). Target gene expression levels were normalized to the AtUBQ5 (AT3G62250). The expression level in WT control was considered to be 1. A minimum of two technical replicates and three biological replicates were used for each experiment. All primers for qRT-PCR are listed in Five or six leaf discs from four-week-old p < 0.05).Statistical analyses were performed using JMP software . For experimental data, at least three independent repetitions were performed. The average value from all of the independent repetitions is shown in the figures. One-way ANOVA Tukey’s test was used followed by Tukey honest difference (HSD) test for statistical analysis. Different letters above the columns were used to indicate differences that are statistically significant (


When seedlings of Arabidopsis thaliana expressing apoaequorin were rotated from the upright position to the upside-down position, a biphasic [Ca2+]c-increase composed of a fast-transient [Ca2+]c-increase followed by a slow [Ca2+]c-increase was observed. We find here a novel type [Ca2+]c-increase, designated a very slow [Ca2+]c-increase that is observed when the seedlings were rotated back to the upright position from the upside-down position. The very slow [Ca2+]c-increase was strongly attenuated in knockout seedlings defective in MCA1, a mechanosensitive Ca2+-permeable channel (MSCC), and was partially restored in MCA1-complemented seedlings. The mechanosensitive ion channel blocker, gadolinium, blocked the very slow [Ca2+]c-increase. This is the first report suggesting the possible involvement of MCA1 in an early event related to gravity sensing in Arabidopsis seedlings.Gravity is a critical environmental factor affecting the morphology and function of plants on Earth. Gravistimulation triggered by changes in the gravity vector induces an increase in the cytoplasmic free calcium ion concentration ([Ca Among these, the [Ca2+]c-increase is presumably involved in an early process of the gravitropic response10.Plants on Earth sense gravity and orient their growth direction with respect to the gravity vector, in a process known as gravitropism. Changes in the gravity vector generate a variety of intracellular signals, including changes in reactive oxygen species2+]c-increases in response to gravistimulation  have been reported repeatedly, e.g., [Ca2+]c-increases in maize coleoptiles were observed using a [Ca2+]c indicator, fluo-311. The [Ca2+]c-increases induced by gravistimulation have been studied in more detail in Arabidopsis  seedlings with aequorin, a luminous Ca2+-reporting protein14. When gravistimulated by turning from an upright to upside-down position, the seedlings showed a biphasic [Ca2+]c-increase in their hypocotyls and petioles14 imaged with an ultrasensitive photon counting camera; a fast-transient [Ca2+]c-increase and a subsequent slow [Ca2+]c-increase. The fast-transient [Ca2+]c-increase depended on the rotational velocity13 but not on the rotational angle, whereas the slow [Ca2+]c-increase depended on the rotational angle but not the rotational velocity14.[Ca2+]c-increase has been demonstrated as a gravistimulation-specific Ca2+-response using μg conditions produced by parabolic flights (PF)13; the μg conditions allowed the rotation of seedlings without gravistimulation, the fast-transient [Ca2+]c-increase was induced by rotation under the μg condition, and the slow [Ca2+]c-increase was initiated by transition from μg to ca. 1.5 g when the µg condition was terminated14, confirming the idea that the fast-transient is principally induced by rotational stimulation, and the slow increase is genuinely induced by gravistimulation.Recently, the slow [Ca2+]c-increase was significantly attenuated by the potential MSCC inhibitors Gd3+ and La3+, and the Ca2+ chelator 1,2-bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), suggesting that it depends on the Ca2+ influx via MSCCs in the plasma membrane. The slow transient [Ca2+]c-increase was also attenuated by the actin-disrupting drugs cytochalasin B and latrunculin B14. These observations agree with the “starch-statolith model”16, which includes MSCCs and actin filament networks; sedimentation of “starch-statolith” increases the stress within the actin filament network and activates MSCCs16. However, the molecular entities of plasma membrane MSCCs responsible for sensing and responding to the gravistimulation have not been elucidated.The slow i increase (OSCA)23. Among these, MCAs, AtTPK4, and OSCA are plasma membrane MS cation channels27.Recent studies identified five families of plant MS channels: the bacterial mechanosensitive channel of small conductance (MscS)-like proteins, MSLs2+ uptake was increased in Arabidopsis seedlings and yeast cells by overexpressing MCA1 in the plasma membrane19. Seedlings of mca1-knockout lines lacked the ability to penetrate their roots into a harder agar19. Membrane stretching elevates [Ca2+]c in CHO cells expressing MCA119, and expression of Arabidopsis MCA1 leads to enhanced mechanosensitive cation channel activity (34 pS) in the Xenopus laevis oocyte plasma membrane25. Elongation growth of hypocotyls was suppressed under the hypergravity condition in wild-type Arabidopsis seedlings, whereas the extent of the suppression was reduced in mca1-knockout seedlings, but was augmented in MCA1-overexpressing seedlings29. These findings suggest that MCA1 is a plasma membrane Ca2+-permeable MS cation channel that is potentially involved in gravity sensing and the subsequent morphological changes in Arabidopsis seedlings.The functional properties of MCA1 have been examined in a variety of studies; Ca2+]c-increases triggered by rotating Arabidopsis seedlings under 1–5 g conditions were investigated. With a backward rotation from an upside-down to an upright position, a novel, “very slow” [Ca2+]c-increase following the biphasic [Ca2+]c-increase was found. In addition, mca1-knockout seedlings showed a reduced amplitude of the very slow [Ca2+]c-increase, and the reduction was partially restored in MCA1-complemented seedlings. This is the first report that characterizes the very slow [Ca2+]c-increase by using 1–5 g gravitational acceleration, and the role of MCA1 in the gravistimulation-induced [Ca2+]c-increase.In this study, [Ca2+]c-changes were monitored. As described previously14, rotation to the upside-down position promoted a biphasic [Ca2+]c-increase, consisting of a fast-transient (white arrowhead with amplitude A0) and a subsequent slow [Ca2+]c-increase (black single arrowhead with amplitude A1) , which is approximately five times longer than the delay of the onset of the slow [Ca2+]c-increase (18 s) in the previous study. The time constant of the decay of the very slow [Ca2+]c-increase (τ2) was also longer than that of the slow [Ca2+]c-increase (τ1)  and the amplitudes of the fast transients  were not changed apparently in mca1-knockout seedlings as shown in Supplementary Table 2+]c-increase was also affected to a lesser extent under 1–4 g, but the difference between WT and mca1 mutants became apparent under 5 g, suggesting the slow [Ca2+]c-increase induced by 180° rotation under 5 g conditions includes activation of MCA1 to some extent.The Ca+c-increaed Figs. , 4D,F. T2+]c-increases and very slow [Ca2+]c-increases under 3 g was decreased to 34% and 39% of wild-type seedlings, respectively, by the presence of MSCC inhibitors Gd3+ , suggesting again that the slow and very slow [Ca2+]c-increases were affected in a different way by prepositioning in the upside-down position. The reduction of the very slow [Ca2+]c-increases may reflect the adaptation of plants to the upside-down positioning. The mean time course of the slow and very slow [Ca2+]c-increases by the standard protocol is superimposed on the [Ca2+]c-increase of the preconditioned WT plants  were nearly the same as those of the very slow [Ca2+]c-increase induced by the standard protocol (with overlap), implying the tail of the slow [Ca2+]c-increase does not affect the kinetics of the very slow [Ca2+]c-increase. The [Ca2+]c-increase in mca1-knockout seedlings showed that the amplitudes of the very slow [Ca2+]c-increases were profoundly decreased by the preconditioned protocol , followed by a molecular interaction and signaling process that were not affected by gravity changes.Once the very slow [Ca2+-permeable channels in the slow [Ca2+]c-increase. The intracellular inositol 1,4,5-trisphosphate (InsP3) level is known to increase within 15 s of gravistimulation in oat shoot pulvini, suggesting the possible involvement of phospholipase C (PLC)-dependent signaling. Thus, once the gravity sensing process is triggered, it can be followed by a signaling process including the activation of PLC, production of InsP3, and InsP3-induced Ca2+-release (IICR) from the ER (or the vacuole), which may be less sensitive to changes in the gravity environment. This explains why the kinetic parameters of the slow and very slow [Ca2+]c-increases were not affected by the different magnitudes of gravitational acceleration  shows that hypergravity suppressed elongation growth of hypocotyls, but this effect was reduced in hypocotyls of mca-null mutants compared with the wild type28, suggesting that MCAs are involved in the sensing of gravity signals in plants. Hyper-gravitropism was not examined in this study, since it may need very high g condition and long observation period according on the above study.Enhanced gravitropism by hypergravity (hyper-gravitropism) is reported in Arabidopsis roots2+]c-increase was presumably a genuine response to rotation, because it was induced by rotatory stimulation even under µg, and its amplitude increases in an angular acceleration-dependent manner. Recently, it was demonstrated that the amplitude of the fast-transient [Ca2+]c-increase (A0 and A0′) is augmented by the gravitational acceleration from μg to 2 g. The present study confirmed these results under a wider range of gravitational acceleration, from 1 to 5 g. In contrast, the amplitude of the slow [Ca2+]c-increase depended on the rotational angle (change in the direction of gravity)14, not on the rotational velocity, and it is a genuine g-change response. These support the idea that the backward rotation induces fast transient, slow and very slow [Ca2+]c-increases as illustrated in Fig. 2+]c-increase.The fast-transient  c-increases in 1–5 g conditions. Thus, plants that have evolved on earth (1 g) may be capable of transducing a wide range of gravitational changes into the gravitropic response by potentiating the amplitude of Ca2+-signaling.Hyper-gravistimulation, achieved by reorienting the specimens 90° under the hypergravity condition , enhances gravitropic curvature in Arabidopsis hypocotyls and roots2+]c-increase was less affected in mca1-knockout seedlings, i.e., multiple MSCCs species may be responsible for the slow [Ca2+]c-increase, and mca1-knockout may only partially affect the slow [Ca2+]c-increase .Our results suggest that plants use MCA1 for detecting the gravity vector from an upside-down to upright position, i.e., changes from an unfavorable position to their accustomed position. On the other hand, plants may use multiple molecular machineries for sensing gravity vector changes . This may be the reason why the amplitude of the slow [Ca2+]c-increase are consistent with the starch-statolith hypothesis: sedimentation of the high-density plastid, and amyloplast in response to the gravity vector, increase stress in the actin filaments, which may activate mechanosensitive Ca2+-permeable channels 16. This idea is supported by the experimental result that mechanical stress in the actin cytoskeleton can activate MS channels36. A centrifuge microscope revealed sedimentary movements of amyloplasts under hypergravity conditions33. In this study backward rotation specifically activated MCA1, leading to the very slow [Ca2+]c-increase, implying that the plant distinguishes the forward and backward rotation. At present the cellular mechanism that distinguishes the forward and backward rotation is not known, however, it might be worth to propose a hypothetical cellular mechanism behind this. The forward rotation will cause the sedimentation of the high-density plastid, which will increase stress in the certain set of actin filaments that is newly encountered with the plastid, and the stress increase in the actin filaments will activate multiple mechanosensitive Ca2+-permeable channels including MCA1. On the other hand, the backward rotation will also cause the sedimentation, but this may increase stress in the “different” set of actin filaments that was used to be associated with the plastid under the upright position before gravistimulation and are primarily connected with MCA1, and the stress increase causes the very slow [Ca2+]c-increase.The characteristics of the c increases are recorded in hypocotyls and petioles of seedlings but not in roots in our experimental condition (coelenterazine-CP does not penetrate the gel substrate).MCA2 is the only paralog of MCA1 in Arabidopsis. Ca2+]c-increase has not been elucidated at present. The Ca2+ signals were collected from hypocotyls and petioles as mentioned in our preceding report14, which shows negative gravitropism. When plants are temporarily rotated and then rotated back to the upright position, plants should terminate the gravitropic responses  induced by the temporal rotation. If the very slow [Ca2+]c-increase might be involved in the termination of gravitropic responses, it affect the Ca2+-related relocalization of PIN auxin transporter proteins or modulate auxin-dependent growth39. These ideas should be examined in future studies.The physiological role of the very slow c-dependent aequorin luminescence was monitored and processed using a photon counter  at 1-s intervals. This configuration enabled the detection of gravistimulation-induced aequorin luminescence generated from approximately 50 seedlings. Four sets of this configuration were used for the experiment; each set was mounted in a light-tight dark box and aequorin luminescence was recorded and stored on a computer. This box was vertically rotated 180° by a computer-controlled DC motor system  at an angular velocity of 6 rpm and an angular acceleration of 2.5 rad s−2, because it induced nearly the maximum [Ca2+]c-increase compared with that by 90° rotation14. The plates were mounted near the rotation axis, producing a centrifugal acceleration of ca. 10–3 g during uniform circular motion; the tangential acceleration at the start of rotation was ca. 10–2 g. These devices were mounted on a custom-made aluminum rack that was fixed on the bucket of a swing type 5 g centrifuge machine  and stored on a computer. The complementation test was conducted as a separate experiment following the WT and mca1 mutant experiments.A seedling-growing plate was mounted below a photomultiplier tube . [Ca3 (10 mM stock) was added to a final concentration of 100 µM to the aequorin-reconstitution medium in a petri dish 1 h prior to the removal of the medium. Plant growth medium containing coelenterazine-CP and GdCl3 was removed from the dish 30 min before the experiment14 to retain the inhibitory action of Gd3+ during the experiment.GdCl2+]c, was used for all analyses, because it was not possible to discharge the remaining aequorin and monitor the signal in the 5 g centrifuge machine. The luminescence ratio was calculated by dividing the aequorin luminescence intensity by the basal luminescence intensity; the average aequorin luminescence intensity before the first gravistimulation at the gravitational acceleration for each experiment  denotes the number of experiments made with independent samples/plates containing approximately 50 seedlings. All experiments were repeated more than three times, and all the data obtained were analyzed.In this paper, the luminescence ratio, rather than the calibrated [CaSupplementary Information


In undergraduate and postgraduate medical education, mentoring offerspersonalized training and plays a key role in continuing medical education andthe professional development of healthcare professionals. However, poorstructuring of the mentoring process has been attributed to failings of the hostorganization and, as such, we have conducted a scoping review on the role of thehost organization in mentoring programs. Guided by Levac et al’s methodologicalframework and a combination of thematic and content analysis, this scopingreview identifies their “defining” and secondary roles. Whilst the “defining”role of the host is to set standards, nurture, and oversee the mentoringprocesses and relationships, the secondary roles comprise of supporting patientcare and specific responsibilities toward the mentee, mentor, program, andorganization itself. Critically, striking a balance between structure andflexibility within the program is important to ensure consistency in thementoring approach whilst accounting for the changing needs and goals of thementees and mentors. Through the provision of personalized training,learning and support, mentoring facilitates “the process by which healthprofessionals keep updated to meet the needs of patients, the health service, andtheir own professional development.”2836 With 2 recentreviews31 attributing ethical issues such as bullying andmisappropriation of the mentee’s work to neglect on the part of the host, it iscritical to scrutinize their role in mentoring programs.3241However, lapses in support and oversight of the mentor-mentee matching process, thenurturing of relationships between the mentee, mentor and the host organization, andthe cultivation of a positive mentoring environment has hindered its fullpotential.120 Perhaps most significanthas been the failure of many reviews in acknowledging and contending with theimpact of mentoring’s evolving, entwined, context-specific, goal-sensitive,mentee-, mentor-, relationship-, and host-dependent nature.4249 This suggests that peer,near-peer, group, mosaic, network, leadership, patient, youth, family, ande-mentoring should not be mistakenly conflated nor intermixed withpreceptorship, supervision, role modeling, and networking which have their ownspecific approach and role in education and training.41 Acknowledging mentoring’s context specific nature, this review will focuson the role of the host in novice mentoring which is defined as the“dynamic, context-dependent, goal-sensitive, mutually beneficialrelationship between an experienced clinician and junior clinicians and/orundergraduates focused upon advancing the development of thementee.”50 Novice mentoring is the dominant form of mentoring in medicaleducation.5160A dearth of data on the role of the host in mentoring has been attributed to anumber of issues.the central sourcesand forms of evidence available” on host organzations.42 The flexible nature of a scoping review allows systematic extraction, synthesis,43 and summarizing44 of actionable and applicable information across a diverse range of studyformats and settings. This circumnavigates the limitations posed by mentoring’snature4550 and a paucity of articleson host organizations.5154A systematic scoping review was adopted to identify “55 adaptations of Arksey and O’Malley’s42 methodological framework for conducting scoping reviews was adopted tosystematically study the potential size, gaps, and scope of available literatureon host organizations in novice mentoring.5660 The PRISMA-P 2015checklist was used to develop the protocol for this study.61Levac et al’s“what is known aboutthe role of the host organization in novice mentoring—particularly inInternal Medicine and its subspecialties as delineated by the AmericanCollege of Physicians?”62 The secondary research question was then determined to be “whatwould make an effective host organization in these disciplines?”Narrowing this scoping review’s focus on novice mentoring in Internal Medicinewas largely determined by the amount of prevailing data already present in thefield of mentoring in undergraduate and postgraduate medical education. Thesequestions were designed on the PCC  elementsof the inclusion criteria64 and presented in a PICOSformat , and prevailing reviews of CPD practices, the 8-member researchteam determined the primary research question to be Sformat .66 The PubMed Search Strategymay be found in Supplemental Appendix A.A search on 7 bibliographic databases  was conductedbetween 24th April and 12th September 2018. A search of gray literatureinvolving GreyLit, OpenGrey, and Web of Science databases was carried outbetween 18th October 2018 and 17th December 2018. In order to update the searchso as to include articles published up to December 2019, a subsequent search ofall 10 databases was conducted between 30th December 2019 and 4th January 2020.Accounts of novice mentoring prior to the year 2000 were omitted given thepropensity of these articles to neglect clear descriptions of mentoring andconflate mentoring approaches.67 approach to “negotiated consensual validation.”Upon completion of the independent searches, each member of the research teamcompiled a list of titles and abstracts to be reviewed. They discussed theirfindings online and at weekly research team meetings, achieving consensus on thefinal list of titles and abstracts by using Sambunjak et al’s68 approach to thematic analysis was adopted in the absence of an apriori framework and a clear definition of the host in novice mentoring.Braun and Clarke’s70 As such, drawing upon theroles of medical education units set out by the “AMEE Education Guide no. 28:The development and role of departments of medical education,”71 2 independent reviewers who were not involved in the thematic analysisadopted Hsieh and Shannon’s72 approach to directed content analysis. This process involves“identifying and operationalizing a priori codingcategories”73 which aligns with the constructivist approach adopted in this scopingreview. This approach circumnavigates the wide range of research methodologiesemployed in the articles and prevents statistical pooling andanalysis.7477 Quantifying the data andtabulating the frequency by which the themes and categories emerge also aids asa proxy indicator of their significance.78In keeping with the reiterative process outlined in Levac et al’s sixth stage,consultations with key stakeholders saw the expert team note that the themesidentified were consistent with descriptions of medical education units whichoversee and support multiple education programs.79 81 to evaluate the quality of the quantitative and qualitative studies includedin this review.Whilst not commonly associated with systematic scoping reviews, quality assessmentswere deemed important to better influence and inform future practice. This sentimentwas shared by the expert team. Two authors thus carried out individual appraisalsusing the Medical Education Research Study Quality Instrument (MERSQI)82 and the STORIES  statement.83The narrative produced was guided by the Best Evidence Medical Education (BEME)Collaboration guideComparisons between the themes and categories identified using thematic and directedcontent analysis revealed significant consistencies.(1) “Defining” and secondary roles83141 These papers suggest thatthe “defining” roles47 of the host include:An overwhelming majority of the included articles defined the host organization bythe roles they play in their respective mentoring programs.86140establishing and/or complying with overarching goals, clinical standards, andcurriculum requirements95135influencing,92122 and overseeing the mentoring program102107designing,90108110 and mentoringrelationships95102and nurturing the mentoring cultureeffective host, in particular, were determinedto be their ability to:Characteristics of an 142144provide consistent leadership142145proactively support mentor and mentee participation146cogently facilitate all mentoring processes146148proactively gather and revert feedback on the mentoring program and thementoring relationships within146and, finally, successfully initiate curricular reform to better meet theneeds of their participantsIn addition, the secondary roles of the host comprise of supporting patient care andspecific responsibilities toward the mentee, mentor, the overall program, and theorganization itself. These are outlined in (2) Balancing structure and flexibilityAlthough not a defining characteristic of the host organization, a key finding whichemerged in numerous papers was the need for the host to balance structure andflexibility within the program.9095123144 Rigorous oversight and thejust provision of support, in turn, influences the mentoring culture.151 It ensurestransparent communication of the program’s mentoring philosophy, mentoring approach,as well as the goals and values of the program.149 It also facilitatesrecruitment and retention of mentors through the provision of financialremuneration,150 opportunities for academic promotion,139 formalrecognition of their time and efforts,150 timely access to facilities,resources,144 research funding,154 and protectedtime.95154Establishing a mentoring structure serves to ensure fairness and consistency in thementoring approach and experience for all.47 within the mentoring program so as to adapt to the evolving mentee-, mentor-,host organization-, and relationship-dependent nature of each individual mentoring relationship.114 This is critical in enhancing the mentee’s sense of autonomy, connectivity,and advocacy.144 It is of note that flexibility is also encapsulated within theclinical standards and codes of conduct set out by prevailing hostorganizations.8692 As outlined in Yet, the host must also allow for flexibility116 with common values, goalsand views on education and clearly delineated roles and responsibilities 9092 who collaborate throughcoordinated lines of communication104, assessment, and reporting 104 in order to realize their“defining” and secondary roles. The “defining” roles of the host includeestablishing, nurturing, and overseeing mentoring relationships whilst ideallyoffering both structure and flexibility within the mentoring program.”86110In addressing its primary and secondary questions, this SSR characterizes the hostorganization as a “team of educators and administrators155 This will provide theprogram leaders with administrative, financial, and training support that will helpstreamline their response to the changing needs of the stakeholders and thementoring process.146 Such resources will ensure that codes of conduct, standards ofpractice, timelines, roles, and responsibilities to be adhered to by mentees,mentors and the educationalists and administrators designing and spearheading theprogram are clearly overseen.8692 A consistent framework is alsocritical in ensuring that mentoring takes place within reasonableboundaries.31 Establishing an implicit or contractual92131 agreement between menteesand mentors on the expectations of the mentoring program would minimize the risk ofmisdemeanors and breaches in ethical conduct.To realize their “defining” roles, the host should design and incorporate thementoring program as part of the formal curriculum.89149 Adaptations should be guidedby consistent evaluations of the mentee’s, mentor’s, and the host organization’schanging needs and goals. In the absence of a specific assessment tool, acombination of tools and assessors may be considered.110116124149153To ensure a flexible approach, the host must adopt adaptable, context sensitive, andstakeholder-specific assessment methods to provide mentees, mentors, and thementoring relationship with personalized, appropriate, specific, timely, holistic,accessible, and longitudinal support.The host should also work to establish an accessible and robust platform for mentorsand mentees to communicate freely and confidentially. Such a platform would alsoencourage mentors and mentees to attune themselves and respond to any changes duringthe course of their mentoring relationship. To facilitate this, they should beprovided with pre-mentoring workshops and longitudinal training programs to developtheir communication and online literacy skills. Such skills will help them tocircumnavigate obstacles such as conflicting schedules amidst urgent deadlines thatmay impede the progress of their mentoring projects.142144, holistic support for thematching,142145 and mentoringprocess146, personalized, specific and comprehensive feedback to allparticipants 146148 and thesuccessful development and execution of crucial curricular reform.146Overall, these considerations will provide the host with the opportunity to deliverconsistent, timely, appropriate, longitudinal leadershipToo narrow a picture of the host organization may have been sketched in this scopingreview given that it was explored in isolation from factors such as the dynamicnature of mentoring relationships, structures, environments, and even mentee-mentormatching processes. Concurrently, given the context-specific nature of the host andtheir roles in mentoring programs, conflation within the included articles ofdifferent healthcare, educational and CPD systems across different national andinternational contexts may prove to be problematic.These limitations are compounded by the scoping review’s focus on articles publishedor translated into English. As a result, much of the data comes from North Americaand Europe, potentially skewing perspectives and raising questions as to theapplicability of these findings in other healthcare settings. In addition, despiteusing the Endnote software to carry out independent searches and consolidation ofthe findings, relevant articles may have been unintentionally omitted.42 Levac et al,55 and Pham et al.157 As a result, we believe that the findings will help to inform the design andoversight of future mentoring programs. We also believe that this review may be ofinterest to educators and program designers in settings beyond the mentoringlandscape due to the potential applicability of the findings to other aspects ofmedical education.However, despite these limitations, this scoping review was carried out with therequired rigor and transparency advocated by Arksey and O’Malley,This scoping review evidences the critical role of the host in mentoring programs andhints at their applicability to undergraduate and postgraduate medical education.Riding on ever improving communication technology and advances in the disseminationof information, the increasing use of technology-enhanced mentoring platforms willalso see ever increasing demands for transparency and accountability. There is aneed to conduct closer evaluations of all intra and interprofessional mentoringinteractions to ensure that personal and professional boundaries are maintained withcodes of conduct and standards of practice strictly adhered to.Prospective studies should be conducted to better understand how balance betweenstructure and flexibility can be better struck to ensure maximum efficacy. However,it is only with the curation and validation of effective assessment tools accountingfor mentoring’s evolving nature that mentoring can realize its true potential in CPDprograms.(a) Mentoring’s role in CPD hinges on effective support and oversight by thehost organization. This may be facilitated through the development of aformal mentoring program that is overseen by the wider education andadministrative team.(b) Collaborative efforts between educators and administrators are requiredto ensure that a clear organizational structure is established with the aimof meeting the critical “defining” roles of the host. These comprise ofestablishing, nurturing, and overseeing the mentoring relationships whilstbalancing structure and flexibility within the program. This process must beguided by clear outcome measures, codes of conduct, standards of practice,and assessment points.(c) Members of the host must be trained, briefed, supported, and appraisedthroughout the mentoring program. Their roles, responsibilities and lines ofreporting should be clearly established.(d) Mentoring in CPD should be run as a longitudinal program that is in turnsupported by mentee and mentor training workshops.(e) Mentoring’s role in CPD is to facilitate personalized social, academic,and leadership development opportunities especially when used in conjunctionwith e-mentoring. However, the effectiveness of such an approach pivots uponthe host’s ability to assess and respond to the evolving needs of thementee, mentor and the mentoring relationship.Click here for additional data file.Supplemental material, Supplementary_material for The Pivotal Role of HostOrganizations in Enhancing Mentoring in Internal Medicine: A Scoping Review byElisha Wan Ying Chia, Kuang Teck Tay, Shiwei Xiao, Yao Hao Teo, Yun Ting Ong,Min Chiam, Ying Pin Toh, Stephen Mason, Annelissa Mien Chew Chin and Lalit KumarRadha Krishna in Journal of Medical Education and Curricular Development