The Mitochondrial Calcium Uniporter (MCU) complex is thought to be the primary pathway for Ca2+ entry into mitochondria in eukaryotes. We investigate the hypothesis that mitochondrial Ca2+ uptake via MCU influences phototransduction and energy metabolism in photoreceptors using a mcu-/- zebrafish and a rod photoreceptor-specific Mcu-/- mouse. Using genetically encoded Ca2+ sensors to directly examine Ca2+ uptake in zebrafish cone mitochondria, we found that loss of MCU reduces but does not eliminate mitochondrial Ca2+ uptake. Loss of MCU does not lead to photoreceptor degeneration, mildly affects mitochondrial metabolism, and does not alter physiological responses to light, even in the absence of the Na+/Ca2+, K+ exchanger. Our results reveal that MCU is dispensable for vertebrate photoreceptor function, consistent with its low expression and the presence of an alternative pathway for Ca2+ uptake into photoreceptor mitochondria.Rods and cones use intracellular Ca Mitochondria have the ability to sequester Ca2+ via the Mitochondrial Calcium Uniporter complex (MCU)2. Mitochondrial Ca2+ uptake via MCU can modulate both cytosolic and mitochondrial Ca2+ levels, meaning that Ca2+-sensitive reactions that occur in both compartments can be affected by MCU activity. In the mitochondrial matrix, increasing Ca2+ can stimulate tricarboxylic (TCA) cycle dehydrogenases and enhance ATP production, although Ca2+ overload in the matrix can trigger cell death4. Mitochondrial Ca2+ uptake can also regulate cytosolic Ca2+ levels and thus influence Ca2+-sensitive cytosolic reactions. Since these Ca2+-sensitive cytosolic reactions vary widely among cell-types depending on each cell’s specific function, disruptions in mitochondrial Ca2+-uptake can have tissue-specific consequences6.Maintaining proper intracellular Ca2+ to regulate both phototransduction and neurotransmission. At the outer segment, intracellular Ca2+ changes in response to light stimulation and this is critical for timely shut-down of the phototransduction cascade and light adaptation7. At the synapse, changes in intracellular Ca2+ modulate vesicle release and neurotransmission10. The outer segment and synaptic Ca2+ pools that control these functions are separated by a cell body filled with a dense cluster of mitochondria, and evidence suggests that these mitochondria may help isolate these pools from each other. Mitochondria from zebrafish cones can prevent Ca2+ in the outer segment from reaching the rest of the cell and conversely, they can prevent Ca2+ in the synapse from reaching the outer segment11. Increasing mitochondrial Ca2+ uptake in zebrafish cones by overexpressing MCU also accelerates cytosolic Ca2+ clearance, which causes cones to recover from light exposure faster12.Photoreceptors, the light-sensitive neurons in the retina, rely on spatially distinct changes in cytosolic Ca2+ into mitochondria could also enhance the ability of photoreceptors to meet their high energy demands. Darkness is the most energetically demanding state of the photoreceptor, and intracellular Ca2+ is at its highest levels in darkness14. If this Ca2+ were to enter mitochondria, it could enhance ATP production by stimulating TCA cycle dehydrogenases3. In line with this hypothesis, overexpressing MCU in zebrafish cones alters the distribution of TCA cycle metabolites in a way which is consistent with enhanced activity of Ca2+-sensitive dehydrogenases12.Efficient uptake of Ca2+ uptake could play an important role in modulating photoreceptor function. Given this, it is surprising that zebrafish cone photoreceptors express extremely low levels of MCU12. MCU has been widely thought to be the sole route of Ca2+ entry into mitochondria in eukaryotes, as loss of MCU completely inhibits mitochondrial Ca2+ uptake in skeletal muscle, liver, heart, brown adipose tissue, and a wide variety of cell lines19. Despite this, it is possible that certain specialized cell types might not rely solely on MCU for mitochondrial Ca2+ uptake, since it also has been observed that brain mitochondria lacking MCU expression do not have a complete loss of mitochondrial Ca2+ uptake20. Thus, the role that MCU plays in vertebrate photoreceptors remains an open question.These observations suggested to us that mitochondrial Ca2+ uptake plays in modulating photoreceptor function, we evaluated the morphological, biochemical and physiological consequences of loss of MCU expression using both a global mcu-/- zebrafish model and a rod photoreceptor-specific Mcu-/- mouse model. Our study demonstrates that MCU is remarkably dispensable for photoreceptor function, which is consistent with our discoveries of very low levels of MCU expression and evidence for an additional mechanism for Ca2+ uptake into photoreceptor mitochondria.To resolve the role MCU-mediated mitochondrial Camcu-/- zebrafish so that we could take advantage of well-established ex vivo retinal Ca2+-imaging tools and techniques to determine the role Mcu plays in modulating cytosolic and mitochondrial Ca2+ dynamics in cone photoreceptors21. Global mcu-/- zebrafish were generated using a CRISPR-Cas9 strategy outlined previously22. A founder carrying a 12-nucleotide deletion in exon 5, which introduces a premature stop codon in exon 6 of mcu was used in this study 11. Retinal slices from mcu-/- gnat2:GCaMP3 and wild-type (WT) gnat2:GCaMP3 siblings were incubated in Krebs–Ringer Bicarbonate (KRB) buffer containing 0 mM CaCl2 and 0.4 mM EGTA for 10 min. Cones were then imaged to establish a baseline fluorescence reading prior to delivery of a 5 mM bolus of CaCl2  was significantly smaller compared to WT (0.02433 s−1), indicating that mcu-/- cones clear cytosolic Ca2+ at a slower rate compared to WT cones. This indicates that Mcu plays a role in mediating mitochondrial uptake of cytosolic Ca2+ in zebrafish cones.To determine what role Mcu plays in clearing cytosolic Ca2+ levels in mcu-/- photoreceptors, we next crossed mcu-/- zebrafish with zebrafish expressing the mitochondrially-targeted Ca2+ sensor GCaMP3 in cones (gnat2:mito-GCaMP3)11. Eyes from live larvae were imaged and we found that there is no difference in basal mito-GCaMP3 fluorescence between WT and mcu-/- cones  to the maximum fluorescence  and minimum fluorescence . Similar to larvae, we found that baseline mito-GCaMP3 fluorescence was not significantly different in adult WT and mcu-/- zebrafish cones . The mcu-/- cone mitochondrial clusters which do respond to sildenafil treatment exhibited a smaller maximum fold change in mito-GCaMP3 fluorescence compared to WT mitochondrial clusters which respond to sildenafil . There is heterogeneity in the timing of the mito-GCaMP3 fluorescence increase from within seconds to many minutes after sildenafil addition in both WT and mcu-/- cone mitochondrial clusters. However, since mcu-/- mitochondrial clusters respond later on average, this suggests that a different biological threshold (such as the size of the cytosolic Ca2+ load or the amount of time it is sustained) must be met in order for an mcu-/- mitochondria to begin taking up Ca2+.In 2+ uptake exhibited by mcu-/- cone mitochondrial clusters in this assay could be attributed to an Mcu-independent mechanism, we repeated this experiment in the presence of the Mcu inhibitor Ru360 . Compared to untreated mcu-/- mitochondrial clusters, mcu-/- + Ru360 mitochondrial clusters do not have a significantly different maximum fold change in fluorescence or TResponse  is not sensitive to Ca2+ uptake in cones, but that Ca2+ can also enter cone mitochondria through an alternative pathway.Taken together, these results show that Mcu contributes to some mitochondrial Camcu-/- mitochondria to take up Ca2+ in cones might lead to metabolic or electrophysiological defects. To assess potential changes in metabolism, we measured total metabolite levels from freshly dissected dark-adapted retinas from mcu-/- zebrafish. We detected no changes in total metabolite levels in mcu-/- retinas, although α-ketoglutarate levels trend slightly but not significantly higher after loss of Mcu , which is attributed to diminished activity of the Ca2+-sensitive phosphatase PDP1c27. We assessed the P-PDH/PDH ratio and found that it is not different between WT and mcu-/- retinas . Recovery kinetics are unaltered by Mcu deficiency  with mice expressing Cre-recombinase under control of a rod-specific opsin promoter (iCre-75) which is active once rods have differentiated32. Immunohistochemistry of adult Mcufl iCre-75 (annotated as Rod Mcu-/-) retinas reveal that MCU expression is ablated in the rod photoreceptors   Fig. B. We obs12. To determine if MCU expression in mouse rods is similarly low, we analyzed MCU expression in WT and Rod Mcu-/- retinas using immunoblot  analysis of Rod Mcu-/- retinas at 6-months old revealed no changes in retinal morphology  and isocitrate dehydrogenase12. To test if the reduced mitochondrial Ca2+ uptake ability of Mcu-/- photoreceptors causes a subsequent increase in enzyme Km, we used gas chromatography–mass spectrometry (GC–MS) to evaluate the influence of MCU on metabolic flux in retinas. We incubated retinas from WT and Rod Mcu-/- light-adapted mice in 5 mM U-13C-glucose for 0, 5, and 30 min and quantified accumulation of unlabeled and labeled metabolites using GC–MS  . In WT retinas, steady-state levels of many metabolites are slightly but significantly lower in darkness relative to light, with α-ketoglutarate and pyruvate levels being the most reduced  . When comparing the fold change in metabolite abundance in darkness relative to light, it appeared as though the influence of darkness in Rod Mcu-/- retinas tended to be smaller than that of WT retinas. This would be expected if Ca2+ uptake via MCU mediated the change in metabolite pool size we observe in darkness relative to light. To test if this were true, we directly compared the fold change in steady-state metabolite levels in darkness and light between WT and Rod Mcu-/- retinas   in freshly dissected and snap-frozen dark-adapted retinas and found it was unchanged in Rod Mcu-/- retinas . A summary of the response parameters in the control and Mcu-/- rods is given in Table p), integration time (Tint), and recovery time constant τrec of the flash response.We determined how much MCU normally contributes to the rod photoresponse recovery by recording ex vivo transretinal ERG responses from Rod ols Fig. A–C. The ols Fig. C, inset.2+ sequestering through MCU affects light adaptation of rods. When illuminated, photoreceptors must adapt to the light exposure in order to retain the ability to respond. Ca2+ levels in the outer segment of photoreceptors modulate the activity of several proteins to mediate this light adaptation38. To test if Ca2+ sequestering through MCU has an effect on light adaptation of rods, we exposed retinas to a series of background light steps of increasing intensity. The peak response to background light of Mcu-/- rods was comparable to controls  remained largely unaffected in Rod Mcu-/- compared to the controls across all background light levels tested  is the only known route for Ca2+ clearance from the outer segment plasma membrane, and it is thought to be the driver of Ca2+ clearance from this compartment. Since MCU expression in mouse rods is extremely low, its contribution to cytosolic Ca2+ clearance may be small compared to that of NCKX1. In an effort to unmask any possible modulation of the rod photoresponse by MCU, we investigated the effect of MCU loss on the rod photoresponse in mice lacking NCKX1. NCKX1-deficiency compromises Ca2+ extrusion in the rod outer segments and it delays photoresponse recovery following a flash stimulus29. If the residual Ca2+ extrusion in NCKX1-deficient rods is mediated by MCU, then it would be expected that the subsequent deletion of MCU would further suppress or completely block Ca2+ extrusion, causing a further delay in photoresponse recovery and suppressing light adaptation.The rod NaNckx1-/- control and Nckx1-/- Rod Mcu-/- mice  is tolerated surprisingly well by photoreceptors. MCU-deficient photoreceptors appear healthy, their TCA cycle activity is largely unaltered, and the photoresponse is preserved. In cones from global mcu-/- zebrafish, cytosolic Ca2+ is cleared more slowly and mitochondrial Ca2+ uptake is reduced in a significant population of mitochondria. However, some mitochondria still display robust increases in mito-GCaMP3 fluorescence in the absence of MCU. Ca2+ uptake in these mitochondria appears to occur more slowly and with a lower magnitude than in WT mitochondria. Consistent with this result, we found that both WT and mcu-/- cones have a population of mitochondrial clusters which still appear to take up Ca2+ even in the presence of the MCU inhibitor Ru360, and the characteristics of this population match those of the responding mitochondria in mcu-/- cones. This is similar to what has been observed in non-synaptic brain mitochondria isolated from Mcu-/- mice, in which uptake of Ca2+ into mitochondria was not blocked but instead occurred at a slower rate compared to controls20. Taken together, these results suggest that that neuronal tissue like retina and brain may have an alternative mitochondrial Ca2+ uptake pathway with different response kinetics and Ca2+ uptake capacity. Future experiments which use other strategies for measuring mitochondrial Ca2+ uptake will be critical for confirming, identifying, and assessing the role of MCU-independent mechanisms for mitochondrial Ca2+ uptake in photoreceptors.Photoreceptors have abundant mitochondria in the compartment of the cell between the nucleus and the outer segment that can influence cytosolic Ca2+ entry have been described but are not as well-characterized as MCU, as MCU is thought to be primarily responsible for mitochondrial Ca2+ influx. However, the viability of Mcu-/- mice and zebrafish, and the extremely mild consequences of MCU loss in photoreceptors we describe here, suggest that there is an alternative uptake mechanism in at least some cell types47. Letm1 is a Ca2+/H+ exchanger on the inner mitochondrial membrane that links Ca2+ influx and efflux to electron transport chain activity and mitochondrial pH48. Letm1 appears to act primarily as a Ca2+ extrusion mechanism, but it is also capable of Ca2+ influx. Knockdown of Letm1 impairs Ca2+ transport into the mitochondria of Flp-In-293 cells and patient-derived fibroblasts, and Letm1+/- mice have impaired ATP production and PDH activity specifically in neuronal tissue50. There is also some evidence that mitochondrial ryanodine receptors may exist in neurons and cardiac cells, but they have yet to be unambiguously identified52. Another candidate is the mitochondrial Na+/Ca2+ exchanger, which is associated with Ca2+ efflux but can run in reverse to promote Ca2+ entry into mitochondria53.Other candidates for Ca2+ transport is that it would link mitochondrial Ca2+ influx to factors other than cytosolic Ca2+ levels. The mitochondrial Na+/Ca2+ exchanger would couple Ca2+ entry to cytosolic Na+, which fluctuates in photoreceptors in response to light. Letm1 would tie Ca2+ entry to mitochondrial pH and ATP production, and Letm1 has even been shown to be upregulated in cancer tissue, which is highly glycolytic like photoreceptors54. Whatever the alternative pathway of mitochondrial Ca2+ uptake might be in photoreceptors, the low expression of MCU in photoreceptors raises the possibility that this alternative pathway could play a significant role in controlling photoreceptor mitochondrial Ca2+ uptake.A possible advantage of relying on these other mechanisms for Ca2+ clearance and subsequent disrupted AMP-activated protein kinase dephosphorylation47. Conversely, in skeletal muscle, loss of MCU causes a metabolic shift towards increased lipid oxidation due to PDH inhibition restricting the availability of pyruvate-derived acetyl-CoA27. These examples suggest that the basal metabolic specialization of a tissue will determine in large part the metabolic consequences of MCU loss. Since photoreceptors rely heavily on aerobic glycolysis to generate ATP, it is not entirely surprising that Rod Mcu-/- mouse retinas are metabolically quite normal and exhibit only a mild accumulation of α-ketoglutarate that does not appear to affect photoreceptor function  is shut down α-ketoglutarate levels are affected by an unknown, matrix CaAn alternative possibility is that α-ketoglutarate levels increase after loss of MCU due to decreased CaMcu-/- rods exhibit a normal photoresponse with no change in the sensitivity and kinetics of the flash response and light adaptation  of Washington University in St. Louis  and the University of Washington in Seattle (protocol number: 2050-01). Zebrafish were maintained and used in accordance with the guidelines of experimental protocols approved by the IACUC of both the University of Washington in Seattle (protocol number: 3113-02) and University of Utah (protocol number: 17-11004).Mcufl/fl iCre-negative littermates. In Fig. Mcu is not floxed. In Fig. Nckx1-/- single knockouts. In Figs. 32. MCUfl mice  were obtained from Jackson Labs. The Nckx1-/- mice used in this study were generated as described in a previous publication29. Mice were maintained on a C57BL/6 J background. Rod Mcu-/- mice used for histology were 6 and 11 months of age  and 7 AM–9 PM (spring–summer) light/dark cycle and had free access to food and water. In Figs. -/- genetic background. All wild-type fish (WT) used in analysis were age-matched siblings to CRISPR-generated mcu-/- zebrafish. Fish used for mito-GCaMP3 slice preparation were between 7 and 11 months of age, fish used for cyto-GCaMP3 slice preparation were 18 months of age, fish used for metabolic analysis were 11 months of age, fish used in ERG analysis were 7 months of age, and fish used for retinal morphology analysis were 11 months of age. Equal numbers of male and female fish were used.All fish used in this analysis were maintained in the University of Washington South Lake Union aquatics facility or the Centralized Zebrafish Animal Resource (CZAR) at the University of Utah at 27.5 °C on a 14/10 h light/dark cycle and were maintained in the Roy12. All primary antibodies were used at 1:1000, with the exception of the custom MCU antibody which was used at 1:100. Secondary antibodies used: IRDye 800CW donkey anti-rabbit IgG (H + L) ; IRDye 680RD donkey anti-mouse IgG (H + L) ; IRDye 680RD donkey anti-rabbit IgG (H + L) ; IRDye 800CW goat anti-mouse IgG (H + L) .Protein was extracted by homogenizing in RIPA buffer  and run on 14% polyacrylamide gels. After running, gels were transferred onto PVDF membranes  and briefly washed with PBS. Primary antibodies were diluted in blocking buffer  and incubated overnight on blots at 4 °C. Membranes were washed twice with PBS containing 0.1% Tween-20 and once with PBS, then incubated with secondary antibodies diluted 1:5000 in blocking buffer for 1 h at RT and washed again before imaging. Membranes were imaged and bands were quantified using the LI-COR Odyssey CLx Imaging System (RRID:SCR_014579). Primary antibodies used: PDH E1 subunit , P-PDH , mtCO1 , SDHB , and Cell Signaling MCU , custom MCU antibody57. Briefly, PBS was placed in a 37 °C water bath and all fixative tubing was flushed repeatedly with PBS to clear any bubbles. Mice were anaesthetized using 270 mg/kg of nebutol. Mice were taped to a dissection board and open-heart surgery was performed in order to insert the perfusion needle into the left ventricle. The right atrium was snipped, and PBS was perfused through the mouse using a peristaltic pump until the outflow was clear. Then, room temperature 4% PFA (prepared from 16% PFA diluted in PBS) was perfused through the mouse. After fixation, eyes were carefully cut out with curved dissection scissors, the sclera was cut off, and eyes were stored in 4% PFA for 2 h. Eyes were then rinsed with PBS, and moved through a sucrose gradient of 5%, 10%, 20%, and 30% sucrose (eyes were transferred to increasing concentrations of sucrose each time they sank to the bottom of the tube). Eyes were embedded in O.C.T. compound and 20 micron sections were cut using a Leica cryostat. For immunostaining, sections were rehydrated with PBS for 10 min and blocked in Normal Goat Serum (NGS) for 1 h. Primary antibodies were diluted in NGS and incubated on sections overnight at 4 °C in a humidified chamber. Sections were washed three times with PBS, and secondary antibodies were diluted in PBS and incubated on sections for 1 h at room temperature. Sections were washed 3 × with PBS, with the middle wash containing 5 μM Hoechst nuclear stain diluted in PBS . Slides were mounted using Fluoromount (Southern Biotech Cat#: 0100-01) and imaged using an Olympus FV1000 Confocal microscope. Antibodies and stains used were: mtCO1 1:500 ; MCU 1:2000 ; PNA 1:200 (after suspending at a concentration of 1 mg/mL in H20) ; Goat anti-Rabbit IgG (H + L) Alexa Fluor 633 .For best immunohistochemistry results, mice were perfused with fixative according published methods2PO4 1.2 mM MGSO4–7H2O, 20 mM HEPES, 2.6 mM CaCl–2H2O, 25.9 mM NaHCO3) optimized for isotopic labeling experiments in retinas was used in these experiments. Mice were euthanized by awake cervical dislocation and eyes were rapidly enucleated into a dish of Hank’s Buffered Salt Solution . For flux measurements, retinas were placed in pre-warmed KRB containing d-[U-13C]-glucose . Retinas were incubated for the specified time points at 37 °C mouse at 5% CO2 and room oxygen, then washed twice in ice-cold PBS and flash frozen in liquid nitrogen. For mouse dark-adapted experiments, mice were dark-adapted for a minimum of 18 h. Samples were collected exactly as above, but in complete darkness under an infrared light using night vision goggles. For zebrafish dark-adapted experiments, zebrafish were dark adapted for 18 h, zebrafish were euthanized using an ice bath, and samples were collected and snap-frozen under dim red light.Krebs–Ringer bicarbonate (KRB) buffer  was added and samples were incubated at 70 °C for 90 min. Metabolites were analyzed on an Agilent 7890/5975C GC–MS using selected-ion monitoring methods described in previous work10. Peaks were manually integrated using MSD ChemStation software (Agilent), and correction for natural isotope abundance was performed using Isocor software58. Raw signals for each metabolite were converted to molar amounts using metabolite standard curves which were run alongside each experiment. Molar amounts were normalized to the total amount of protein (determined using a BCA assay) for each sample to determine the molar amount per μg of cellular protein. Statistical analysis and figure preparation was performed with GraphPad v 8.4.3 .Metabolites were extracted from retinas using ice-cold 80% MeOH. 150 μL extraction buffer was added to each sample and tissue was disrupted by sonication. Samples were then spun at maximum speed, the supernatant transferred to a new tube, and the pellet saved for protein quantification. The supernatant was lyophilized at room-temp until dry. Extracted metabolites were derivatized using a two-step process: (1) 10 μL of 20 mg/mL Methoxyamine HCl  dissolved in pyridine  was added and samples were incubated at 37 °C for 90 min, then (2) 10 μL of 12. Larvae were maintained in embryo media containing 0.0003% 1-phenyl 2-thiourea  starting at 20 h postfertilization for confocal imaging. Larvae were analyzed at 6 days postfertilization (dpf) by embedding in 0.5% low melting point agarose containing embryo media with 0.02% (w/v) Tricaine . The agarose was submerged in embryo media containing 0.0003% PTU and 0.02% (w/v) tricaine. Imaging was performed using an Olympus FV1000 with a 40 × water objective in conjunction with Olympus FluoView FV10-ASW software (RRID:SCR_014215). The excitation/emission wavelengths used for mito-GCaMP3 were 488/510 nm. Images of total eye mitochondrial clusters were collected at a z-depth of 2 µm, and blinded quantification was performed using ImageJ + Fiji software (SCR_002285).Larvae were imaged as described previously21. For cyto-GCaMP3 measurements, retinal slices were incubated in KRB buffer (containing 0 mM CaCl2 and 0.4 mM EGTA) for 10 min. For basal cyto-GCaMP3 determination, slices were imaged every 2 s for 24 s to establish baseline cyto-GCaMP3 fluorescence. A bolus of CaCl2 was then injected in the imaging chamber to bring the final [Ca2+]free in the chamber to 5 mM. Retinal slices were imaged for a total of 10 min to monitor Ca2+ clearance. For basal mito-GCaMP3 determination, 15 z-slices of 2 µm thickness were collected every 30 s. Retinas were incubated in KRB buffer (containing 2 mM CaCl2) for 5 min, then the chamber was injected with ionomycin to a final concentration of 5 µM  for another 5 min of image collection. An excess of EGTA (5 mM) was then injected and images were collected for another 5 min. For mitochondrial Ca2+ uptake experiments, retinas were incubated in KRB buffer containing 100 µM KB-R7943 for 10 min. After this preincubation, baseline measurements of mito-GCaMP3 fluorescence were taken for 3 frames (30 s each) before treatment with 25 µM sildenafil for a total of 15 min of imaging.Slices were prepared as described previously60. Fixed ROIs were used to quantify average fluorescence signal across the mitochondrial cluster (for mito-GCaMP3) or cell body (for cyto-GCaMP3) at every time point. Statistical analysis and figure preparation for all GCaMP experiments was performed with GraphPad v 8.4.3 .For analysis of all GCaMP3 experiments, any cell bodies or mitochondrial clusters where the maximum fluorescence signal was completely saturated were excluded. For basal mito-GCaMP3 fluorescence analysis, mitochondrial clusters that did not respond to ionomycin treatment were not included in the analysis. Analysis was conducted blinded (masked) to sample identity. The excitation/emission wavelengths used for mito- and cyto-GCaMP3 were 488/510 nm. Timelapses were analyzed using ImageJ + Fiji software (SCR_002285) and were corrected for X–Y drift using the MultiStackReg plugin of ImageJ2 incubation. Eyes were enucleated under dim red light immediately after euthanasia followed by dissection under infrared illumination. The retinas were gently detached from posterior eye cups and were stored in dark in a dish containing oxygenated Ames medium at room temperature until recording. Recordings were conducted using previously described methods61. The retinas were mounted photoreceptors facing up in a closed chamber and were continuously superfused with oxygenated Ames medium (Sigma) at a flow rate of 3–5 mL/min. For isolating the a-wave of ERG, 50 μM DL-AP4 (Tocris) and 100 μM BaCl2 (Sigma) were included in the Ames medium. The recording chamber was maintained at 35–36 °C and retinas were allowed to adapt to the chamber temperature for at least 15 min before experiments. Ex-vivo transretinal ERG recordings were made in scotopic conditions by presenting light flashes produced by LEDs (Thor Labs). The ERG signals were amplified using a differential amplifier (Warner Instruments), low-pass filtered at 300 Hz (Krohn Hite Corp.), digitized using digidata 1440 (Molecular Devices), and were recorded at a sampling frequency of 10 kHz using pClamp 10 software.Mice were dark adapted overnight prior to the day of experiment and were euthanized by CO2, 1.6 mM MgCl2, 10.0 mM HEPES, 10.0 mM Glucose) with pH adjusted to 7.8 with NaOH. The eyes were hemisected and retinas isolated from the eyecup. All procedures after the dark adaptation were performed under dim red light. To ensure ex vivo ERG signal was predominantly cone responses, dark adaption was limited to ~ 30 min to allow cone photopigment regeneration but not provide enough time for full rod photopigment regeneration, and experiments were carried out during the day (between 11 AM and 4 PM) when rod contributions to retinal responses are at their lowest due to the circadian regulation of photoreceptor biology in the zebrafish retina. Ex vivo ERG recordings were performed as described previously62. Isolated retinas were mounted photoreceptor side up onto the specimen holder and perfused with Modified Salamander Ringer’s solution, supplemented with 40 µM DL-AP4 (Tocris Bioscience) and 40 µM CNQX (Tocris Bioscience) to isolate the photoreceptor component of the ERG signal (a-wave). The rate of perfusion was ~ 5 mL/min and the experiments were conducted at room temperature (~ 23 °C). ERG signal was first amplified (100 ×) and low-pass filtered at 300 Hz by a differential amplifier , and data was further amplified (10 ×) and acquired at 10KHz using an integrated amplifier/digitizer . A High Power LED light source , with filter for red light  and LED driver  were used to provide the flashes of light stimuli, durations ranged from 5 to 100 ms. The SutterPatch software  drove both stimulus generation and data acquisition via the IPA amplifier’s analogue output and input, respectively. Light stimuli were calibrated before experiments using a calibrated photodiode  and flash intensities converted to photons/µm2. Data analysis, including statistical analysis and figure preparation, was performed with GraphPad v 8.0.0 . Normalized responses were calculated for each retina by dividing the response amplitude data by the maximal amplitude measured at the peak/plateau of the response to the brightest flash. To quantify the gain of phototransduction activation, we fitted the Lamb–Pugh model to the initial leading edge of the dim flash response for each retina, and compared the average amplification constant (A) between WT and mcu-/- siblings63.Zebrafish were approximately 7 months of age for all electrophysiology experiments. Zebrafish were briefly dark adapted (~ 30 min), before euthanasia by ice water immersion. Eyes were enucleated into Modified Salamander Ringer’s solution (110 mM NaC, 2.5 mM KCl, 1.0 mM CaClSupplementary Information 1.Supplementary Information 2.


However, the specific activation mechanism and drugs that can effectively activate UPRmt in the cardiac muscle are yet to be elucidated. The aim of this study was to determine the regulation role of UPRmt on preventing pathological cardiac hypertrophy by tetrahydrocurcumin (THC) and explore its underlying molecular mechanism. Male C57BL/6J wild-type (WT) mice were divided into a control group and subjected to sham treatment for 4 weeks, and a test group which was subjected to transverse aortic constriction (TAC) surgery. Animals in the control and test group were orally administered THC (50 mg/kg) for 4 weeks after TAC procedure; an equivalent amount of saline was orally administered in the control sham-treated group and the TAC group. Subsequently, oxidative stress and UPRmt markers were assessed in these mice, and cardiac hypertrophy, fibrosis, and cardiac function were tested. Small interfering RNA (siRNA) targeting proliferator-activated receptor-gamma coactivator (PGC)-1α and activating transcription factor 5 (ATF5) were used to determine the UPRmt activation mechanism. THC supplement partly upregulated UPRmt effectors and inhibited TAC-induced oxidative stress compared with TAC-operated WT mice, thereby substantially attenuating contractile dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, PGC-1α knockdown blunted the UPRmt activation and the cardioprotective role of THC. The interaction between PGC-1α and ATF5 was tested in neonatal rat cardiac myocytes under normal conditions. The results showed that PGC-1α was an upstream effector of ATF5 and partly activated UPRmt. In vitro, phenylephrine- (PE-) induced cardiomyocyte hypertrophy caused ATF5 upregulating rather than downregulating corresponding to the downregulation of PGC-1α. The PGC-1α/ATF5 axis mediated the UPRmt activation and stress-resistance role of THC in vitro. Collectively, the present study provides the first evidence that PGC-1 and ATF5 can form a signaling axis to partly activate UPRmt that mediates the cardioprotective role of THC in pathological cardiac hypertrophy.Mitochondrial unfolding protein response (UPR Pathological cardiac hypertrophy involves a series of gene expression changes, epidermal morphology, and cardiac function produced by hypertrophic factors such as stress load, inflammation, and oxidative stress . Increasmt is activated under various conditions of mitochondrial dysfunction such as ROS overproduction and unfolded protein accumulation in mitochondria  for 48 hours at 37°C with 5% CO2. Next, the NRCMs were incubated with PE at a concentration of 50 μM for 24 h to induce cardiomyocyte hypertrophy . This study demonstrated the effect THC exerted on TAC-induced cardiac fibrosis. Masson stained heart section graphs  are shown in Figure α in mediating the anticardiac hypertrophic role of THC was confirmed in this study. LV carrying siRNA against PGC-1α was administrated in the hearts of the mice. The EF% was decreased, the HW/BW ratio, thickness of left ventricular wall, hypertrophy degree of cardiomyocytes, left ventricular collagen volume, and the expression of hypertrophic and fibrotic markers were increased after TAC, in PGC-1α knockdown mice as shown in Figures α.The role of PGC-1α in PGC-1α knockdown mice was markedly decreased, and the expression of ATF5 was decreased correspondingly, as shown in Figures α knockdown, as evidenced by the upregulation of gp91 phox and NOX 4  induced by THC was also abolished by PGC-1α siRNA, as shown in α siRNA group and PE+THC+PGC-1α siRNA group.PGC-1α and ATF5 that played a role in the regulation of UPRmt activation was further elucidated by transfecting NRCMs with adenovirus overexpressing PGC-1α. As shown in Figures α. Under PE treatment, although ATF5 expression increased and PGC-1α expression decreased, overexpression of PGC-1α further significantly upregulated ATF5. Meanwhile, the mRNA expression of ClpP and Lonp1 also synchronized with the changes of ATF5 expression, as shown in α under normal conditions, while PE treatment downregulated PGC-1α but upregulated the expression of ATF5 and UPRmt effectors, as shown in Figures mt in PE-treated NRCMs.The interaction of PGC-1α protein expression, as shown in Figures β-MHC mRNA were exaggerated by ATF5 knockdown, while the protective role of THC against hypertrophy was also blunted by ATF5 knockdown, as shown in α, as shown in Figures α enhanced the cardioprotective effect of THC, further activating ATF5 and downstream UPRmt, as shown in NRCMs were treated with ATF5 siRNA, and the knockdown effect of ATF5 siRNA was confirmed as there was no significant change in PGC-1α and ATF5 can form a signaling axis to partly activate UPRmt. Furthermore, PGC-1α/ATF5/UPRmt mediates the protective role of THC in TAC-induced cardiac hypertrophy and oxidative stress. The UPRmt activating effect of the PGC-1α/ATF5 axis and the cardioprotective effect of THC was also verified in PE-induced hypertrophic NRCMs. This study found a new mechanism for the regulation of UPRmt activation and proved THC to be a new drug activating UPRmt to inhibit mitochondrial dysfunction.Hypertension is a major global health problem in present times with no significant improvement seen in managing or controlling its effects and incidence . Hypertemt response. Activation of UPRmt involves a signal transduction pathway from mitochondria to nucleus, activating specific nuclear transcription factors. As a result, the expression of several genes protecting the mitochondria, especially those coding for molecular chaperones (like hsp10 and hsp60), antioxidant enzymes, and proteases located in the mitochondrial matrix (ClpP and Lonp1), is upregulated and helps in cell survival [α) activates the expression of CHOP, ATF4, and ATF5, thereby enhancing the mitochondrial protective action, although the relationship between CHOP, ATF4, and ATF5 is not entirely clear [mt activation significantly improved mitochondrial function and myocardial contractility, reduced myocardial cell mortality, and alleviated pathological myocardial fibrosis and remodeling. The positive regulation by myocardial cell UPRmt is expected to be an important target for the treatment of stress-induced myocardial injury [mt in the heart needs further detailed evaluation.Under the stimulation of different neurohumoral or mitochondrial stress, the protein-folding machinery in mitochondria is damaged, thereby leading to the production of misfolded and dysfunctional proteins. Dysfunction of the mitochondrial respiratory chain is accompanied by an increase in ROS and oxidative stress, which interferes with the protein integrity and folding process, leading to a vicious cycle of mitochondrial damage and cardiac dysfunction . The mitsurvival . Recent ly clear . Furtherl injury . Howevermt by THC for protection against pressure overload-induced cardiac hypertrophy has been investigated in this study. ROS production and accumulation has previously been confirmed to be involved in the promotion of pathological cardiac hypertrophy and HF in humans and animal models [α, ATF5, and several UPRmt effectors. This led to the possibility of the existence of any potential THC facilitated UPRmt activation modes which may exert a protective effect against pathological cardiac hypertrophy.THC, a major metabolite of curcumin, exhibits a stronger antioxidant activity than curcumin . Previoul models , 33. Morl models . Hypertrl models . THC exel models , 35. In α-SMA and oxidative stress in rats with chronic kidney disease (CKD) [α-MHC can enhance the supply of energy to the heart by improving the myocardial contractile velocity, while the increased expression of β-MHC can reduce the energy consumption of cardiomyocytes [α-MHC decreases while expression of β-MHC increases in cardiomyocytes [α-MHC in cardiomyocytes exerts a certain important role in improving cardiac function [α-MHC decreased, and the expression of β-MHC increased; however, THC treatment blunted this expression trend, indicating a good prognosis in pathological cardiac hypertrophy. This led to the inference that THC treatment effectively inhibits the progression of pathological cardiac hypertrophy.Therefore, the effect that THC exerts in pathological cardiac hypertrophy was investigated. The pathological cardiac remodeling including cardiomyocyte apoptosis and abnormal fibrosis formation with reduced cardiac function results in heart failure . This stse (CKD) . This stmyocytes . When hymyocytes . A previfunction . Followimt via a novel ATF5-dependent way, supported by the fact that the effect of enhanced UPRmt activation on the mitochondrial dysfunction of stressed cardiomyocytes depends on the enhanced expression of ATF5 [α was also upregulated by THC, the interactions between PGC-1α and UPRmt were further investigated. PGC-1α mediates the anticardiac hypertrophic role of several medicines [α exerts an effect in the activation of UPRmt remains unclear. PGC-1α siRNA was used to inhibit the cardioprotective role of THC both in vivo and in vitro. Meanwhile, the ATF5 and UPRmt effectors activated by THC were also downregulated by PGC-1α knockdown. The interaction between PGC-1α and ATF5 was confirmed by transfecting NRCMs with PGC-1α siRNA or Ad- PGC-1α under normal conditions. The results showed that ATF5 protein expression was significantly regulated in line with PGC-1α expression. However, when NRCMs were treated with ATF5 siRNA, PGC-1α expression did not change significantly under normal conditions. This confirmed that PGC-1α functions as the upstream regulator of ATF5 in the cardioprotective effect of THC treatment, and that the effect on UPRmt activation is blunted by PGC-1α siRNA and ATF5 siRNA. Based on our results, we conclude that the novel PGC-1α/ATF5 axis can activate UPRmt in cardiomyocytes, and it can be activated by THC treatment. Interestingly, when NRCMs were treated with PE, PGC-1α was downregulated, but ATF5 was upregulated. Therefore, it can be concluded that there exist other modes of PE-mediated upregulation of UPRmt which warrant further in-depth research.In the present study, THC treatment activated ATF5 expression, besides CHOP and ATF4. THC can therefore activate UPR of ATF5 . Since Pedicines , 38. It edicines , 40. Howmt effectors in cardiomyocytes under different stimuli such as pressure overload, isoproterenol, nicotinamide riboside, G-TPP, and Paraquat and concluded that different stimuli activates UPRmt in different modes. For example, pressure overload mainly upregulates ATF5, ClpP, and Lonp1, while G-TPP (10 μmol/l for 8 hrs) treatment upregulates CHOP, Lonp1, Hsp60, and Hsp10 [mt effectors were upregulated under pressure overload in vivo or with PE treatment in vitro, THC treatment further upregulated Lonp1 and ClpP mRNA expression via the PGC-1α/ATF5 axis. However, it is indicated that some mechanisms by which THC and PGC-1α/ATF5 axis regulate gene expression mode of UPRmt effectors, remains unknown and needs more in-depth research.Smyrnias et al. also studied the expression levels of UPRnd Hsp10 . In thisα/ATF5 axis can partly activate UPRmt and mediate the protective role of THC against pathological cardiac hypertrophy and oxidative stress induced by pressure overload. In vitro experiments elucidated the upstream and downstream relationship of this signal axis and the role of media in stress-resistance of THC in PE-induced hypertrophy of cardiomyocytes. This study also confirms a new mode of UPRmt regulation in cardiomyocytes, by which THC exerts its well-known antioxidant role. These results provide the possibility to treat pressure overload caused by cardiac hypertrophy and heart failure by therapeutic THC supplement and also provides a new possibility of treating oxidative stress. However, further studies are needed to explore the mechanism and mode of UPRmt activation under different conditions.This study primarily verified that the newfound PGC-1


Time-lapse imaging was performed in CHO, BPAE, and HEK cells loaded with Fluo-4, Calbryte, or Fluo-8 dyes. Ca2+ and Ba2+ did not change fluorescence in intact cells, whereas their entry after nsPEF increased fluorescence within <1 ms. The threshold for one 300-ns pulse was at 1.5–2 kV/cm, much lower than >7 kV/cm for the formation of larger pores that admitted YO-PRO-1, TO-PRO-3, or propidium dye into the cells. Ba2+ entry caused a gradual emission rise, which reached a stable level in 2 min or, with more intense nsPEF, kept rising steadily for at least 30 min. Ca2+ entry could elicit calcium-induced calcium release (CICR) followed by Ca2+ removal from the cytosol, which markedly affected the time course, polarity, amplitude, and the dose-dependence of fluorescence change. Both Ca2+ and Ba2+ proved as sensitive nanoporation markers, with Ba2+ being more reliable for monitoring membrane damage and resealing.The principal bioeffect of the nanosecond pulsed electric field (nsPEF) is a lasting cell membrane permeabilization, which is often attributed to the formation of nanometer-sized pores. Such pores may be too small for detection by the uptake of fluorescent dyes. We tested if Ca Permeabilization of the cell membrane by intense electric field pulses, or electroporation, has been known for decades and has found numerous applications in gene and drug delivery, cancer ablation, membrane biophysics research, as well as in food processing and decontamination ,2,3,4,5.(1)Whole-cell patch clamp, which is arguably the most sensitive and quantitative technique. It is, however, most laborious (one cell at a time) and prone to diverse artifacts due to the nsPEF pickup by the amplifier. It requires either a temporary disconnection of the amplifier when nsPEF is applied , or nsPE(2)Measurements of changes in the cell or bleb volume, which result from the colloid-osmotic water uptake or loss ,15,26. T(3)Fluorescence detection of the entry or leakage of ions or dyes is by far the most common method of detection and quantitation of membrane electropermeabilization ,28,29,30The need to understand the nature of nanopore formation by nsPEF and their properties is emphasized by the fact that nanopores give rise to a wide range of downstream effects, from the activation of second messenger signaling, depolarization, and excitation, to cell swelling, blebbing, actin disassembly, loss of the mitochondrial membrane potential, and necrotic or apoptotic cell death. There are three principal techniques which enable the analysis of different nanopore features ,21:(1)Wh+-sensitive fluorescent dye and monitor Tl+ entry from the outside as a measure of nanopore transport [+ precipitates in solutions with Cl−, which puts unusual restrictions on the formulation of extracellular media. Also, Tl+ can be admitted “spontaneously” into intact cells through constitutively open K+ channels (and perhaps some other channels), so the dye fluorescence gradually goes up and limits the time interval when the entry by nanopores can be reliably measured.However, the size of the dye molecules  restricts their passage through membrane pores. YO-PRO-1 is smaller than Pr and is a better marker for nanopore detection, but there are still smaller pores which do not admit either dye. Earlier, we proposed to load cells with a Tlransport ,31. This2+ entry as a marker of nanoporation does not require special media, a variety of Ca2+- sensitive fluorophores are available, and the detection sensitivity is similar to Tl+ entry [2+ level is tightly controlled, hence, in quiescent cells, the dye fluorescence can be stable for a long time. However, Ca2+ entry through nanopores should be separated from the activation of cation channels which can admit Ca2+ [2+ entry [2+ entry through the electropermeabilized membrane may trigger an overwhelmingly strong response by activating calcium-induced calcium release (CICR), followed by massive active pumping of excess Ca2+ from the cytosol [2+ level depends on many Ca2+- and time-dependent processes [2+ flow through nanopores and obstruct quantitative measurements.Using Cal+ entry ,33,34,35mit Ca2+ ,34,35,362+ entry . Moreoverocesses , which c2+, are not involved in the intracellular signaling, may be detected with a Ca2+ sensitive fluorophore and serve as more quantitative markers of nanoporation. We found that Ba2+ entry, as recorded by time-lapse fluorescence imaging with Calbryte dye, has nsPEF sensitivity similar to that of Ca2+ uptake, and much better than either YO-PRO-1 or Pr uptake. At the same time, Ba2+ does not trigger CICR and accumulates in the cytosol, making it more suitable than Ca2+ for quantitative measurements.The goal of this study was to test if other divalent cations, which, unlike Ca2+-sensitive fluorophore Calbryte or Fluo-8. Time-lapse imaging of the dye fluorescence started when cells were bathed in a standard physiological solution with 2 mM Ca2+ (see Methods). In a few minutes, this solution was replaced with the same solution but containing 2 mM of Cd2+, Zn2+, or Ba2+. To control for possible mechanical disruption, we replaced the initial Ca2+-containing solution with the same Ca2+-containing solution. We found that switching to Cd2+ or Zn2+ solutions strongly increased the emission of both dyes , whereas Zn2+ triggered a series of brief spikes, probably due to the mobilization of the intracellular Ca2+ (2+ and Zn2+ (which was beyond the scope of this study), these two ions were regarded unsuitable as nanoporation markers.To serve as a useful nanoporation marker, an ion should not elicit a dye signal in intact cells that have not been exposed to nsPEF. CHO cells, which typically do not express any endogenous voltage-gated channels ,17,23,39oth dyes A,B. The lar Ca2+ C. Regard2+- or Ca2+-containing solutions, even when Ba2+ concentration was increased to 5 mM .2+ level in our extracellular solutions formulated without adding Ca2+ and without Ca2+ chelators was at 2–5 μM [2+ in quiescent cells. Therefore, the Calbryte traces in 2+ entry, could reflect some Ca2+ entry as well. The addition of Ca2+ chelators to Ba2+-containing solutions was avoided, because it would have reduced Ba2+ concentration as well and make data interpretation difficult. Instead, the contribution of the nominal Ca2+ was evaluated by comparing the traces of Calbryte fluorescence in CHO and BPAE cells exposed to nsPEF in different solutions (2+-containing solution was by far the strongest (to 1900% in CHO cells and 700% in BPAE cells), due to its amplification by CICR. The same nsPEF exposure in the solution with nominal Ca2+ caused a weak (by up to 8–15%) but statistically significant (p < 0.01) response, without signs of CICR. The nsPEF effect in the solution with 2 mM Ba2+ was several times higher  and was clearly dominated by Ba2+ entry .The nominal Cat 2–5 μM , which iolutions . Both ce2+ solution partially blocked the response to nsPEF  to the nominal Caan 20 nM , suggestby nsPEF ,33. Inde 4–5 min . Weaker  4–5 min A.2+ could contribute to the fluorescent signal caused by Ba2+ entry. This contribution was negligibly small at low electric field strengths, but a transient Ca2+ peak at the highest electric field strengths could be comparable to Ba2+ response.To summarize, both the nominal and store Ca2+ entry reached a stable value in about 2 min after nsPEF. In contrast, high Ca2+ transients continued to decline throughout the period of observation (300 s) without reaching a stable level that could be preferentially chosen to quantify the electroporation of the cell membrane. Therefore, we wanted to test if a stable level can be reached at longer time intervals, up to 30 min, after intense nsPEF treatments.In 2+ transients after 12.1 and 19 kV/cm nsPEF continue to decline even in 30 min, albeit slowly  caused faster Ca2+ clearance from the cytosol, yielding disproportionally low Ca2+ levels by the time CICR was over. In contrast, Ba2+ response increased almost linearly with the electric field  did not impact the results.In CHO cells, the response was measured at the end of a 5-min recording, when it was less distorted by CICR see A,B. Noneic field A, and tw2+ response [p < 0.05, one-sample t-test). Ba2+ response in HEK cells was already significant at 1.8 kV/cm (p < 0.05). Surprisingly, Ba2+ response did not increase at 3 kV/cm, which was first regarded as a “statistical glitch.” However, the same peculiarity was also seen in CHO cells (2): The amplitude of the response was the same for 1.8, 2.3, and 2.6 kV/cm exposures . This result may be indicative of the involvement of at least two mechanisms of membrane permeabilization, differing in the dose dependence; however, more studies are needed to confirm it. Also, Ba2+ response did not further increase when the electric field was increased from 10 to 14 kV/cm, possibly because of Calbryte dye leakage from excessively damaged cells , but Ca2+ response at the anodic pole was locally amplified and extended by Ca2+ store disruption, CICR, store-operated Ca2+ entry, or by some other process [2+ response at the cathodic pole could be attenuated by fast Ca2+ sequestration into the endoplasmic reticulum [2+ responses would occur at just one but not the opposite pole of the cell. The identification of the exact mechanism involved could shed new light on the nanoporation phenomenon and its immediate consequences for cell function, but it was beyond the scope of this study. The fact that two similar approaches yielded qualitatively different results emphasizes the complexity of the nanoporation process and the necessity of combining different techniques for its analysis.Unexpectedly, this asymmetry of the response to nsPEF disappeared almost entirely when the external Cafor Ba2+ C,D,E. Thfor Ba2+ E. The st process . Alternaeticulum ,52. It i2+ ions. This method was compared to fluorescence detection of Ca2+ and of the uptake of membrane-impermeable nucleic acid stains YO-PRO-1, TO-PRO-1, and Pr. Different degrees of the cell membrane permeabilization in CHO, BPAE, and HEK cells were achieved by the electroporation with 300- or 600-ns electric pulses at various electric field strengths, from 1.8 to 19 kV/cm.We have introduced and characterized a new semi-quantitative method of analysis and monitoring of the permeabilized state of the cell membrane by the fluorescence detection of entry of Ba2+ and Zn2+ ions, Ba2+ did not penetrate into intact cells and did not change fluorescence in cells preloaded with Ca2+-sensitive fluorophores Calbryte or Fluo-8. A disruption of the cell membrane with a sufficiently strong nsPEF (over 1.5–2 kV/cm) could be detected within less than 1 ms by the fluorescence detection of Ba2+ or Ca2+ entry. This electric field strength matched earlier observations in diverse cell types, which consistently reported 1–2 kV/cm thresholds for a single pulse (200- to 600-ns duration) when the electroporation was detected by patch clamp, Tl+ uptake, or Ca2+ uptake [In contrast to Cd+ uptake ,36,41,53+ uptake ,27,28,30+ uptake ,30. For + uptake . This di2+ and Ca2+ ions have approximately the same size and water mobility, the entry of Ca2+ into the cytosol triggers a myriad of active biological responses, which by themselves change the cytosolic Ca2+ level and may obscure the nanopore transport. The initial Ca2+ entry may be magnified by CICR disproportionally to the extent of the membrane damage, and the subsequent reduction of the cytosolic Ca2+ may reflect its active pumping out of the cell rather than the restoration of the membrane integrity. For example, CICR in CHO cells can increase the cytosolic Ca2+ level 10 times more than its increase solely due to Ca2+ entry through the electroporated membrane [2+ can be estimated only in the presence of CICR inhibitors.While Bamembrane , so the 2+ was a more quantitative marker of the membrane electropermeabilization than Ca2+. It did not evoke CICR and was not appreciably cleared from the cytosol within 5 to 30 min of observation, which is consistent with prior reports that Ba2+ ions are at best only poorly pumped out by the plasma membrane calcium ATPase [2+ entry  the fluorescence signal had approximately the same amplitude in cells with and without Ca2+ channels [2+ channels was mediated by nanopore formation and the resulting loss of the resting membrane potential . This indirect mechanism of activation of Ca2+ channels was also suspected in adrenal chromaffin cells exposed to 5-ns pulses, since the Ca2+ entry could be inhibited entirely with Ca2+ channel blockers, but was mediated by Na+ entry through nanopores [2+ evoked by 150–400-ns pulses could be isolated by applying a cocktail of inhibitors of voltage-gated Ca2+ channels [The possible entry of Cal+ entry , or celll+ entry , or patcl+ entry ,16,60,61channels . The thrchannels , possiblanopores . In contchannels .2+ and Ca2+ entry  and human epithelial kidney cells (HEK 293) were obtained from the American Type Culture Collection  and propagated as recommended by the supplier. Bovine pulmonary artery endothelial cells (BPAE) were a kind gift of Dr. J. Catravas . These three cell lines were propagated, respectively, in Ham’s F-12 K medium ; EMEM with 1.5 g/L sodium bicarbonate, non-essential amino acids, L-glutamine and sodium pyruvate ; and a low-glucose DMEM with 2.5 µg/mL amphotericin B . The media were supplemented with 10% fetal bovine serum  and antibiotics . Cells were grown in flasks at 37 °C, 5% CO2, and 1–2 days prior to experiments were passaged onto glass coverslips (BPAE and HEK) or into glass-bottomed 35-mm Petri dishes .2, 2 CaCl2, 10 glucose, and 10 HEPES . Modifications to this solution included: The omission of CaCl2; the omission of CaCl2 and the addition of 0.5 or 1 mM EGTA; and the substitution of CaCl2 with 2 mM of ZnCl2 or CdCl2, or with 2 or 5 mM of BaCl2. These modifications are indicated in the figures and respective sections of Results. Ba2+-containing solutions for most experiments were prepared with the ultrapure BaCl2 salt . For several sets of experiments : 140 NaCl, 5.4 KCl, 1.5 MgCleriments , the solNucleic acid stains YO-PRO-1 and TO-PRO-1 , and Pr iodide  were utilized to detect membrane permeabilization ,21,26,302+-sensitive indicator preloaded into cells. In most experiments, cells were loaded with 5 µM Calbryte™ 520 AM  in the physiological solution, for 15–25 min at room temperature and protected from light. Multiple experiments, especially those in 2+ ions. As indicated in the Results section above, in isolated sets of experiments we also tested other Ca2+ indicators, namely Fluo-8 AM (AAT Bioquest) and Fluo-4 AM ; they were loaded into cells for 30 min at 10 or 5 μM, respectively. Cells were rinsed several times with the unmodified or modified physiological solution, and left in the same solution on the microscope stage for the duration of the experiments with nsPEF (up to about 60 min). In a single series of experiments without nsPEF exposure, the bath solution was changed during the time-lapse imaging, as described in In separate experiments, we measured a change in the fluorescence of a CaCell membrane permeabilization by nsPEF and recovery were monitored by time-lapse fluorescence microscopy at three different workstations based on IX71, IX81, and IX83 inverted microscopes .The IX71-based workstation, equipped with an iXon Ultra 897 back-illuminated CCD Camera , pE-340 Fura LED Fluorescence Light Source , and Solis interface (Andor), was used for ultrafast imaging in HEK cells . A singlAll other experiments in HEK cells and those in BPAE cells were carried out at the IX81-based workstation with an FV1000 confocal attachment and utilizing FluoView software (Olympus). All experiments in CHO cells were performed with the IX83-based system equipped with an Orca Flash 4.0 V.3 sCMOS camera , a 488-nm excitation laser , and utilizing MetaMorph v. 7.10.2.240 software . A small group of cells in the center of the field of vision was imaged with a 40×, N.A. 0.95 air objective. The total recording time was varied from 100 s to over 30 min, with a 2- to 60-s interval between sequential images. For data analysis, ROI were manually drawn around the perimeter of each cell.0). For fluorescence dyes that were preloaded into cells , the intensity measured in each frame after the exposure (F) was first corrected for the background (B), as measured in each frame, in a cell-free area of the image, and then normalized to the baseline as (100% × (F−B)/(F0−B0)). In some series of experiments, this formula was simplified to (100% × F/F0), either because the background intensity was negligible, or because the cell-free area was too small (in the cropped sensor recordings). For fluorescence dyes that were present in the extracellular solution during the experiment and entered the cell as a result of permeabilization by nsPEF , the entry was quantified in arbitrary units as (F-F0) [Each workstation was equipped with an nsPEF generator and delivery system (see below), which were triggered and synchronized with the time-lapse image acquisition by a TTL pulse protocol using a Digidata board  and Clampex software (Molecular Devices). Imaging always started before the exposure to nsPEF. Fluorescence intensity within the defined ROI was quantified using the MetaMorph or ImageJ software . For eacs (F-F0) ,15,26,30The concept of how nsPEF is delivered to individual selected cells or groups of cells on a microscope stage and the numerical simulation of the electric field distribution were reported by our group earlier ,33,36,53A pair of tungsten rod electrodes  connected to a pulse generator were positioned within the microscope field of vision so that the selected cells were centered between their tips, and then were lifted precisely to 50 µm above the coverslip surface. The precise positioning was accomplished with a robotic manipulator . The electric field at the cell location was calculated by 3D numerical simulations using a finite element analysis software COMSOL Multiphysics, release 5.0  . The exaAll protocols were designed to minimize potential biases and to ensure the accuracy and reproducibility of results. All series of experiments included a sham-exposed parallel control group, in which cells were subjected to all the same manipulations, but the nsPEF amplitude was set to zero. The nsPEF exposures at various electric field strengths and sham exposures were alternated in a random manner. In all experiments, each cell or group of cells was subjected to nsPEF exposure only once. With experiment protocols which lasted 5–15 min or less, we performed several exposures, at different electric field strengths, of spatially separated areas on the same coverslip (or in the same glass-bottomed Petri dish). For any type of nsPEF exposure, we performed from 6 to as many as 15 independent experiments, with a single cell (fast imaging) or a cluster of 4–6 cells per experiment. The number of cells used for statistical analysis for each type of treatment is indicated in figure captions.The data from sham-exposed cell samples were used to determine any “baseline” fluorescence changes resulting from factors other than nsPEF. Such factors included dye bleaching and/or sequestration and removal from the cytosol (for dyes preloaded into cells) or the unforced uptake or “sticking” to the cell surface (for dyes present in the extracellular solution during experiments). nsPEF exposure data were corrected by subtracting the “baseline” fluorescence change as averaged from all sham-exposed samples in each series. The same correction was applied to the sham exposure data itself, which expectedly yielded a constant “100%” or a “0 a.u.” value; nonetheless, the corrected sham exposure data were included in graphs to show the error bars.n cells; for clarity, the error bars are often plotted in one direction only. Student’s t-test with Dunnet’s correction when applicable [p < 0.05 (two-tailed) was considered statistically significant. Due to multiple statistical comparisons made between different groups and at multiple time points, we minimized the use of special symbols to mark the statistical significance in the graphs. Instead, the statistical significance can be estimated from the gap between the error bars of the compared groups: A gap exceeding the length of the error bar indicates a significant difference at p < 0.05 or better [Data are presented in graphs as mean values ± s.e. for plicable ,67 was er better .


Penicillins and other β-lactam antibiotics are the most common elicitors of allergic drug reaction. However, data on the pattern of clinical reaction types elicited by specific β-lactams are scarce and inconsistent. We aimed to determine patterns of β-latam allergy, i.e. the association of a clinical reaction type with a specific β-lactam antibiotic.We retrospectively evaluated data from 800 consecutive patients with suspected β-lactam hypersensitivity over a period of 11 years in a single German Allergy Center.β-lactam hypersensitivity was definitely excluded in 595 patients, immediate-type (presumably IgE-mediated) hypersensitivity was diagnosed in 70 and delayed-type hypersensitivity in 135 cases. Most  immediate-type anaphylactic reactions were induced by a limited number of cephalosporins. Delayed reactions were regularly caused by an aminopenicillin  and usually manifested as a measles-like exanthem . Intradermal testing proved to be the most useful method for diagnosing β-lactam allergy, but prick testing was already positive in 24 out of 70 patients with immediate-type hypersensitivity (34.3%). Patch testing in addition to intradermal testing did not provide additional information for the diagnosis of delayed-type hypersensitivity. Almost all β-lactam allergic patients tolerated at least one, usually several alternative substances out of the β-lactam group.We identified two patterns of β-lactam hypersensitivity: aminopenicillin-induced exanthem and anaphylaxis triggered by certain cephalosporins. Intradermal skin testing was the most useful method to detect both IgE-mediated and delayed-type β-lactam hypersensitivity. Among commonly used drugs, β-lactam antibiotics, particularly penicillins and cephalosporins, are most frequently associated with an allergic hypersensitivity reaction . Up to 1The sheer number of cases impedes thorough diagnostic work-up of all patients claiming to be allergic to β-lactam antibiotics by an allergy specialist. Once an acute infection requires immediate antibiotic treatment, there is usually no time for testing, and/or equipment and specialist knowledge are not available. The clinical picture and diagnosis of an allergic reaction induced by penicillin or other β-lactam antibiotics is comprehensively presented in reviews and guidelines , 6. HoweWith the present data evaluation in a single Allergy Center, we aimed: (i) To identify clinical reaction patterns and assess their association with specific β-lactam antibiotics. (ii) To assess the utility of currently available diagnostic methods for different reaction types. (iii) To evaluate whether patients with confirmed penicillin or cephalosporin allergy tolerate an alternative β-lactam.We retrospectively evaluated 800 patients referred to our Allergy Center from January 2009 to December 2019 for diagnostic work-up of an immediate-type or delayed-type hypersensitivity reaction attributed to a β-lactam antibiotic by the treating physician. The severity of immediate reactions (i.e. anaphylaxis) was classified as mild, moderate or severe as specified in Additional file A/L was considered significant only in patients with a convincing history of an IgE-mediated reaction, i.e. the anaphylaxis spectrum.Immunoglobulin E binding to benzyl penicilloyl, phenoxymethyl penicilloyl, amoxicilloyl, ampicilloyl, and cefaclor was determined by ImmunoCAP  . A valueSkin testing was performed according to international guidelines and included reading at 15 min in suspected immediate reactions and additional readings on days two, three, and four in delayed reactions . PatientPatients with negative β-lactam-specific IgE and negative skin test results underwent diagnostic challenge. Challenge testing was also performed to identify an alternative tolerated β-lactam antibiotic in patients with proven β-lactam allergy. General principles of our protocol are based on guideline recommendations : (i) ProIn 205 out of total 800 evaluated patients, a diagnosis of β-lactam hypersensitivity could be established in our Allergy Center, based on an overall assessment including history, the clinical reaction and results of testing. β-lactam hypersensitivity could be definitely excluded in the remaining 595 cases by negative serum IgE and negative skin testing followed by challenge of the incriminated β-lactam antibiotic (data not shown). The time interval between the β-lactam associated reaction and allergy testing of all 800 patients was ≤ 1 year in 491 cases, > 1 to 5 years in 79, > 5 to 10 years in 30, > 10 years in 181, unclear or not sufficiently documented in 19.Out of 205 cases with β-lactam hypersensitivity, an immediate-type (presumably IgE-mediated) reaction was diagnosed in 70 34.1%) . Out of these, 117 developed a measles-like exanthem (86.7%) and 12 a SDRIFE reaction pattern (8.9%) , and the diagnosis was confirmed by challenge which triggered an anaphylactic reaction Table . In 17 sIn 177 out of 205 cases with β-lactam hypersensitivity (86.3%), we identified at least one tolerated alternative β-lactam antibiotic by challenge testing  immediate-type hypersensitivity reactions to β-lactam antibiotics in our series. Single shot intravenous administration of cephalosporins has accordingly been identified as an important elicitor of intraoperative anaphylaxis leading to hypotension and increased ventilation pressure , 16.The different clinical forms of delayed-type β-lactam hypersensitivity are comprehensively described and discussed in a number of recent reviews , 6, 17. According to international standards, we used a commercial immunoassay to determine allergen-specific IgE , 20. CefThe most powerful method to detect β-lactam allergy in our data evaluation was intradermal testing. As a requirement, the respective β-lactam must be available for intravenous administration, and 1:10 or a higher dilution is needed to obtain a non-irritating test concentration. The intradermal test concentrations used in our series are within the range of published standards . Prick tAccording to current recommendations, oral β-lactam antibiotics were preferably used for challenge testing in our series if available. Intravenous challenge, however, has the clear advantage that the infusion can be stopped immediately in case of an anaphylactic reaction . The optCo-sensitization to numerous or even all different β-lactam antibiotics seems to be extremely rare. The side chain structure of the β-lactam ring of penicillins (R at C6) and cephalosporins (R1 at C7) seems to be the most important antigenic determinant . The strData were retrospectively extracted from patient records, resulting in a certain methodological inhomogeneity, e.g. not all patients were examined with all skin test methods. The frequency of certain β-lactam antibiotics as a trigger of an allergic reaction depends on general usage and prescription rate. Consequently, our data from a single German Allergy Center may not be generalizable to other regions or study centers.i.In this patient series, immediate-type β-lactam hypersensitivity was most commonly triggered by single cephalosporins including cefuroxime, cefazolin, and ceftriaxone, frequently as full-blown anaphylaxis.ii.The aminopenicillins amoxicillin and ampicillin were leading elicitors of delayed-type β-lactam hypersensitivity. They mainly caused measles-like exanthem; in contrast to other series, other forms of a delayed drug reaction including SDRIFE, FDE, and DRESS were far less common.iii.Intradermal testing of β-lactam antibiotics in a non-irritating dilution was the most powerful method for the diagnosis of both immediate and delayed-type β-lactam hypersensitivity. Preliminary prick testing detected IgE-mediated allergy in almost one third of cases. In our hands, additional patch testing does not add to the overall utility of skin testing.iv.Almost all our patients with proven β-lactam allergy tolerated at least one, usually several alternative substances out of the β-lactam group.Additional file 1. Grading the severity of anaphylaxis [modified from (7)].Additional file 2. Concentration of β-lactam antibiotics for skin testing and sequence of increasing doses used in single-blinded challenge. * For prick and patch testing, tablets were ground in a mortar and suspended with 1 mL physiological saline solution. iv, intravenous; n.d., not done.


Mentoring’s success in enhancing a mentee’s professional and personal development, and a host organisations’ reputation has been called into question, amidst a lack of effective tools to evaluate mentoring relationships and guide oversight of mentoring programs. A scoping review is proposed to map available literature on mentoring assessment tools in Internal Medicine to guide design of new tools.The review aims to explore how novice mentoring is assessed in Internal Medicine, including the domains assessed, and the strengths and limitations of the assessment methods.Guided by Levac et al.’s framework for scoping reviews, 12 reviewers conducted independent literature reviews of assessment tools in novice mentoring in PubMed, Embase, Scopus, ERIC, Cochrane, GreyLit, Web of Science, Open Dissertations and British Education Index databases. A ‘split approach’ saw research members adopting either Braun and Clarke’s approach to thematic analysis or directed content analysis to independently evaluate the data and improve validity and objectivity of the findings.9662 abstracts were identified, 187 full-text articles reviewed, and 54 full-text articles included. There was consensus on the themes and categories identified through the use of the split approach, which were the domains assessed and methods of assessment.Most tools fail to contend with mentoring’s evolving nature and provide mere snap shots of the mentoring process largely from the mentee’s perspective. The lack of holistic, longitudinal and validated assessments propagate fears that ethical issues in mentoring are poorly recognized and addressed. To this end, we forward a framework for the design of ‘fit for purpose’ multi-dimensional tools.Most tools focus on the mentee’s perspective, do not consider mentoring’s evolving nature and fail to consider mentoring holistically nor longitudinallyA new tool capable of addressing these gaps must also consider inputs from all stakeholders and take a longitudinal perspective of mentoring Mentoring in medicine helps shape a mentee’s professional identity and personal development, and enhances the career, progress and satisfaction of mentors and mentees , 2. It aCartesian reductionism and Newtonian principles of linearity” [However, mentoring’s ability to provide consistent personalised support is suspect given the lack of robust assessments of mentoring processes that can detect problems and direct timely and appropriate support to the mentee and mentor , 28. Thinearity” , 45. Thenearity” , 43.To address gaps in assessing mentoring tools, a scoping review is proposed to map “how is mentoring processes, support, relationships, outcomes and the oversight of the mentoring programs assessed?”. Acknowledging mentoring’s nature and recognizing that mentoring assessment tools need to be specific and contextualised for each particular form of mentoring, data form this review promises to inform design of a assessment framework that could act as a template for the construction of individualised mentoring tools –36.“a dynamic, context-dependent, goal-sensitive, mutually beneficial relationship between an experienced clinician and junior clinicians and/or undergraduates focused upon advancing the development of the mentee” [This scoping review seeks to map prevailing tools published in peer-reviewed and grey literature –52, iden mentee” .Levac et al. ’s refineThe 12-member research team (henceforth the research team) discussed the research question with medical librarians from the Yong Loo Lin School of Medicine (YLLSoM) and the National Cancer Centre Singapore (NCCS) and sought advice from educational experts and clinicians at the NCCS, YLLSoM, the Palliative Care Institute Liverpool and Duke-NUS Medical School (henceforth the expert team).what tools are available to assess mentoring in novice mentoring in Internal Medicine?” The secondary research questions were “What domains are evaluated by available mentoring assessment tools?”, “When and how are these tools deployed?” and “Are prevailing tools to assess novice mentoring validated?”.Guided by the expert team, the research team determined the primary research question to be “Envisioning that the findings of this scoping review will guide design of mentoring tools; the comprehensiveness and feasibility of prevailing tools were also considered given the longitudinal nature of the mentoring process. This is outlined in Given novice mentoring’s context specific nature, this review scrutinises accounts of novice mentoring in all subspecialties of Internal Medicine as defined by the Accreditation Council for Graduate Medical Education (ACGME) . Given tWith guidance from the expert team, the search terms were expanded using Boolean operators to include MeSH and Keywords for all relevant concepts. . The broThe research team carried out pilot searches of PubMed and OpenGrey databases to determine the appropriateness of the search terms in the pilot searches.The members of the research team carried out independent searches of Embase, Scopus, ERIC, Cochrane, GreyLit, Web of Science, Open Dissertations and British Education Index databases using similar search strategies. All searches were carried out between 24th April 2018 and 18 October 2018, and 17 December 2019 to 14 February 2020.Each member of the research team compiled their own list of articles to be included and compared their results in online discussions with other members of the research team. Sambunjak et al. (2010) ’s “negotmethod triangulation whilst having each reviewer independently analyse the same data provides investigator triangulation [To enhance the comprehensiveness of this approach, data from the manuscripts were analyzed using Krishna’s split approach. Krishna’s split approach sees concurrent and independent use of Hsieh and Shannon (2005) approach to directed content analysis approach  and Braugulation . Triangugulation . In addiThese two processes are elaborated in turn below:Thematic analysis. Braun and Clarke [a priori framework of mentoring [d Clarke ’s approad Clarke ’s approad Clarke , 61. A tentoring , 62–67.In phase 1 of Braun and Clarke’s approach, 8 members of the research team  carried out independent reviews, ‘actively’ reading the included articles to find meaning and patterns in the data.In phase 2, ‘codes’ were constructed from the ‘surface’ meaning , 59, 68 In phase 3, the categories were organised into themes that best depict the data.In phase 4, the themes were refined to best represent the whole data set and discussed.Negotiated consensual validation” was used to determine a final list of themes approach [In phase 5, the research team discussed the results of their independent analysis online and at reviewer meetings. “approach .when a theory exists about a phenomenon that needs further refinement or development through qualitative research” [In consultations with the key stakeholders as part of the phase 6 of Levac et al. ’s methodesearch” . As a reesearch” , Sng et esearch”  and Tan esearch”  in additesearch” .Directed content analysis. Hsieh and Shannon (2005) approach to directed content analysis approach [approach  was emplapproach –73.Using deductive category application , 74, thenegotiated consensual validation” to resolve any differences in the coding [Stage 2 saw the two reviewers using the ‘code book’ to independently extract and code the relevant data from the included articles. Any relevant data not captured by these codes were assigned a new code that was also described in the code book. In keeping with deductive category application , coding e coding . The fine coding  based one coding , 77.Comparisons between the themes identified using Braun and Clarke ’s approaThe split approach adopts an iterative process which meant that any new codes identified was reviewed to verify the classification and ensure complete data extraction. Analysis of all included articles was carried out and discussed online and face-to-face meetings by the independent reviewers. The consensus decisions on the final categories from Hsieh and Shannon (2005) approach to directed content analysis approach  and themThe characteristics of all 54 articles included in this scoping review were tabulated. Details on the author(s), year of publication, study location, Intervention type, and comparator (if any); duration of the intervention, study populations , aims of the study, methodology, outcome measures and important results were compiled . In keep9,662 unique titles were identified from the nine databases, 187 full-text articles were retrieved, and 54 articles were included in this scoping review. The two categories identified using the split approach include the domains assessed and assessment methods.The 5 domains assessed were the communication, the mentoring process, the mentee’s growth, the mentor and perception of the program.Mentoring relationships pivot upon effective communications. Nine studies evaluated communications between the mentee and mentor –86. The Five studies evaluated mentee’s and/or mentor’s understanding and expectations of the mentoring process , 89–92. 15 studies evaluated the mentee’s personal growth by inquiring about their “personal wellbeing”, “development of personality” and “self-perception of their own abilities” , 99–108.The mentor’s impact upon the mentoring process was evaluated using self-reported changes in practice and the mentee’s perception of the mentor’s abilities. 10 studies , 118 ass14 studies assessed the success of the mentoring process by gauging the mentee’s general level of satisfaction with the mentoring program , 121. NiAssessment methods encapsulated the type of study, the means of collecting data, the mode of measurement, the number of points of evaluation throughout the study, the validity of the tool employed and the target of the tool’s assessment.Stakeholders were consulted on the findings to garner their inputs on the relative importance and viability of implementing the findings and upon the focus of future studies.In meetings its objectives, this scoping review highlights the variability in the construct, content, timing, participants involved and focus of prevailing assessment methods in novice mentoring. It also reveals that there is also little by way of consistency in the tools as evidenced by the presence of 49 tools used to assess mentoring. Whilst it is clear that this lack in consistency has a knock-on effect upon practice and oversight of mentoring programs, relationships and processes, in truth it also reflects a more fundamental failure in understanding the mentoring process. In some cases, however, variations in mentoring assessment methods are the result of adaptations to mentoring’s context-dependent and evolving nature whilst others differ by virtue of their settings and goals. These differences often reflect the notion that mentoring is complex and difficult to study holistically or longitudinally predisposing to piecemeal evaluations of ‘areas of interest’ in key areas of mentoring such as communications, the mentee, the mentor, the host and their mentoring relationships. This underpins the presence of makeshift measures in different formats involving specific stakeholders at different times of the mentoring journey.Reliance upon a constructivist approach and a relativist lens to pull the various socially constructed perspectives together however fails to fully render an effective assessment of the mentoring relationship nor fully capture the changing nature of the mentoring process, the maturing mentoring relationship and the impact of the external influences such as the mentoring environment upon the mentoring environment. This suggests a lack of holistic appraisals of mentoring. It also reaffirms a lack of clarity on the purpose of these assessments.There is also little by way of explanations of the ‘conceptual foundations’ of the tools , compromIndeed, the purpose for the assessments and the role that data from these measures take in influencing the mentoring process also vary. Unsurprisingly, there is little determination as to whether these assessments are formative or summative and few establish the goals of these assessments and how they influence the mentoring process. With little psychometric data, the evidence-based underpinnings and validity of these tools remain questionable particularly given the continued reliance upon self-assessment data at the end of the mentoring process and little by way of frameworks to inculcate formal and informal feedback.Concurrently, with only 8 out of the 49 tools choosing to evaluate the mentoring process, prevailing data on mentoring progress and the health of mentoring relationships are suspect. This is especially so when the methods used to assess the various domains of the mentoring relationship and program are reliant upon makeshift measures that have not been validated or found to be fit for purpose. Recording of the data collected is also variable and dependent upon whose opinion is sought. There is no consistency in the format of the tool nor little data by way of their validity, feasibility and reliability or their design and theoretical underpinnings.Furthermore, the majority of studies evaluated mentoring at a single time point, often at the end of the mentoring process where recall bias and the halo effect brought on by a successful mentoring process potentially biases responses.These findings do little to dissuade concerns that mentoring assessments are flawed, piecemeal and fail to provide an effective picture of the mentee’s, mentor’s and the relationship’s progress and overall condition. These gaps have significant implications upon novice mentoring and mentoring as whole and underline the need for urgent attention.However, despite prevailing limitations, a preliminary framework may be constructed drawing upon the data accrued thus far. Here, there are guiding ‘considerations’ to consider.1. Guiding considerationsThe goals of the assessments need to be ascertainedMixed methods approach should be used that captures the participant’s demographics, social, academic, personal, clinical and research backgrounds, experience, goals and motivations. It must also consider the mentoring context and the mentoring goalsThe assessments should occur throughout the mentoring journey and should include multi-modal assessments, such as surveys, questionnaires, journals and/or interviews, at various junctures of the mentoring process, acknowledging both the various stages of the mentoring process but also the perspectives of ALL the various stakeholders involved2. Assessing mentoring dynamics:a. Mentoring Process:consider the expectations, motivations and preferred mentoring approach.Assess the knowledge, skills and attitudes of the mentees and the mentorsHow mentor training and matching of mentors and mentees occursDetermine how mentoring process is initiated and the approach to mentoring.b. Mentoring Relationship:how mentors and mentees communicate- the form, the frequency, duration and purpose of the meeting.In the face of an evolving mentoring process, there is a need to assess the quality of the mentoring relationships.3. Assessing stakeholders:of multisource feedback collected longitudinally from mentors, mentees and the host organisation to determine how the different stakeholders perform at specific time points and over time.use 4. Assessing outcomes:This would require longitudinal assessment by the host organisation of the mentoring relationship and mentoring goalsAssess satisfaction -feedback in an informal or formal manner, mentor and mentees’ development and growth by self-evaluation and against objective measures.Without being drawn into prevailing controversies regarding definitions and the role of scoping reviews , 125–128However despite these limitations, this scoping review was carried out with the required rigour and transparency advocated by Arksey and O’Malley (2005) , Levac eThe gaps in the depth and focus of existing tools identified in this scoping review underscore the need for a systematic review to provide a focused and in-depth analysis of mentoring assessment tools , 54, 130As such, a modified Delphi is proposed in order to better understand how best to assess the stakeholders , the dynamics between them as well as the outcomes of mentoring. This will the next phase of this research project.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S1 Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.