Here, we found that calbindin knockout (CBKO) mice exhibited dentate GC hyperexcitability and impaired pattern separation, which co-occurred with reduced K+ current due to downregulated surface expression of Kv4.1. Relatedly, manipulation of calbindin expression in HT22 cells led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO mice. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 mice to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that the regulation of CaMKII signaling by Ca2+ buffering is crucial for neuronal excitability regulation.Calbindin, a major Ca Regulation of cytosolic calcium in neurons is crucial for normal brain functions. Neuronal signaling is governed in part by dynamiic changes in calcium concentrations, and calbindin acts as a ‘buffer’ that binds calcium ions to shape the spatiotemporal extent of changes in free calcium concentrations. Prior studies have noted reduced calbindin levels in certain neurological disorders, and researchers led by Won-Kyung Ho at Seoul National University, and Jong-Sun Kang at Sungkyunkwan University, South Korea, have uncovered a potential mechanism underlying these observations. Loss of calbindin in mouse models resulted in excessive firing by certain cells in a brain structure called the dentate gyrus. This structure is important to cognition, and the mice showed impaired performance in discriminating similar contexts, so-called “pattern separation”, highlighting the critical importance of calcium homeostasis in the brain. Cytosolic Ca2+ buffers are essential components for the maintenance of Ca2+ homeostasis and the shaping of Ca2+ signals1. Calbindin-D28k (CB) is a major Ca2+ buffer of mature granule cells (GCs) in the dentate gyrus (DG) of the hippocampus2, where half of the Ca2+ buffering is attributable to CB3. CB is typically a mobile and fast-acting Ca2+ buffer that helps to shape the spatiotemporal extent of Ca2+ signals4. Increased CB levels in response to nerve growth factor5 and a lack of CB in the degenerated substantia nigra6 suggest that CB plays a protective role against excitotoxicity. Interestingly, the expression of CB in the DG is markedly reduced in various pathological conditions that are accompanied by cognitive dysfunction, including Alzheimer’s disease (AD)9 and schizophrenia/bipolar disorder12. Downregulation of CB in hippocampal excitatory neurons by early life stress is implicated in increased susceptibility to stress-induced memory deficits13. However, the current mechanistic understanding of the pathophysiological roles of CB reduction is poor.Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that is highly concentrated in the brain, and activity-dependent activation of CaMKII plays a central role in synaptic plasticity14 and neuronal excitability16. Abnormal CaMKII activity has been implicated in various neuronal diseases, including schizophrenia11, intellectual disability17, and AD18, highlighting the importance of CaMKII in neuronal function. Calcineurin, a Ca2+-dependent protein phosphatase, plays a critical role in the activity-dependent alteration of synaptic transmission19. Intriguingly, Ca2+ buffers affect Ca2+-dependent protein kinase and phosphatase activity, thereby regulating neuronal function. Overexpression of CB was found to promote neuronal differentiation, which was concurrent with CaMKII activation and inhibited by a CaMKII inhibitor20, suggesting a link between CaMKII and CB in hippocampal progenitor cells. It is of great interest whether the interplay between CB and CaMKII plays a role in mature neurons.Ca23. The sparse activity of GCs has been regarded as essential for the computational function that makes pattern separation possible24. We have recently discovered that Kv4.1 is a key ion channel for the low-frequency firing of GCs, and mice with Kv4.1 depletion in the DG show impaired pattern separation25, highlighting the role of Kv4.1 in DG functions. The regulatory mechanisms of Kv4.1 are an intriguing topic worthy of further investigation.The DG of the hippocampus has long been postulated to mediate pattern separation by transforming similar inputs into distinct neural representationsIn this study, we discovered that the GCs in CB knockout (CBKO) mice show increased neuronal excitability along with reduced Kv4.1 activity and CaMKII activation. CB deficiency results in impaired CaMKII activation, which in turn reduces the surface localization of Kv4.1. CaMKII interacts with and phosphorylates the Kv4.1 protein at serine 555 (S555), which regulates its targeting to the membrane and its channel activity. Furthermore, CBKO mice showed impaired pattern separation. Our data collectively suggest that CB reduction predisposes patients to cognitive dysfunction at least partially by disrupting the CaMKII-dependent regulation of Kv4.1.28k knockout (CBKO) mice (on a C57BL6/J genetic background) and control C57BL6/J mice aged 1–2 months old. CBKO mice were kindly provided by Dr. Schwaller . The average ages of CBKO and control C57BL6/J mice used in the present study were 5.6 (n = 36) and 5.7 weeks (n = 10), respectively. Experiments for dentate GCs were mostly conducted using mice at postnatal week (PW) 4 to PW 7, while experiments for CA1-PCs were conducted using mice at PW 3 to PW 4. Mice were killed by decapitation after being anesthetized with isoflurane, and the whole brain was immediately removed from the skull and chilled in artificial cerebrospinal fluid (aCSF) at 4 °C. Transverse hippocampal slices (350-μm thick) were prepared using a vibratome . Slices were incubated at 35 °C for 30 min and subsequently maintained at 32 °C until in situ slice patch-clamp recordings and fluorescence microscopy. All experimental procedures were conducted in accordance with the guidelines of the University Committee on Animal Resource at Seoul National University .Brain slices were prepared from calbindin-Ds) after the establishment of the whole-cell configuration was between 10 and 15 MΩ. The pipette solution contained the following (in mM): 143 K-gluconate, 7 KCl, 15 HEPES, 4 Mg–ATP, 0.3 Na–GTP, 4 Na-ascorbate, and 0.1 EGTA/or 10 BAPTA, with the pH adjusted to 7.3 with KOH. For the antibody blocking experiments, patch pipettes were dipped into an internal solution containing anti-Kv4.1 antibodies at a concentration of 0.3 µg/mL and were then back-filled with that solution. The bath solution (or aCSF) for the control experiments contained the following (in mM): 125 NaCl, 25 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2, 1 MgCl2, 20 glucose, 1.2 pyruvate, and 0.4 Na-ascorbate, pH 7.4 when saturated with carbogen (95% O2 and 5% CO2). In all bath solutions, 20 μM bicuculline and 10 μM CNQX were included to block synaptic inputs. In voltage-clamp experiments to record K+ currents, we added TTX (0.5 µM), CdCl2 (300 µM), and NiCl2 (500 µM) to block Na+ and Ca2+ channels, and membrane potentials were depolarized to a maximum of 30 mV for 1 s in 10 mV increments from the holding potential of −70 mV. In current-clamp experiments to analyze neuronal excitability, the following parameters were measured: (1) the resting membrane potential (RMP), (2) the input resistance  for the given hyperpolarizing current  input), (3) the F–I curve (firing frequencies (F) against the amplitude of injected currents (I), for DG; 100–600 pA, 100 pA increment, 1 s duration, for CA1 pyramidal cells (CA1-PCs); 50– 250 pA, 50 pA increment, 1 s duration), and (4) the AP onset time at 300 pA (the delay from the start of 300 pA injection to the beginning of the upstroke phase of the 1st evoked AP). All chemicals were obtained from Sigma , except for CNQX, bicuculline, and TTX, which were obtained from Abcam Biochemicals .Hippocampal GCs of the DG were visualized using an upright microscope equipped with differential interference contrast (DIC) optics . Electrophysiological recordings were made using the whole-cell clamp technique with an EPC-8 amplifier . The experiments were performed at 32 ± 1 °C. After break-in, we waited 5 min for the neurons to stabilize. The perfusion rate of the bath solution and the volume of the recording chamber for slices were 2.2 mL/min and 1.2 mL, respectively. Patch pipettes with a tip resistance of 3–4 MΩ were used. The series resistance . The slices were illuminated using a polychromatic light source , which was coupled to the epi-illumination port of an upright microscope  via a quartz light guide and a UV condenser, and images were captured with an air-cooled slow-scan CCD camera . The monochromator and the CCD camera were controlled by a PC and ITC18, running a custom-made software program written in Microsoft Visual C++ (version 6.0). Details of the calibration method have been described previously26.Cytosolic  transients in the mature dentate GCs. Normally, κE is estimated according to the single compartment and linear approximation model28. When two different Ca2+ buffers, a Ca2+ indicator dye (B) and an endogenous Ca2+ buffer (E), exist in the compartment, the increments of total and free calcium have the following relationship:B and κE are the calcium-binding ratios of B and S, respectively. Ca2+ transients (Δ[Ca2+](t), CaTs) following short pulses of Ca2+ influx can be described by the following equations:A is the initial amplitude, d[Ca2+]T is the total intracellular Ca2+ increase evoked by the influx, γ is the Ca2+ extrusion rate, and κB is the Ca2+ binding ratio of the Ca2+ indicator dye (fura-2). The calcium-binding ratio of a buffer X is defined byT and Kd are the total concentration of X and the dissociation constant of X for Ca2+, respectively28. When κX is not constant over the dynamic range of [Ca2+] in a Ca2+ transient, the incremental Ca2+-binding ratio, κ‘X, should be used. This ratio is defined as2+]i,rest and [Ca2+]i,peak represent the [Ca2+]i value before and at the peak perturbation, [X]T is the total concentration of Ca2+ buffer X, and Kd is the Ca2+ dissociation constant of X28. According to Eqs.  and transiently transfected with a combination of DNA plasmids by using Lipofectamine 3000 (Invitrogen). To examine CB knockdown effects, HT22 cells were infected with a lentivirus carrying the control scrambled-shRNA (5′-CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′) or CB-specific shRNA (5′-CCGGGATTGGAGCTATCACCGGAAACTCGAGTTTCCGGTGATAGCTCCAATCTTTTTG-3′) with polybrene (Sigma) for 2 days. To induce differentiation, cells at ~70–80% confluence were switched to Neurobasal medium (Gibco) containing N2, B27, GlutaMAX, 5 ng/mL BDNF, and 50 ng/mL NGF (Thermo Fisher). The constructs used in this study were as follows: pCI-neo, pCI-neo-CB, Kv4.1-GFP  and GFP-C1-CaMKIIα . To examine the role of Kv4.1 phosphorylation, a serine-alanine mutation was introduced at serine residue 265, 555, or 568 of Kv4.1 by using a mutagenesis kit (Stratagene).HT22 (Sigma-Aldrich #SCC129) and HEK293T (ATCC CRL-3216) cells were cultured, as previously described2PO4, 11.1 glucose, 0.5 MgCl2, 1.8 CaCl2, pH 7.4 after adjustment with NaOH. The pipette solution was as follows (in mM): 135 K-aspartate, 2 MgCl2, 3 EGTA, 1 CaCl2, 4 Mg–ATP, 0.1 Na–GTP, 10 HEPES, pH 7.4 after adjustment with KOH. Currents were analyzed and fitted using Patchmaster (HEKA Instrument) and Origin 6.1  software. All values are given as the mean ± SEM. Current–voltage (I/V) relations were obtained by plotting the outward current at 1 s into the test pulse as a function of the test potential. Current densities (pA/pF) were obtained after normalization to cell-surface area as calculated by Patchmaster.The Kv4.1 currents from HEK293T cells were measured with the whole-cell patch-clamp technique. A voltage clamp was performed by using an EPC-10 amplifier , and a 10 kHz filter was applied. The patch pipettes  were made with a Narishige puller . The patch pipettes used in this study had a resistance of 2–3 MΩ when filled with the pipette solutions listed in the next sections. All recordings were carried out at room temperature. The normal external solution for HEK293T cell recording was as follows (in mM): 143 NaCl, 5.4 KCl, 5 HEPES, 0.5 NaH31. Briefly, cells were lysed in RIPA lysis buffer (iNtRON) containing a complete protease inhibitor cocktail (Roche), followed by SDS-PAGE and incubation with primary and secondary antibodies. Primary antibodies against calbindin, p-CaMKIIα, CaMKIIα , Kv4.1, Kv4.2 (Alomone Lab), GFP (Abcam), and β-tubulin (Sigma) were used.Western blot analysis was performed as previously described32. Briefly, precleared cell extracts were incubated with anti-phosphoserine antibodies (Santa Cruz) overnight at 4 °C, followed by incubation with protein G-agarose beads (Roche) for 1 h. Subsequently, the beads were washed three times with cell extraction buffer, and the precipitates were subjected to western blotting.Immunoprecipitation was performed as previously described33. In brief, the surface proteins of HT22 cells or mouse dentate gyrus were biotinylated by exposure to 1 mg/mL NHS-LC-biotin (Thermo) for 30–60 min at 4 °C. After a quenching reaction with 100 mM glycine, cells or dentate gyrus were lysed and sonicated in RIPA lysis buffer (iNtRON) with a proteinase inhibitor (Roche), followed by incubation with streptavidin–agarose beads (Pierce) and western blotting. Immunoreactivity was determined by enhanced chemiluminescence . The images were captured using a bioimaging analyzer  and analyzed with the Multi-Gauge program .Surface biotinylation analysis was carried out as previously described34 to conduct the contextual fear discrimination task. Mice were trained to discriminate between two similar contexts, A and B, through repeated experience in each context. Context A (the conditioning context) was a chamber  consisting of a metal grid floor, aluminum sidewalls, and a clear plexiglass front door and back wall. Context A was indirectly illuminated with a 12 W light bulb. The features of Context B (the safe context) were the same as those of Context A, except for a unique scent (1% acetic acid), a dimmer light (50% of A), and a 15° slope in the floor. Each context was cleaned with 70% ethanol before the animals were placed in. On the first 3 days , the mice were placed in Context A for 3 min to explore the environment and then received a single footshock . The mice were returned to their home cage 1 min after the shock. On days 4–5, mice of each genotype were divided into two groups: one group was exposed to Context A on day 4 and Context B on day 5, while the other group was exposed to Context B on day 4 and Context A on day 5. On days 4–5 , neither group received a shock in Context A or B, and the level of freezing behavior was measured for 5 min only in Context A. We defined freezing behavior as behavioral immobility except for respiratory movement35. We observed videos for 2 s bouts every 10 s (18 or 30 observation bouts for 3 min or 5 min of recording time) and counted the number of 2 s bouts during which the mouse displayed freezing behavior (referred to as a freezing score). The percentage of freezing was calculated by dividing the freezing score by the total number of observation bouts (18 or 30). The mice were subsequently trained to discriminate between these two contexts by being placed in the two contexts daily for 8 days . The mice always received a footshock 3 min after being placed in Context A but not B. Discrimination ratios were calculated as FA/(FA + FB), where FA and FB are the freezing scores in Contexts A and B, respectively. All experiments and analyses were performed while blinded to the genotypes of the mice.Contextual fear conditioning was performed with male mice between 15 and 16 weeks of age, including 8 CBKO mice. We modified the protocol of McHugh et al.For one trial of contextual fear conditioning, an experiment was performed with male mice between 14 and 19 weeks of age (eight control mice and eight CBKO mice) in a pair of very distinct contexts (A and C). The aforementioned Context A was used as the conditioning context. The distinct context (Context C) was a white acrylic cylinder with a blind end  standing vertically on the metal grid floor of Context A, and the bottom of the cylinder was covered with cage bedding, on which the mice were placed. The chamber and cylinder were cleaned using 70% ethanol between runs. On day 1 (acclimation), mice were placed in Context A and then placed in Context C an hour later. Mice were allowed to freely explore in both contexts for 5 min. On day 2 (conditioning), all groups of mice were placed in Context A and received a single footshock  3 min later. Mice were left in Context A for 1 min after the shock. On day 3 (assessment), mice were separated into two groups; the mice of each group were placed in Context A or C for 3 min without footshock, during which their freezing scores were measured.36. Open-field exploration was performed with male mice aged 16 weeks (six control mice and six CBKO mice). Mice were handled before the open-field exploration behavior test (OFT). Mice were picked up by the tail and supported without restraint in the palm of an experimenter’s hand for 15 min/day for 4 days. Following handling, each mouse was placed in a holding cage until all mice had been handled. They were then returned to their home cage. During mouse handling, handlers wore latex gloves, which they changed between mice. After the handling day, we executed the OFT using a modified version of the protocol of Kim et al.36. The open-field box was made of white plastic (40 × 40 × 40 cm) and divided into a center zone (20 × 20 cm) and an outer field. The entire field was illuminated with diffuse light, and white noise was played in the background. Individual mice were placed in the center zone, and the path of each animal was recorded with a video camera. In a 20 min of observation period, the total distance traveled during the total period and the time spent in the center zone in the initial 5 min were analyzed using the program EthoVision XT .An open-field exploration test was used to assess locomotor activityn) used in each experiment. The statistical significance was evaluated using Student’s t test, and the level of significance is indicated by the number of asterisks . P > 0.05 was regarded as not significantly different (N.S.). Data from behavioral studies were analyzed using IgorPro and Origin . Comparisons between multifactorial statistical data were made using a two-way analysis of variance (ANOVA). The differences in time-dependent changes in behavioral parameters between the two genotypes were evaluated using two-way repeated-measures ANOVA.Data were analyzed with IgorPro  and are presented as the mean ± SEM with the number of cells or mice  lower than 200 MΩ. We confirmed that the average resting membrane potential  and Rin values  were not significantly different between control and CBKO GCs. Interestingly, we found that the firing frequency in response to long depolarizing pulses was significantly higher in the CBKO GCs . Both had the same metal grid floor, but B had a unique odor (1% acetic acid), dimmer lighting (50% of A), and a 15° slope on the floor. As shown in Fig. F = 0.30, P = 0.58, context: F = 1.17, P = 0.29, genotype × context: F = 0.03, P = 0.86). The mice were subsequently trained to discriminate these contexts by visiting the two contexts daily for 8 days in 2-h intervals (from day 6 to 13), always receiving a footshock 180 s after being placed in Context A but not in Context B. The daily discrimination ratio was calculated as the ratio of freezing during the 180 s in Context A to the total freezing during both visits (A and B). On day 6, neither genotype could distinguish between contexts  = 0.005, P = 0.94, context: F = 0.001, P = 0.97, genotype × context: F = 0.32, P = 0.57); thus, the discrimination ratio was ~0.5. As the experiment progressed, the control mice gained the ability to discriminate Context B from Context A effectively, and the discrimination ratio increased. However, CBKO mice exhibited significant deficits in the acquisition of discrimination ability  = 6.71, P = 0.01, context: F = 52.99, P < 0.0001, genotype × context: F = 25.60, P < 0.0001). To examine whether the impaired discrimination between similar contexts in CBKO mice was due to a problem with memory acquisition, we examined the context specificity of the conditioning by assessing freezing behavior using a distinct pair of contexts (A and C). This distinct context (context C) evoked significantly lower levels of freezing (similar in the two genotypes) than Context A  = 0.002, P = 0.96, context: F = 30.34, P < 0.0001, genotype × context: F = 0.002, P = 0.96). These data imply that CBKO mice have no deficit in learning or discriminating between very distinct contexts but have difficulty discriminating between similar contexts (pattern separation). We also tested the locomotor activity and anxiety levels of CBKO mice using the open-field test. Control and CBKO mice showed similar exploratory patterns, as they moved the same distance in the open-field box and spent a similar amount of time in the center zone  from a holding potential of −70 mV. TTX, Cd2+/Ni2+, bicuculline, and CNQX were added to the external solution to block Na+ channels, Ca2+ channels, GABAA receptors, and AMPA/kainate receptors, respectively. Total K+ currents were dissected into TEA-sensitive (ITEA) and 4-AP-sensitive K+ currents (I4-AP)  showed no significant difference from that of control GCs, as reported previously25; both the peak  and steady-state currents  were comparable between genotypes  was more pronounced than the reduction in Ipeak (36%). I4-AP in CBKO GCs was similar to I4-AP in the control GCs in the presence of anti-Kv4.1 antibodies, as reported previously25. This suggests that the outward K+ current component reduced in the CBKO GCs is likely to be the Kv4.1 current.We then investigated the ion channel mechanisms responsible for the hyperexcitability of CBKO GCs. Since the increased firing was not associated with a change in the AP threshold, the involvement of NaAP) Fig. . The ITEpes Fig. . In contIss Fig. , which ants Fig. 25. The aGCs Fig. . The red+ current component reduced in CBKO GCs is the Kv4.1 current, we monitored changes in the outward current amplitude during perfusion of anti-Kv4.1 antibodies through a patch pipette after patch break-in. Anti-Kv4.1 antibodies significantly reduced the outward K+ currents in the GCs of control mice but did not induce changes in CBKO GCs  was significantly reduced in the CBKO DG, while the level of total Kv4.1 protein (Kv4.1-T) was not significantly altered relative to the control DG Fig. . These r2+ signaling pathway implicated in diverse cellular processes, including the control of neuronal excitability38. The level of p-CaMKIIα was substantially reduced in CB-depleted HT22 cells . Thus, it appears to be paradoxical that CaMKII inhibition shows a phenotype similar to that of CBKO. To understand how p-CaMKII is downregulated by CB knockdown, we analyzed the effect of a Ca2+-dependent phosphatase inhibitor, FK506, on p-CaMKII and the surface localization of Kv4.1 in CB-depleted HT22 cells , which was comparable to the previously reported Kv4.1 current amplitude in control GCs (619.6 ± 66.4 pA)25. In contrast, KN-93 did not show a significant effect in CBKO GCs  on outward Knts Fig. . In contGCs Fig. , which wGCs Fig. . These rA sequence analysis to predict the phosphorylation sites in Kv4.1 by CaMKII using the PhosphoSite database revealed three potentially phosphorylated serine residues at positions 265, 555, and 568 of Kv4.1 Fig. . Thus, wn = 4) for WT Kv4.1, and it decreased to 254.86 ± 49.34 pA/pF  for S555A Kv4.1. Furthermore, CaMKIIa inhibition with KN-93 decreased WT Kv4.1 activity to 350.36 ± 10.57 pA/pF , while it did not further reduce S555A Kv4.1 activity . The S555A mutation and KN-93 treatment did not change the inactivation kinetics , we loaded GCs with fura-2 (250 µM) using whole-cell patch pipettes (RS = 20.5 ± 1.0 MΩ), and evoked Ca2+ transients (CaTs) by applying voltage pulses  at different time points during fura-2 loading  and increased decay time constants (τ). To estimate the κE value, we plotted τ against κB and obtained the x-intercept from the extrapolation of the linear fit , while that in Tg2576 GCs was 206.8 ± 4.7 (n = 7), which was significantly lower than that in WT GCs  production is increased in the dentate GCs of 1- to 2-month-old Tg2576 mice, leading to depolarization of the mitochondrial potential, which was reversed by the antioxidant Trolox26. To test the possibility that oxidative stress induces the oxidation of CB to compromise the Ca2+ buffering capacity, we examined whether the decreased κE in Tg2576 GCs was restored by antioxidant treatment. When hippocampal slices obtained from Tg2576 mice were incubated in Trolox (500 µM) for 1 h, the κE value of Tg2576 GCs was 322.98 ± 30.2 . To confirm whether the effect of Trolox on κE in the Tg2576 GCs was indeed attributable to the protection of calbindin from oxidation, we tested the effect of Trolox on κE in the CBKO GCs. Incubation of hippocampal slices obtained from CBKO mice with Trolox treatment for 1 h did not affect κE in CBKO GCs (Fig. 2+ buffer capacity originating from Ca2+ buffers other than calbindin. Reduced Ca2+ buffering and its restoration by Trolox in Tg2576 GCs suggest that CB function is impaired by oxidative stress.CB has two N-terminal cysteine residues that undergo redox-driven structural changes, and oxidized CB has a lower CaGCs Fig. , suggest2+ buffers in the brain have been investigated mostly by analyzing the phenotypes of mice either lacking or overexpressing one of the Ca2+ buffers1. Mice with reduced CB expression show deficits in memory and hippocampal long-term potentiation41, reduced short-term facilitation42, and impaired motor coordination43. In contrast, the overexpression of CB in DG GCs was shown to alter mossy fiber presynaptic function and impair hippocampal-dependent memory44. These data suggest that proper levels of CB are critical for neuronal activity control. However, the mechanisms underlying the involvement of CB in these phenotypes are largely unknown. In this study, we investigated the cellular and molecular mechanisms underlying the excitability changes in dentate GCs in CBKO mice and found that Ca2+ dysregulation caused by the loss of CB induces Kv4.1 downregulation, leading to hyperexcitability in dentate GCs. In addition, we observed impaired pattern separation in CBKO mice. These results, together with previous results showing that mice with selective depletion of Kv4.1 in the DG exhibit impaired pattern separation25, highlight the importance of the low excitability of dentate GCs in pattern separation.The roles of endogenous Ca8. CB loss is also a well-known feature of temporal lobe epilepsy47. Since neuronal hyperactivity is also associated with AD, it has been suggested that CB loss is a consequence of neuronal hyperactivity. However, the possibility that CB loss is not merely an epiphenomenon of these diseases but causally related to DG dysfunction remains to be elucidated. In this respect, CBKO mice can be a useful model for studying the pathophysiological significance of CB loss in various diseases. In this study, we proposed a causal link between Ca2+ buffering deficits and hyperexcitability of the DG, which leads to cognitive deficits. Furthermore, we identified the molecular mechanism by which Ca2+ buffering deficits lead to neuronal hyperexcitability. We showed that CB depletion or overexpression, respectively, in HT22 cells decreased or increased the level of surface Kv4.1 along with p-CaMKII levels (Fig. 2+ buffering inhibited CaMKII activity, since reduced Ca2+ buffering was expected to increase cytosolic Ca2+ levels, and CaMKII activity was expected to increase at high Ca2+. To investigate this, we focused on another Ca2+-dependent enzyme that can act in the opposite way to kinase, calcineurin, a Ca2+-dependent phosphatase, and confirmed that reduced CaMKII activity and reduced Kv4.1 surface expression were restored, at least in part, with the pharmacological inhibition of calcineurin (Fig. 2+ dysregulation caused by CBKO resulted in an abnormal balance between Ca2+-dependent protein phosphatase and Ca2+-dependent protein kinase, causing a decrease in Kv4.1 phosphorylation, which is crucial for the surface localization of Kv4.1.CB loss in dentate GCs has been shown to be tightly linked to AD-related cognitive deficitsels Fig. . Furtherels Fig. , suggestrin Fig. . These r2+-dependent kinases and phosphatases play key roles in the activity-dependent regulation of neuronal functions. In particular, their roles in synaptic plasticity are well-known. A small increase in Ca2+ activates phosphatases, leading to long-term depression, while a higher increase in Ca2+ activates kinases, leading to long-term potentiation48. These results imply that Ca2+ signals are translated into the balance between Ca2+-dependent kinases and phosphatases, thus determining the phosphorylation states of synaptic proteins to regulate synaptic plasticity. Activity-dependent regulation of ion channels through the balancing of Ca2+-dependent kinases and phosphatases has also been well documented. Activity-dependent downregulation of Kv1.2 activity in CA3 pyramidal neurons is mediated by Ca2+- and Src family kinase-dependent endocytosis, which is facilitated by Zn2+-mediated suppression of tyrosine phosphatase50. The surface expression of Kv4.2 and endogenous A-type K+ currents in hippocampal neurons was increased by the introduction of constitutively active CaMKII51. Taken together, these and our results suggest that the phosphorylation-dependent surface localization of Kv channels, rather than their expression, might be an important regulatory mechanism modulating neuronal activity. However, we found that the levels of surface Kv4.2 proteins were not altered by the CB levels (Fig. 2+ influx, while previous studies mainly focused on the role of Ca2+-dependent kinases and phosphatases in activity-dependent regulation. Therefore, our results represent the impact of the balance between Ca2+-dependent kinases and phosphatases on the phosphorylation states of Kv4.1 at resting Ca2+ levels. It remains to be investigated how the Kv4.1 phosphorylation state is changed upon stimulation, contributing to the activity-dependent regulation of GC excitability.Protein phosphorylation and dephosphorylation are two essential mechanisms that regulate many functional proteins, such as enzymes, receptors, and ion channels. In the brain, Caels Fig. . Further52. Key findings include reduced autophosphorylation of CaMKII in the hippocampus and frontal cortex of patients with AD53, alterations in the subcellular localization of autophosphorylated CaMKII in the CA3 and DG of the AD-affected brain18, and inhibition of CaMKII activation by amyloid β54. However, it is still unclear how this dysregulation occurs. In a recent study, we showed that Tg2576 mice exhibited impaired pattern separation and hyperexcitable DG at the early preclinical stage39. These findings are comparable to those observed in CBKO mice in the present study. The similar phenotypes of Tg2576 and CBKO mice suggest that they share a common mechanism, and we found that Ca2+ buffering capacity is severely reduced in GCs from Tg2576 mice (Fig. 2+ buffering capacity in Tg2576 GCs may contribute, at least in part, to dysregulated CaMKII signaling in AD. It is interesting to note that mice lacking CaMKII or calcineurin exhibit multiple abnormal behaviors related to schizophrenia55. These results together with the present study support the view that Ca2+ buffering deficits are causally related to hyperexcitable DG and cognitive deficits. However, abnormal excitability and CB depletion in the DG were also reported in CaMKII-deficient mice11, suggesting the possibility that CB expression is regulated by CaMKII. It is possible that CaMKII activity is regulated by Ca2+ and that activated CaMKII, in turn, regulates various mechanisms that regulate Ca2+ homeostasis, including CB expression. Close interrelationships between Ca2+ homeostasis and the balance between excitation and inhibition may be the most important features underlying normal brain functions. Considering that numerous Ca2+ buffers are present in neurons, future studies should examine the role of different Ca2+ buffers in this relationship.It has been established that CaMKII is dysregulated in the hippocampus of patients with AD


Colpodella species are free living bi-flagellated protists that prey on algae and bodonids in a process known as myzocytosis. Colpodella species are phylogenetically related to Apicomplexa. We investigated the life cycle of Colpodella sp. (ATCC 50594) to understand the timing, duration and the transition stages of Colpodella sp. (ATCC 50594). Sam-Yellowe’s trichrome stains for light microscopy, confocal and differential interference contrast (DIC) microscopy was performed to identify cell morphology and determine cross reactivity of Plasmodium species and Toxoplasma gondii specific antibodies against Colpodella sp. (ATCC 50594) proteins. The ultrastructure of Colpodella sp. (ATCC 50594) was investigated by transmission electron microscopy (TEM). The duration of Colpodella sp. (ATCC 50594) life cycle is thirty-six hours. Colpodella sp. (ATCC 50594) were most active between 20–28 h. Myzocytosis is initiated by attachment of the Colpodella sp. (ATCC 50594) pseudo-conoid to the cell surface of Parabodo caudatus, followed by an expansion of microtubules at the attachment site and aspiration of the prey’s cytoplasmic contents. A pre-cyst formed at the conclusion of feeding differentiates into a transient or resting cyst. Both DIC and TEM microscopy identified asynchronous and asymmetric mitosis in Colpodella sp. (ATCC 50594) cysts. Knowledge of the life cycle and stages of Colpodella sp. (ATCC 50594) will provide insights into the development of intracellular parasitism among the apicomplexa. Colpodella species are free-living terrestrial, fresh water or marine predators that feed on protists and algae. Trophozoite and cyst stages have been described in the life cycles of Colpodella species [Colpodella species possess a pellicle and apical complex organelles, like pathogenic apicomplexans, that include rhoptries, micronemes, pseudo-conoid, polar rings and microtubules, which facilitate predation. Trophozoites possess hetero-dynamic flagella that originate from separate flagella pockets and possess transversal plates in the transitional zone. A thin wall cylinder lies over the transversal plate. [Colpodella species possess tricho-cysts which are organelles that are ejected in response to stimuli [ species . Trophoz species ,2. Colpol plate. . Some Co stimuli .Colpodella vorax, C. unguis, C. turpis and C. pugnax have been reported [Colpodella species would benefit from knowing when specific life cycle stages occur in culture to facilitate isolation of specific stages and subcellular organelles. Feeding is initiated when the predator attaches onto the prey using its rostrum where the apical complex organelles are located. A tubular tether forms between the predator and prey and is used for aspirating cytoplasmic contents from the prey. The prey’s contents are aspirated into a posterior food vacuole which enlarges during and after feeding. This type of feeding is known as myzocytosis [Colpodella sp. (ATCC 50594) forms cysts that divide to contain more than four juveniles. Cysts containing uneven number of juveniles, as low as three to seven juveniles, were observed [Colpodella sp. (ATCC 50594) were found to be asymmetric and asynchronous, with juveniles having different rates of development within the cysts [The mechanisms of encystation, excystation and transformation of life cycle stages is unknown among colpodellids and has not been described in culture or in the environment. The life cycles of reported ,3. Howevreported ,3. Studiocytosis . The posocytosis ,3. Immatsp. ATCC 0594 formColpodella gonderi and C. tetrahymenae are described as ectoparasites and remain attached to their prey for long periods of time [Colpodella tetrahymenae form cysts with four juveniles following feeding [Single or multiple predators can attach to a single prey on any side.  of time ,7. Colpo feeding . Among c feeding ,2,3.Colpodella-like species have been recently reported as potential infectious agents. Two cases of opportunistic human infections believed to be caused by Colpodella-like species were reported [Colpodella infection. Although polymerase chain reaction (PCR) amplification of blood, cerebrospinal fluid and tick samples identified Colpodella sp. DNA, life cycle stages of Colpodella species were not identified in host tissue. Furthermore, Colpodella species’ DNA was identified from ticks infecting cattle and from raccoons [Colpodella gonderi and Colpoda steinii were identified in the urine of an individual with multiple chronic diseases. Both protists were not found to contribute to urinary infections in the reported case [Colpodella sp. is phylogenetically related to Apicomplexan parasites such as Plasmodium species and the plastid containing Chromera velia [Colpodella sp. (ATCC 50594) are related to Colpodella pontica (renamed Voromonas pontica), Colpodella tetrahymenae and the pathogenic apicomplexans, Cryptosporidium serpenti, C, muris and Toxoplasma gondii [Colpodella sp. (ATCC 50594) in the life cycle [Colpodella species.reported ,9. Infecreported  and Jianraccoons ,11,12. Sted case . Howeverra velia ,15. Phyla gondii ,16. In aColpodella sp. (ATCC 50594) in detail. In order to gain insights into how intracellular parasitism developed among the apicomplexans, the life cycle stages involved in myzocytosis, encystation and excystation in Colpodella species (ATCC 50594) were investigated. In previous studies, antibodies specific to RhopH3, a rhoptry protein of Plasmodium species, were shown to cross react with Colpodella sp. (ATCC 50594) [P. falciparum. In addition, antibodies against a food vacuole protein of P. falciparum and inner membrane complex (IMC) proteins of Toxoplasma gondii were also investigated for cross reactivity against Colpodella sp. (ATCC 50594) proteins in the different life cycle stages. Our hypothesis is that Colpodella sp. (ATCC 50594) has life cycle stages representative of the known cyst forming Colpodella species. Recently described trichrome staining protocols facilitated differentiation of cyst stages from predator and prey [In the current study we employed time course experiments to investigate the duration, timing and stage transformations in the life cycle of C 50594) . In the and prey .Colpodella sp. (ATCC 50594) were maintained in Hay medium as a di-protist culture. Hay medium was bacterized with Enterobacter aerogenes in tissue culture flasks as described [Bodo caudatus (renamed Parabodo caudatus Dujardin) in the di-protist culture served as prey for Colpodella sp. (ATCC 50594). Cultures were examined using an inverted microscope to observe different stages of trophozoites and cysts as described [escribed . Ten mL escribed .Colpodella sp. (ATCC 50594) in di-protist cultures were fixed using 5% formalin as described [escribed ,19. Brieescribed . The fixescribed . FollowiColpodella sp. (ATCC 50594) and Parabodo caudatus. All stained smears were examined under oil immersion at ×1000 magnification and images were captured using an Olympus BX43 compound microscope  attached to an Infinity HD Lumenera digital camera and Olympus U-TV0.35xc-2 adapter using Infinity HD Capture software.For light microscopy, formalin fixed cells were stained with Giemsa, Kinyoun’s carbol fucshin (KCF), and Sam-Yellowe’s trichrome stains . Giemsa Colpodella sp. (ATCC 50594) in Hay medium culture, a time course analysis of Colpodella sp. (ATCC 50594) development was performed. Subculture of 500 μL resting cyst stages of Colpodella sp. (ATCC 50594) and P. caudatus was performed in 10 mL of Hay medium in each T25 flask (nine flasks), to initiate the time course. The initial subculture of cells was collected at T = 0 and then cells were collected every four hours for 36 h . The first time course experiment was performed in four replicates. Each replicate was performed for 36 h using slide culture chambers for the first two replicates and tissue culture flasks for the second two. The four replicates were performed to determine the reproducibility of the life cycle and stage transitions. Slide culture chambers contained 2 mL cultures in duplicate per slide. Encysted cultures were also collected 5 and 7 days after encystation to identify the morphology of the resting cyst stages. In order to observe cells in the most active period of the life cycle, an additional time course experiment was performed for 40 h. Cells were collected for fixation and staining every four hours up until twenty-two hours. At twenty-two hours cells were collected every hour until 30 h. Then cells were collected for fixation and staining every two hours until 40 h. A final time course experiment was performed to identify the predominant cyst stage of Colpodella sp. (ATCC 50594) in resting cultures. Cells were collected every 24 h for eight days and formalin-fixed for staining with Sam-Yellowe’s trichrome staining [Colpodella sp. (ATCC 50594) life cycle stage present in resting cultures.In order to identify life cycle stages of staining . Days fiColpodella sp. (ATCC 50594) proteins as described [Immunofluorescence assay (IFA) was performed on cells from the di-protist culture fixed using 5% formalin. Formalin fixed cells were permeabilized with 0.1% Triton X-100 then blocked with 3% bovine serum albumin (BSA). After blocking, incubation occurred with primary antibodies specific for antigen. This incubation was followed by three washes with 1× dPBS and then the smears were incubated with species specific secondary antibody conjugated to an Alexa fluorophore. Alexa fluorophores used include Alexa 488 and Alexa 647. Antibodies specific to apical complex organelle proteins were used to identify escribed . Antiserescribed  specificescribed , Anti-plescribed , anti-EBescribed , anti-AMescribed , anti-IMescribed ,27,28 anescribed . In someColpodella sp. (ATCC 50594) in culture medium was added to an equal volume of 8% paraformaldehyde in 0.1 M cacodylate buffer and spun down for 10 min at 3500 rpm. The cell pellet was fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer. The fixation continued in the same fixative solution for a total of 2 h at room temperature. The pellets were thoroughly rinsed in 0.1 M cacodylate buffer, and then postfixed for 2 h in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide. After rinsing with distilled water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in an EMbed 812 embedding media . Thin sections (70 nm) were cut on a RMC MT6000-XL ultramicrotome. These were mounted on T-300 mesh nickel grids  and then sequentially stained with acidified methanolic uranyl acetate and stable lead staining solution. They were then coated on a Denton DV-401 carbon coater , and observed in a FEI Tecnai Spirit (T12) transmission electron microscope with a Gatan US4000 4k × 4k CCD.An aliquot of Colpodella sp. (ATCC 50594) from di-protist cultures. Two major life cycle stages occur in Colpodella sp. (ATCC 50594) and the staining protocol distinguishes these. The stages are trophozoites and cysts of predator and prey. Colpodella sp. (ATCC 50594), and Colpodella sp. (ATCC 50594) trophozoites attached to P. caudatus prey. Black arrows show young demilune cysts of Colpodella sp. (ATCC 50594) . Colpodella sp. (ATCC 50594) cysts have a large clear zone surrounding the cyst, separating the cysts from bacteria. Mature cysts of Colpodella sp. (ATCC 50594) are identified by black arrowheads  and P. caudatus cysts are identified by the red arrowhead (panel A). Predator (yellow arrow)-prey (red arrow) in myzocytosis with tubular tethers of varying lengths (open black arrows), used for attachment, are shown in panels E and F. A large posterior food vacuole (Fv) is formed in the predator as cytoplasmic contents are aspirated from the prey. Multiple predators can attach to one prey at a time for feeding in the process of myzocytosis. Colpodella sp. (ATCC 50594) cysts were identified, still attached at the anterior ends , time course experiments were performed. We sought to determine at what time point each life cycle stage occurred and to identify the predominant cyst stage during the resting stage of the life cycle. Three time course experiments were performed. In the first experiment, four replicates each lasting 36 h was performed. Cells were collected every four hours, formalin-fixed and stained with Sam-Yellowe’s trichrome to observe the development of Colpodella sp. (ATCC 50594) trophozoite and cyst stages (P. caudatus and Colpodella sp. (ATCC 50594) cysts were present. From 4 to 8 h, there were predominantly P. caudatus trophozoites, and only few Colpodella sp. (ATCC 50594) trophozoites. From 8 to 20 h the number of Colpodella sp. (ATCC 50594) trophozoites increased along with mature Colpodella sp. (ATCC 50594) cysts. By 24 h most Colpodella sp. (ATCC 50594) trophozoites observed were in myzocytosis with P. caudatus, representing the most active time point of the life cycle (Colpodella sp. (ATCC 50594) and pre-cyst stages of Colpodella sp. (ATCC 50594). Young trophozoites and cysts of Colpodella sp. (ATCC 50594) were observed at 36 h.In order to identify the timing and stage transitions within the life cycle of t stages . At zerofe cycle . Cells iColpodella sp. (ATCC 50594) life cycle. The four replicates of time course 1 were very similar in the timing and life cycle stages represented, as shown in Parabodo caudatus trophozoites and Colpodella sp. (ATCC 50594) in myzocytosis were identified at 20 h  pre-cysts and cysts were identified (panels C and D), with the active feeding by myzocytosis observed from 22 (T = 6) to 30 h (T = 13) (Colpodella sp. (ATCC 50594) encysted. Pre-cysts of Colpodella sp. (ATCC 50594) were observed (panels 3C and 3J). In cells undergoing myzocytosis, young and mature cysts were observed up to 30 h (T = 13). By 32 h, more Colpodella sp. (ATCC 50594) cysts were observed and young trophozoites excysting from cysts were observed (panels 3U to 3Y). By 36 h, cyst stages of both Colpodella sp. (ATCC 50594) and P. caudatus were present in the culture and remained predominant up to 40 h (T-18). Clear zones separating Colpodella sp. (ATCC 50594) cysts from bacteria were observed (panels 3D and 3U).Time course two was performed to understand life cycle stage differentiation at the most active stages of the h T = 5, A,B, Colp(T = 13) E–T. CellColpodella sp. (ATCC 50594) cyst stage in a resting culture. Cells were formalin-fixed for staining every 24 h for eight days. Additionally, cysts from day five and cysts from day seven resting cultures were each pooled and formalin-fixed for trichrome staining. The results show that the predominant Colpodella sp. (ATCC 50594) life cycle stage in resting cultures for up to eight days were mature cysts with a single nucleus. Cysts on days seven and eight were the same stage. A few mature cysts were observed with two or more nuclei and some young demilune cysts were also observed (Colpodella sp. (ATCC 50594) and P. caudatus trophozoites excysted during these time points.A third time course was performed to identify the predominant observed A’–H’. Inobserved . A few CColpodella sp. (ATCC 50594) (yellow arrows) feeding on P. caudatus (red arrow) were identified. DAPI staining identified the kinetoplast and nucleus of P. caudatus and the central nucleus of Colpodella sp. (ATCC 50594) trophozoites (white arrow). DIC microscopy and DAPI staining are shown in Colpodella sp. (ATCC 50594) trophozoite (yellow arrow) feeding on P. caudatus (red arrow) in myzocytosis is shown by DIC and DAPI staining in Colpodella sp. (ATCC 50594) is prominent. The tubular tether joining predator and prey was identified (open white arrow). DAPI stained aspirated cytoplasmic contents from the prey are shown in Colpodella sp. (ATCC 50594) by the grey arrow. Yellow arrowheads identify Colpodella sp. (ATCC 50594) flagella and red arrowheads identify P. caudatus flagella. The tubular tethers are flexible and of varying lengths (Colpodella sp. (ATCC 50594) are shown in We performed DIC microscopy and DAPI staining to identify the morphology of life cycle stages and the number of nuclei present in trophozoites and cysts, respectively. Single or double  lengths . DIC micColpodella sp. (ATCC 50594) life cycle stages. Colpodella sp. (ATCC 50594) trophozoites were identified in Colpodella sp. (ATCC 50594) trophozoites (yellow arrows) and P. caudatus (red arrows) in myzocytosis were also identified in Colpodella sp. (ATCC 50594) to the plasma membrane of P. caudatus. The point of attachment is indicated by blue arrows. Microtubular organization in the cytoskeleton in areas of close proximity to the point of attachment is seen in panels 6 D to G (Colpodella sp. (ATCC 50594) (white arrow) was observed (Colpodella sp. (ATCC 50594) (blue arrows) with foci of microtubules is shown (P. caudatus were observed in the cytoplasm.Cells from di-protist cultures were prepared for TEM to investigate the ultrastructure of 6 D to G . The flo6 D to G . The floobserved . The plaobserved  and exteis shown  in a twois shown . BacteriColpodella sp. (ATCC 50594) (yellow arrow) is seen in P. caudatus being pulled into the predator surrounded by the plasma membrane of the predator (Formation of the large posterior food vacuole (Fv) in predator  and FiguColpodella sp. (ATCC 50594) trophozoite disintegrates resulting in loss of the flagella and organelles to form the pre-cyst formed from the posterior food vacuole (Fv), remnant cytoplasm and nucleus (Colpodella sp. (ATCC 50594) with developing trophozoites (DT) are shown in Colpodella sp. (ATCC 50594) cysts can have both asymmetric and symmetric division as both odd and even-numbered juvenile trophozoites were observed within mature cysts. In Following myzocytosis, the anterior end of the  nucleus K. The yoP. caudatus is shown in P. caudatus (Colpodella sp. (ATCC 50594).The thin cyst wall is indicated by black arrows. A trophozoite of caudatus U can be Colpodella sp. (ATCC 50594), we performed immunofluorescence assay (IFA) using Plasmodium sp. and Toxoplasma gondii specific antibodies (Colpodella sp. (ATCC 50594). In order to determine if proteins associated with host cell invasion in pathogenic apicomplexans are shared by the free-living tibodies . We inveColpodella sp. (ATCC 50594) trophozoites (red arrow) and the kinetoplast (gold arrowhead) and nucleus (grey arrowhead) of prey P. caudatus  and antiserum FL against the RhopH3 protein of P. berghei (red) reacted with structures in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites. Antibody reactivity was also observed in the tubular tether with what appears to be a spherical structure in the tube. Anti-RhopH3 antibody reactivity was also observed in the cytoplasm of the prey  trophozoites and within the cytoplasm of the trophozoite (Colpodella sp. (ATCC 50594) identified by antibody reactivity as particulate circular structures could be seen . Antisera were diluted at 1:50, 1:100, 1:200, 1:500, and 1:1000. Anti-IMC3 FLR which recognizes the full-length antigen was reactive with proteins in the cysts and trophozoites of Colpodella sp. (P. caudatus. Colpodella sp. (ATCC 50594) two-way cysts were reactive with anti-IMC3FLR (yellow arrows). Red arrows show weak to no reactivity in P. caudatus. Colpodella sp. (ATCC 50594) trophozoites were reactive with the antibody  trophozoite reacted with anti-IMC3 FLR. Both flagella are shown . IMC7 is another inner membrane complex protein identified in apicomplexan parasites. There was no reactivity with anti-IMC7 antibody as the reactivity was observed as background reactivity (Colpodella sp. (ATCC 50594)  and prey (red arrow) Colpodella sp. trophozoites and weakly on P. caudatus trophozoites  E–H. The hozoites A–H.Colpodella sp. (ATCC 50594) trophozoites was observed towards the apical end (Colpodella sp. (ATCC 50594) trophozoites was identified by white arrows and the trophozoites showing strong anti-AMA1 reactivity are identified by yellow arrows. Parabodo caudatus trophozoites (red arrows) showed weak to no reactivity with the antibody. DAPI stained cytoplasmic contents aspirated from the prey were identified  at the apical end of the cell and weakly in P. caudatus (P. caudatus and the nucleus of Colpodella sp. (ATCC 50594) trophozoite (white arrow) is shown in Colpodella sp. (ATCC 50594) trophozoites (Parabodo caudatus with weak reactivity was identified (red arrows) and a group of four Colpodella sp. (ATCC 50594) (indicated by yellow arrowhead) feeding on a single prey (red arrow) were identified. Antibody reactivity was strongest at the apical ends of Colpodella sp. (ATCC 50594) trophozoites  pre-cyst stages (Colpodella sp. (ATCC 50594) reacted with anti-plasmepsin II  trophozoite (yellow arrow) attached to P. caudatus prey (red arrow)  trophozoite (white arrow) were identified by DAPI staining.There was no reactivity observed with normal mouse C,G and rd arrow) A,E. The Colpodella sp. (ATCC 50594) was grown in tissue culture flasks in Hay medium [Colpodella sp. (ATCC 50594) cultures. These four replicates revealed that P. caudatus excysts much earlier than the predator approximately four hours after subculturing and dominates in the culture until about 20 h when majority of Colpodella sp. (ATCC 50594) trophozoites begin to egress from their cysts. Young trophozoites of Colpodella sp. (ATCC 50594) emerge and begin myzocytosis lasting between 20 and 30 h in culture. At 28 h, P. caudatus trophozoites begin to encyst with Colpodella sp. (ATCC 50594) trophozoites beginning to encyst at 30 h. By 36 h the culture is mostly quiet with only a few predator and prey trophozoites remaining. Clear zones surrounding Colpdella sp. (ATCC 50594) cysts, separating them from bacteria were observed. It is unclear if these zones represent anti-bacterial activity or artifacts of fixation. Additional investigations will be required to understand the identity and significance of these structures. These type of experiments have not been performed previously for any Colpodella species. Recent investigations in our lab identified previously undocumented life cycle stages in the di-protist culture using Sam-Yellowe’s trichrome stains, confocal and DIC microscopy and TEM [Colpodella sp. (ATCC 50594) shows an irregular dual-colored, dark blue-purple and white colored cyst which we designated a demilune cyst [Colpodella sp. (ATCC 50594) cysts stained dark-red blue and contained two or more juveniles [In this study, y medium ,20 and usp. ATCC 0594 was y medium  stains tune cyst . The stauveniles . These dColpodella sp. (ATCC 50594) cysts were single nuclei mature cysts. Not all Colpodella sp. (ATCC 50594) cysts were mature with a single nucleus, and some had multiple asymmetric nuclei or symmetric nuclei with two or more juvenile trophozoites. Early Colpodella sp. (ATCC 50594) demilune cysts were also counted but there only seemed to be a relatively small number. Even though these cultures were primarily resting, free swimming predator and prey trophozoites, encysting and excysting during these time points, were also detected. The most active part of the life cycle was between 20 and 30 h, with peak activity observed between 20 and 28 h. The cultures looked the same from 36 to 40 h. Knowledge of a general life cycle reflecting transition times in culture will aid future investigations of Colpodella sp. (ATCC 50594) and help in the identification of infective stages in opportunistic human infections caused by Colpodella species. Furthermore, Colpodella species in ticks that feed on animals, with potential for zoonotic infections in humans, can be identified.The second and third time course experiments focused on the most active time period of the life cycle and identification of the predominant cyst stage present in resting cultures, respectively. Predator and prey remain encysted until they are sub-cultured and are viable up to 14 days. The cells must be sub-cultured by 14 days or the cysts start to deteriorate, and the cell yield is low. Smears from di-protist cultures stained from pooled day five and seven cultures were counted to determine the percentage of each cyst stage present in the resting culture. Eighty to ninety percent of the Colpodella species [Colpodella sp. attaching to one prey may be connected to the smaller number of prey available when the majority of Colpodella sp. (ATCC 50594) trophozoites egress from cysts. Colpodella sp. (ATCC 50594) trophozoites exceed the number of P. caudatus trophozoites because of early prey encystation. This has only been observed in culture and may be different in the natural environment and in different culture conditions as summarized in C. vorax shows one to two trophozoites feeding on P. caudatus resulting in cysts that contain two to four juveniles [C. turpis and C. pugnax were shown to have conjugation in their life cycles along with the formation of cysts containing two or four juveniles [C. unguis, oblique-transverse fission was identified in the life cycle [Colpodella sp. (ATCC 50594). Knowledge of the timing of life cycle stage transitions will also facilitate investigations of organelles by isolating them. Colpodella species from natural habitats and their life cycle timing and stages can be investigated.Single predator prey attachments were the most common pairings observed, similar to reports in other  species ,3,5. Twouveniles . In C. ufe cycle ,14. In tColpodella sp. (ATCC 50594) life cycle stages. Asymmetric and asynchronous multinucleate cyst stages were identified and Colpodella sp. (ATCC 50594) trophozoites in myzocytosis with P. caudatus prey were identified. Different stages of predator-prey attachment were observed, from initial attachment of the pseudo-conoid to the prey’s plasma membrane and reorganization of microtubules and apical complex organelles at the point of attachment, after which the membrane of the prey is engulfed, pulled into the predator, degraded, and cytoplasmic contents of the prey flow into the predator’s cytoplasm toward the posterior food vacuole. The extended plasma membrane of the predator with foci of microtubular organization was observed. Myzocytosis in Colpodella vorax also showed microtubules at the point of attachment of predator and prey [In the current study, transmission electron microscopy was performed to investigate the ultrastructure of and prey . In the Colpodella sp. (ATCC 50594) and P. caudatus cysts. The cysts of Colpodella sp. (ATCC 50594) have a thin cyst wall and a remnant food vacuole associated with undifferentiated trophozoites. The electron microscopy images confirmed that cysts of Colpodella sp. (ATCC 50594) can have asymmetric division as shown previously [Colpodella sp. (ATCC 50594) cysts is asynchronous. Not all the trophozoites develop and differentiate at the same rate within the cyst. Inside the cysts Colpodella sp. (ATCC 50594) trophozoites that are more developed can be visualized showing a pseudo-conoid, mitochondria, and defined nucleus. P. caudatus cysts can be differentiated by the thickness of their cyst wall. They are also seen to have a nucleus and kinetoplast. Both protists show parts of their flagella inside the cysts.Electron microscopy images identified the ultrastructure of sp. ATCC 0594 and Colpodella sp. (ATCC 50594) are summarized as shown from steps A–J’ in Colpodella sp. (ATCC 50594) trophozoites attach to P. caudatus prey (A) and feed by myzocytosis, after which trophozoites begin to undergo encystation by first forming the pre-cyst  which differentiates into the early demilune cyst (D). The most active stage of the life cycle in culture is between 20–30 h after subculture with peak activity at 20–28 h. Colpodella sp. (ATCC 50594) encyst in culture to form transient and long-term cysts. Transient cysts excyst and release trophozoites in the active phase of the culture. Long term (mature resting) cysts can survive in culture for up to 14 days (E). The demilune cysts (D) mature and divide to form two trophozoites (F) then divide to form more than two trophozoites . Multinucleate cysts can contain up to ten nuclei. Cysts with four and seven nuclei are depicted . Trophozoites egress as individual trophozoites  or egress as a pair still attached at the anterior end with incomplete cytokinesis (J’). The duration of the life cycle in culture is 36 h.Based on the life cycle stages identified by Sam-Yellowe’s trichrome staining, DIC microscopy, DAPI staining, ultrastructure investigated by TEM and time course experiments, the transitions through the life cycle of Colpodella species.Not much is known about the process of cell division inside the cysts of Colpodella sp. (ATCC 50594) trophozoites within the cyst, with immature and mature juveniles present within the same cyst. Furthermore, flagella were also identified within the cyst. Mitosis in C. vorax was described as semi-open resulting in two and four trophozoites [Chromera velia also produce more than four juveniles [Closed mitosis is characteristic of apicomplexans where the nuclear envelope remains intact and the mitotic spindle is intranuclear ,31,32,33uveniles , with thuveniles .P. falciparum in Colpodella sp. (ATCC 50594) life cycle stages, we performed immunofluorescence and confocal microscopy. Antibodies specific for apical and non-apical complex proteins of P. falciparum and non-apical complex proteins of Toxoplasma gondii was used in IFA with DAPI used to stain the nuclei of the predator and prey. The morphology of each protist was identified by DIC microscopy. Antiserum 686 specific for the RhopH3 rhoptry protein reacted with Colpodella sp. (ATCC 50594) trophozoites in free swimming trophozoites and in trophozoites attached to P. caudatus in myzocytosis, similar to previous reports [Colpodella sp. (ATCC 50594), at the points of attachment of predator and prey and within prey attached to Colpodella sp. (ATCC 50594). The microneme specific antibodies against AMA-1 [Colpodella sp. (ATCC 50594) trophozoites. The IFA data showing cross reactivity of apical complex protein specific antibodies is suggestive of a similar use of apical complex proteins for myzocytosis as reported for zoite invasion into host cells in parasitic apicomplexa. Additional investigation using Colpodella sp. (ATCC 50594) specific antibodies and molecular characterizations will determine if the predator–prey interactions observed use similar mechanisms to the zoite–host cell interactions recognized in pathogenic apicomplexan where intracellular host cell infections are dominant. The use of microneme and plasmepsin II specific antibodies in immunofluorescent assay studies to characterize Colpodella species proteins has not been performed previously. The specific structures containing the cross-reactive proteins are unknown. However, the cross reactivity of antibodies against apical complex proteins with Colpodella sp. (ATCC 50594) proteins suggests that events of myzocytosis may have preceded events that led to zoite internalization within host cells in intracellular parasitism. Discrete spherical and particulate structures were identified with RhopH3 specific antibodies in Colpodella sp. (ATCC 50594). There are no antibodies against apical complex or food vacuole proteins of Colpodella species. Antibodies against IMC3 cross reacted with proteins on Colpodella sp. (ATCC 50594) with a more diffuse staining pattern on the cells. There was no reactivity with antibodies against IMC7 and Py235. Interestingly, the antibodies against plasmepsin II, an aspartate protease found in the food vacuole of P. falciparum, reacted with proteins in the pre-cyst stages of Colpodella sp. (ATCC 50594) suggestive of a similar localization to the posterior food vacuole of Colpodella sp. (ATCC 50594) [Colpodella sp. (ATCC 50594) [In order to identify cross reactivity of antibodies specific for invasion and food vacuole proteins of  reports . The sphst AMA-1  and EBA1st AMA-1  reacted sp. ATCC 0594 lifeColpodella sp. (ATCC 50594) are unknown. Identifying genes conserved in Colpodella sp. (ATCC 50594) that have been identified among the parasitic apicomplexans can help with phylogenetic studies. Reactivity of antibodies against the microneme proteins, EBA175 and AMA1 used for merozoite invasion in P. falciparum will need to be confirmed by identifying the genes encoding these proteins in Colpodella sp. (ATCC 50594). The gene encoding AMA1 is highly conserved among the parasitic apicomplexans [Perkinsus marinus, identified liver stage antigen 3 and merozoite surface protein 3 of P. falciparum and Py235 rhoptry protein of P. yoelii [Plasmodium sp. cross-reacted with apical proteins in Colpodella sp. (ATCC 50594) and Voromonas pontica [The type of genes activated and what proteins are expressed during each stage of the life cycle of mplexans ,36,37. A. yoelii . In prev pontica ,40. Thes


The non-genomic actions of T3 have been reported but are not yet completely understood. Acute T3 treatment significantly enhanced basal, maximal, ATP-linked, and proton-leak oxygen consumption rates (OCRs) of primary differentiated mouse brown adipocytes accompanied with increased protein abundances of uncoupling protein 1 (UCP1) and mitochondrial Ca2+ uniporter (MCU). T3 treatment depolarized the resting mitochondrial membrane potential (Ψm) but augmented oligomycin-induced hyperpolarization in brown adipocytes. Protein kinase B (AKT) and mammalian target of rapamycin (mTOR) were activated by T3, leading to the inhibition of autophagic degradation. Rapamycin, as an mTOR inhibitor, blocked T3-induced autophagic suppression and UCP1 upregulation. T3 increases intracellular Ca2+ concentration ([Ca2+]i) in brown adipocytes. Most of the T3 effects, including mTOR activation, UCP1 upregulation, and OCR increase, were abrogated by intracellular Ca2+ chelation with BAPTA-AM. Calmodulin inhibition with W7 or knockdown of MCU dampened T3-induced mitochondrial activation. Furthermore, edelfosine, a phospholipase C (PLC) inhibitor, prevented T3 from acting on [Ca2+]i, UCP1 abundance, Ψm, and OCR. We suggest that short-term exposure of T3 induces UCP1 upregulation and mitochondrial activation due to PLC-mediated [Ca2+]i elevation in brown adipocytes.Thyroid hormones, including 3,5,3′-triiodothyronine (T Adipose tissues are traditionally divided into two types: brown adipose tissue (BAT) and white adipose tissue (WAT), based on the differences in stem cell origin, anatomy, morphology, cell structure, and function . While tUCP1 is highly expressed in BAT. This channel is located on the mitochondrial inner membrane and allows protons to translocate from the intermembrane space to the matrix. The proton currents via UCP1 dissipate energy as heat production, which is uncoupled with substrate oxidation, electron transport chain (ETC) activities, and ATP production. In BAT, UCP1 abundance accounts for about 10% of the mitochondrial membrane protein, while UCP2 and UCP3 constitute only about 0.01~0.1% ; therefo4) and its more active form 3,5,3′-triiodothyronine (T3), have been widely accepted and applied in clinical practices because of their wide spectrum of effects on the regulation of cellular metabolism, cell structure, and membrane transport i increase in a Krebs–Ringer bicarbonate (KRB) solution containing 1.5 mM Ca2+. The response was attenuated but maintained in a Ca2+-free KRB -induced ER Ca2+ release, transient receptor potential channels (TRPC), and store operated Ca2+ entry (SOCE) i elevation. It has been reported that T3 increases [Ca2+]i within several minutes in myoblast cell lines and thyroid hormone receptor βA1-expressing oocytes [3 was reported to participate in T3-induced [Ca2+]i increase, there has not been any direct evidence demonstrating the involvement of PLC as a downstream signal of non-genomic T3′s action i-bound CaM led to the upregulation of mitochondrial protein abundance. Additionally, direct stimulation of mitochondrial metabolism by MCU-mediated mitochondrial Ca2+ uptake also participated in the activation of mitochondrial respiration. These two signals originated from T3–induced [Ca2+]i elevation due to PLC-mediated intracellular Ca2+ release and extracellular Ca2+ entry.Acute treatment of T2+ solution activates the entire mitochondrial oxidative phosphorylation pathway in a dose-dependent manner. In particular, at the optimal concentration of Ca2+ (840 nM), the conduction of complex IV increases 2.3-fold, complexes I and III 2.2-fold, and ATP synthase is 2.4-fold [3-induced high [Ca2+]i activates not only the electron transport chain but also ATP production. However, the increase in the proton leak current in brown adipocytes may occur as a secondary consequence of increased UCP1 abundance and the phosphorylation of AMPK and its downstream acetyl-CoA carboxylase (ACC) i changes and mitochondrial activities.According to the OCR measurement results, T3 increases mitochondrial protein abundance and activates mitochondrial respiration in a PLC-mediated [Ca2+]i elevation-dependent manner in brown adipocytes. This Ca2+ signaling regulates not only mitochondrial metabolism via MCU-mediated Ca2+ uptake but also autophagic protein degradation leading to the accumulation of mitochondrial proteins, possibly contributing to acute thermogenic stimulation. Identifying the binding partners of T3 to induce these activations could be an effective therapeutic strategy for treating metabolic and age-related diseases i was analyzed with MetaFluor 6.1 software . At the end of each measurement, 10 μM ionomycin dissolved in KRB was perfused as a positive control.The immortalized cells were seeded on poly-473Ser) , p-AKT (308Thr) , AKT , and PPARγ  from Cell Signaling Technology  and β-actin  and PGC1α  from Abcam . All primary antibodies were diluted to 1:1000 in 5% BSA dissolved in a solution of 0.1% Tris-buffered saline and Tween 20 (TBST). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody against mouse or rabbit IgG  diluted in 6% skim milk in TBST for 1 h at room temperature (23 °C). Immunoreactive bands were detected by using ECL solution  and Chemi Doc XRS+ imaging system and were analyzed using Image Lab software 6.0 .The cells were washed with cold PBS on ice and lysed with cold Pro-prep buffer  supplemented with phosphatase and protease inhibitors . Lysates were centrifuged at 15,000 rpm for 20 min at 4 °C to collect supernatants. Protein concentration was measured using a BCA protein assay kit  before being electrophoresed on SDS-PAGE gels with an equal amount of protein in each well. The proteins were then electroblotted onto a polyvinylidene difluoride membrane . The membrane was blocked in 6% skim milk for 1 h at room temperature before being incubated with a primary antibody at 4 °C for 18 h. The following primary antibodies were used: p-p70S6K (Thr389) , p70S6K , p-ACC (Ser79) , ACC , p-AKT  by following the manufacture’s protocol.Brown adipocytes were lysed by RiboEx . The lysates were mixed with chloroform and centrifuged to collect the upper aqueous phase containing mRNA. A rough mRNA pellet was collected by centrifuging the mixture of this aqueous phase with isopropyl alcohol after incubating at −80 °C for 1 h. The mRNA was roughly washed with 75% ethanol to purify and re-dissolved in 0.1% diethylpyrocarbonate (DEPC)-treated water. Complementary DNA was synthesized from total RNA with ReverTra AceActb (β-actin) was used as an internal control. qPCR experiments were conducted with Power SYBR Green PCR Master Mix  by following the manufacturer’s protocol and real-time PCR system . Data analysis was performed by the ∆∆CT method.The mRNA levels of brown adipocytes were checked by quantitative real-time PCR (qPCR) using sequence-specific primers , a lipophilic cationic dye. Due to positive charges and lipophilic characteristics, JC-1 monomers concentrate to mitochondria where the membrane potentials are extremely negative (about −150 to −200 mV) and aggregate into polymers. While the monomers were excited at a wavelength of 490 nm and emitted 535 nm green fluorescence, the excitation and emission wavelengths of the polymers were 535 nm and 590 nm (red fluorescence), respectively. 3 solution (in KRB) for 30 min before the measurement. The fluorescence signals were recorded by a fluorescence microplate reader . The ∆Ψm were estimated by a ratio of red/green (590 nm/535 nm).Brown adipocytes were seeded in 96-well black polystyrene microplates  a day before the experiment. The cells were incubated with 2 μM JC-1 in KRB solution for 30 min and then changed to Tt-test and one-way ANOVA (Tukey’s posthoc test) using GraphPad Prism 9.0 . Data were presented as mean ± SEM. p values below 0.05 were considered statistically significant.The statistical analysis was conducted using two-tailed unpaired Student’s 3 increases [Ca2+]i via PLC activation. Elevated [Ca2+]i increases mitochondrial respiration directly through mitochondrial Ca2+ uptake and metabolic activation and also through the increased abundance of mitochondrial proteins such as UCP-1. We suggest that [Ca2+]i bound CaM activates mTOR signaling and inhibits autophagic degradation leading to increased mitochondrial protein abundance.In this study, we demonstrated that the acute exposure of brown adipocytes to T


Results: After 7 days of I/R, the cell contraction was significantly lower in cardiomyocytes compared to sham-treated mice. It was accompanied by a significant decrease in both systolic Ca2+ transients and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) activity measured as Ca2+ transients decay. Moreover, the incidence of pro-arrhythmic events, measured as the number of Ca2+ sparks, waves or automatic Ca2+ transients, was greater in cardiomyocytes from mice 7 days after I/R than from sham-treated mice. Ca2+ mishandling related to systolic Ca2+ transients and contraction were recovered to sham values 15 days after I/R, but Ca2+ sparks frequency and arrhythmic events remained elevated. Conclusions: Renal I/R injury causes a cardiomyocyte Ca2+ cycle dysfunction at medium (contraction-relaxation dysfunction) and long term (Ca2+ leak), after 7 and 15 days of renal reperfusion, respectively.Background: Acute renal failure (ARF) following renal ischemia-reperfusion (I/R) injury is considered a relevant risk factor for cardiac damage, but the underlying mechanisms, particularly those triggered at cardiomyocyte level, are unknown. Methods: We examined intracellular Ca Acute renal failure (ARF) caused by renal ischemia-reperfusion (I/R) injury is a complex clinical entity related to a variety of clinical and surgical settings, including sepsis and kidney transplantation ,2,3, andThe complex physiological relationship that exists between the kidneys and the heart has long been appreciated, and damage to one organ may induce damage to the other, which has been termed the cardiorenal syndrome. This coexistence of cardiac and renal dysfunction has been classified into 5 types of cardiorenal syndrome based on the initial injured organ and the range of the conditions (acute or chronic) . AccordiRenal ischemia typically occurs in the setting of renal infarction, sepsis or kidney transplantation. It is often followed by an inflammatory state, redox imbalance and changes to the structure and function of renal cells, including apoptosis . These f2+ handling that mediates membrane excitation and contraction (EC)-coupling during the cardiomyocyte contraction-relaxation. EC-coupling transforms electrical signals into mechanical force, which relies on tightly coordinated Ca2+ release and reuptake in cardiomyocytes [2+ entry through L-type Ca2+ channels from the extracellular medium. This small increase in cytosolic Ca2+ concentration triggers a pronounced release of Ca2+ from the sarcoplasmic reticulum (SR), given the opening of ryanodine receptors type 2 channels (RyR2), in a physiological process termed Ca2+-induced Ca2+ release [2+ is transported back from the sarcoplasm to the SR. This process occurs principally by Ca2+-ATPase 2a pump (SERCA2a Ca2+) re-uptake and also by Ca2+ extrusion to the extracellular medium by the Na+/Ca2+ exchanger [2+ handling are related to direct cardiac injury during HF by stimulating ventricular dysfunction and arrhythmia [2+ homeostasis are well known during HF, playing a principal role in causing mechanical dysfunction and arrhythmia, little is known about the potential cardiac Ca2+ imbalance in ARF. Heart function is tightly controlled by the intracellular Camyocytes . Cardiac release , inducinxchanger . Alteratrhythmia . While d2+ handling would be clinically useful to better understand the extrarenal physiological consequences of ARF on the heart and may allow early intervention focused on protecting the myocardium in patients after I/R injury. Accordingly, the aim of the present study was to investigate whether ARF triggers relevant cardiac features related to cardiomyocyte dysfunction and intracellular Ca2+ handling in an experimental model of unilateral I/R injury for 7 and 15 days.Deciphering the intracardiomyocyte alterations linked to Cap < 0.001). In contrast to LKW, the right kidney weight (RKW)/BW ratio was significantly higher in the 7 d I/R and 15 d I/R groups than in the sham group , and RKW/TL was significantly increased in the 15 d I/R group (p < 0.05). These results indicate hypertrophy of the right kidney, likely because of compensational mechanisms to maintain renal function. In the same line, the HW/BW and HW/TL ratios were significantly higher in mice after 7 (p < 0.05) and 15 (p < 0.05 and p < 0.01) days of renal I/R than in sham mice. We used both BW and TL in order to ensure that we normalized with respect to the growth of the animal (TL) and not only with BW. As the tibia is a non-variable measurement at short-term, the changes are more accurately quantified by relating heart/kidney weight to tibia length than to body weight after the I/R surgery.We employed an experimental model of ARF to comprehensively analyze the cardiac consequences of acute unilateral renal I/R through the occlusion of the left renal pedicle. Macroscopic parameters of each experimental group are shown in p < 0.05, respectively). FGF-23 levels were significantly higher in the 15 d I/R group than in the sham group (p < 0.01), and FGF-23 levels were also significantly elevated when compared with levels in the 7 d I/R group (p < 0.01). Likewise, phosphate levels were significantly higher in the 15 d I/R group than in the 7 d I/R group (p < 0.05).We next examined renal damage by analyzing the plasma levels of urea, BUN, FGF-23 and phosphates . Urea anp < 0.001; p < 0.001; p < 0.001) and were closer to those observed in cardiomyocytes from sham mice  was significantly lower in cardiomyocytes from the 7 d I/R mice than from the equivalent sham mice  in cardiomyocytes was significantly longer in the 7 d I/R group than in the sham group  than in cells from sham mice. Analysis of the morphological properties of Ca2+ sparks showed that the amplitude (peak F/F0) and full width was significantly lower in cardiomyocytes from 7 d I/R mice than from sham mice . Both parameters recovered 15 days after renal I/R and were similar to those of sham cardiomyocytes . No differences were found in the duration of Ca2+ sparks between the three experimental groups  in quiescent cardiomyocytes. Two representative examples of Ca2+ waves are shown in 2+ events revealed that the occurrence of SCR was significantly higher after ARF, both at 7 (p < 0.01) and 15 (p < 0.001) days. There is a direct association between altered intracellular Ca2+ handling and a predisposition to fatal ventricular arrhythmias.We next analyzed spontaneous Ca2+ waves or transients evoking extra-contraction and missing Ca2+ transients in ventricular cardiomyocytes stimulated at 2 Hz for three cycles, each separated with a non-pacing period. Representative line-scan images of a normal cellular response corresponding to a cardiomyocyte from a sham-treated mouse is shown in 2+ dependent pro-arrhythmic events were significantly greater in cardiomyocytes from the renal I/R group (more than 30% of cells showed events) than cardiomyocytes from sham mice, both at 7 and 15 days following renal I/R (p < 0.001).Accordingly, we next evaluated the presence of pro-arrhythmic behavior as spontaneous Ca2+ mishandling, manifested as cardiomyocyte contractile dysfunction 7 days after left unilateral renal occlusion, which recovers 15 days after the renal I/R. Likewise, ARF increases the incidence of cardiomyocyte pro-arrhythmic Ca2+ events in the heart after 7 and 15 days in vitro, indicating that aberrant diastolic Ca2+ leak, and thus the risk of arrhythmic events, continues in the long term. The findings of this study are corroborated by other studies from our group that demonstrated the efficiency of renal I/R to promote arrhythmogenic events in vivo using the isoproterenol challenge after 15 days of reperfusion [In the present study, we show that ARF induces intracellular Ca2+ handling are an important platform to investigate cardiomyocyte contractility. Even though they are very often described in other cardiac diseases, such as HF, the mechanisms surrounding Ca2+ handling dysfunction have not yet been studied much in the context of acute cardiorenal syndrome type 3. ARF is associated with an unacceptably high rate of mortality despite being recognized for more than 40 years ,16. As y2+ handling in ventricular cardiomyocytes in an experimental model of ARF at early (7 days) and later (15 days) stages following renal I/R injury. Our results establish an early impairment in cardiomyocyte contraction-relaxation processes after 7 days of renal I/R that is chiefly due to: (1) a decrease in systolic Ca2+ release, observed as a reduction in Ca2+ transient amplitude; (2) an increase in the decay time constant of Ca2+ transients; and (3) a depressed SR Ca2+ load. The Ca2+ handling abnormalities, together with the compromised contraction-relaxation cycle in cardiomyocytes observed 7 days post-renal I/R, are similar to what is observed in several models of HF [2+ reuptake into the SR is impaired in ventricular cardiomyocytes after 7 days of renal I/R, which is considered an early phase of kidney injury. This could be explained by the reduction in expression or activity of SERCA2a. Similar to that which occurs in HF [2a function might explain the depletion of Ca2+ stores in the SR, the lower transient amplitudes and the impaired cellular contractility. These activities recovered 15 days after renal I/R in mice and were similar to cardiomyocytes from sham mice. A plausible explanation for this recovery is that ARF after 15 days is considered a transition period between the acute and chronic state of post-renal injury, according to the guidelines of the Kidney Disease: Improving Global Outcomes (KDIGO) organization [2+ mishandling in systole seem to be set before the 7th day. Analyzing the adult cardiomyocytes in previous timepoints could provide us a superior understand about the acuteness of the cardiac dysfunction after renal I/R.The present study shows—for the first time, to the best of our knowledge—relevant dysfunctional Cals of HF ,20,21. Onization , and it 2+ leak during diastole is a known and shared Ca2+-dependent pathological mechanism associated with the HF progression and arrhythmias [2+ leak is a phenomenon defined as an inappropriate release of Ca2+ from the SR [2+ from SR during diastole . This increased release of Ca2+ leak reduces its availability for the subsequent contraction, thereby impairing contractility as it was observed after 7 days of renal I/R. However, despite the evident cardiac improvement in terms of systolic Ca2+ handling and contraction after 15 days of renal I/R, diastolic Ca2+ leak remains abnormally elevated.An increase in SR Cahythmias ,23. The m the SR . In path2+ leak is potentially a pathological substrate for the induction of arrhythmias, as the released Ca2+ from the SR can diffuse to neighboring RyR2 clusters to induce pro-arrhythmogenic Ca2+ events that, together with automatic contractions, ultimately trigger ventricular arrhythmias. Gambardella et al. reinforced that the RYR2 represents the central target of many pathways dysregulated in cardiac pathological conditions, including metabolic disorders, ROS production and inflammation [2+ handling and subsequent impairment in cardiac contraction. The study of stabilization of RYR2 seems to be the main target for clinical practice, being studied in models of genetic mutation in human stem cells derived cardiomyocytes [2 protein expression would be enlightening to provide a better understanding for the cardiorenal syndrome type 3 and its consequences to the heart.The increase in diastolic Caammation . All themyocytes  and as amyocytes . Evident2+ handling in adult ventricular cardiomyocytes [2+ leak from the SR in the form of Ca2+ SCR and triggered in vitro pro-arrhythmogenic activity as automatic systolic Ca2+ transients and extra-contractions. Therefore, our results suggest that the vulnerability to ventricular arrhythmias at early and longer term after I/R injury might be explained, at least in part, by the elevated and maintained circulating FGF-23 levels.A possible explanation for these results is that renal injury was sustained or worsened after 15 days of I/R, which is supported by the finding that the highest levels of FGF-23 were observed 15 days after renal I/R. FGF-23 is one of the most important components of mineral and bone metabolism and its excess is linked to the progression of renal dysfunction . FGF-23 myocytes ,35. Anal2+ cycle dysfunction in ventricular cardiomyocytes. Despite the resolution of the initial contraction-relaxation dysfunction, the Ca2+ leak observed during diastole remains as an important sequela  of experimental animals and the directive of the European Union (2010/63/EU) on the protection of animals used for scientific purposes. The General Directorate of Agriculture and the Environment of the Environment Council of Madrid approved the study (PROEX 186.5/20). Male C57BL/6 mice  were purchased from Charles River Laboratories International . Mice were group housed in cages (maximum of five) at the Experimental Animal Center, Hospital Universitario 12 de Octubre. Mice were maintained on a 12-h light/dark artificial cycle, at a constant ambient temperature of 23–25 °C, with ad libitum access to water and a standard diet.Mice underwent sham or I/R surgery using a previously described procedure ,12,15. IThe Urea Assay Kit  was used to measure plasmatic urea and blood urea nitrogen (BUN) levels. The Phosphate Assay Kit  was used to measure plasmatic phosphate. Lastly, the FGF-23 Dosing Kit  was used to measure plasmatic FGF-23 levels by ELISA. All assays were used according to the manufacturers’ recommendations.2+-free Tyrode’s solution was used to retrograde perfuse the hearts. After that, the hearts were digested using a type II collagenase solution with 1 mg/mL , as previously described [g and suspended in Tyrode’s solution. Only rod-shaped, excitable under electrical pacing and Ca2+-tolerant cardiomyocytes with clear cross-striations were selected and used for intracellular Ca2+ imaging studies.Mice were anesthetized with sodium pentobarbital-heparin  and the heart was rapidly removed. It was connected to a Langendorff perfusion system through the ascending aorta . A Ca2+-escribed . Then, h2+ was imaged by confocal microscopy  at the speed of 1.5 ms/line (1000 lines per image) for recording Ca2+ transients, Ca2+ sparks and Ca2+ wave images, and at 3 ms/line  for caffeine and arrhythmia protocols. All Ca2+ images were corrected for background fluorescence. For Ca2+ transients, the fluorescence value (F) was normalized by baseline fluorescence (F0) to obtain F/F0. The decay time constant of the Ca2+ transients, Tau, was obtained by adjusting the decay trace of the fluorescence to a single exponential. The measurement of the cell shortening was made using the difference of cardiomyocyte length between electrical stimulation and resting period. It was displayed as the percentage cell shortening length. The Ca2+ load of the SR was estimated in intact ventricular myocytes by a quick challenge with 10 mmol/L caffeine to deplete Ca2+ stores. Caffeine induces Ca2+ release by reducing the threshold for luminal Ca2+ activation of the RyR, causing a total Ca2+ release from intracellular Ca2+ stores [2+ load [2+ sparks, Fluo-3AM-loaded cardiomyocytes were maintained without electrical stimulation and Ca2+ spark regions were determined as the specific sites where the fluorescence signal briefly increased by at least four times the standard deviation of the fluorescence. Abnormal spontaneous Ca2+ release, manifested as Ca2+ waves or automatic Ca2+ transients without electrical stimulation, was quantified by the percentage of occurrence, and globally referred to as spontaneous calcium release (SCR). Three cycles of a 2 Hz electrical stimulation field were applied to record pro-arrhythmogenic activity. This consisted of seven pulses with a resting period in each cycle. Arrhythmic Ca2+ events were considered to be any spontaneous abnormal release of Ca2+ as waves, missing transients or automatic contractions corresponding to spontaneous Ca2+ transients during the protocol. All Ca2+ images were analyzed using home-made routines with IDL  and Image J  softwares.An intracellular fluorescent calcium dye Fluo-3AM  was used to load the isolated ventricular cardiomyocytes. They were field stimulated by two electrodes at 2 Hz. Intracellular Ca+ stores . This me[2+ load ,38,39. T2a antibodies at 1:2.000 dilution  and anti-GAPDH antibodies at 1:200,000 . Secondary antibodies used were anti-mouse at 1:5000 dilution .Total protein was isolated from hearts by lysing the tissue with an extraction buffer . Protein content was quantified using a BCA kit . Next, 20 μg of the extracted proteins was loaded in a 12% SDS-page gel and was electrophoresed for 180 min at a constant voltage of 120 V. Next, the proteins resolved on the gel were transferred onto a PVDF membrane using a semi-dry transfer system . The membranes were blocked with 5% skimmed milk in TBST buffer  for 1 h and incubated overnight at 4 °C with primary antibodies. After washing, the membrane was incubated with a corresponding peroxidase-conjugated secondary antibody for 1 h at room temperature. Finally, the membrane was analyzed using an ECL system . Protein bands were visualized using ImageQuant LAS4000 and band densities were measured using ImageJ software . Primary antibodies used were anti-SERCA2 test or analysis of variance (ANOVA), with a Newman-Keuls multiple comparison test used when appropriate. It was performed with OriginPro 9.0  or GraphPad Prism v6.0 . Differences with p-values < 0.05 were considered statistically significant.Data are here stated as mean ± standard error of the mean (SEM). Statistical significance was accomplished applying an un-paired Student’s test, χ


Research productivity is expected of academic faculty, and mentoring can facilitate it. This paper presents a framework for using mentoring to develop researchers in health disciplines.We utilized recent literature reviews, and experience developing researchers at an emerging research institution within the Research Centers for Minority Institutions (RCMI) program, to propose a precision mentoring (PM) framework for research development.how much improvement was due to the PM framework, over the 4 years of our program, the quality and quantity of pilot project proposals (PPP) has increased, the number of external proposals submitted and funded by PPP investigators has increased, and the number of faculty participating in our program has increased. Surveys distributed to our 2021–22 PPP applicants who did not receive funding (n = 5/6 or 86.7%) revealed that new investigators most frequently sought mentoring related to career guidance , grant proposal basics , and identifying funding opportunities.Although we cannot precisely determine We recommend shifting the mentoring paradigm such that: (a) mentees are pre-screened and re-screened for their current skill set and desired areas of growth; (b) mentoring occurs in teams vs. by individuals; (c) mentors are trained and rewarded, and (d) attention is paid to enhancing institutional culture. Research productivity, , is one of the most important components of success in higher education. Mentoring is an essential component for helping faculty succeed in higher education, and numerous literature reviews and meta-analyses have espoused the benefits of mentoring . Althoughttps://grants.nih.gov/grants/esi-status.pdf. Mid-career faculty were not included in our literature review, but they are important to include.In a recent literature review covering the 10 years of mentoring research, we examined personal, interpersonal, and institutional barriers and facilitators related to research productivity in new investigators (NI), early-stage investigators (ESI) and underrepresented minority faculty (UMF), also known as underrepresented minorities (URM) , for best practices for training research mentors.As soon as mentoring needs are identified and a plan is developed, a team of mentors is recruited. In addition to identifying members of mentoring teams, mentors should be trained in ‘best practices of mentoring’ and regularly evaluated. Readers are referred to the National Research Mentoring Network website (see: ograms/) ,14, for During months 2 and 3, those who are selected to submit full proposals have a meeting with senior faculty for an assessment of strengths and weaknesses of their proposal. Over the course of the next few months, a second meeting is scheduled with mentor teams who have content knowledge, statistical/methodological expertise, and community connections. An NIH grant writing workshop is presented to help investigators put their proposals into final format.After pilot proposals are selected, monthly meetings are held with the mentee and mentoring team to assess progress, make recommendations, and solve problems. Investigators with funded pilot projects are encouraged to submit full NIH proposals, and publish papers related to their grants. Re-assessment of mentoring goals, strategies, successes and failures occurs regularly, and re-assignment of mentors occurs when necessary. Individuals who do not receive pilot funding are queried about the mentoring they received, and they are encouraged to continue with mentoring.Researchers should continue to test the PM framework. It is important to determine whether regularly screening and individualizing the mentoring approach, having a team of mentors, training mentors, and being attentive to campus research culture will result in more successful research development. We should also study the impact of mentoring within specific disciplines and varying faculty ranks. We know that mentoring is beneficial, yet we need to go further to answer ‘how’ mentoring works. Developing additional scales to measure mediators and moderators of mentoring success would help us determine which facilitators are most important, specific to discipline, institution, and resources.