Hypera postica and its parasitoid Bathyplectes curculionis. Although classical biological control efforts have largely suppressed H. postica in the eastern United States, it remains problematic in the western United States. We sampled 20 production alfalfa fields in southeastern Wyoming to estimate H. postica density, parasitism rates by B. curculionis, and vegetation at local scales. We used remotely sensed imagery to characterize both landscape composition and configuration surrounding each sampled field. We used a hypothesis-driven modeling approach to determine which model was most predictive of H. postica and parasitism rate by B. curculionis. Landscape composition was the best model to predict H. postica densities. Host density was the best predictor of parasitism rates by B. curculionis. Production fields that had received insecticide applications in the last 5 years had higher weevil densities than fields that had not received insecticide applications. Stand age was not associated with weevil density or parasitism rate. In conclusion, we found local, landscape, and management components to be important in this system.It is widely recognized that both local and landscape-scale factors can be important drivers of crop pests, natural enemies, and biocontrol services. However, recent syntheses have found that landscape effects are inconsistent across study systems, highlighting the need for system-specific research to guide management decisions. In particular, studies conducted in perennial crops and that examine landscape configuration, not just composition, are especially lacking. We studied the impact of local and landscape factors on alfalfa weevil  Medicago sativa L. , where disturbances are less severe  was first found in the United States in the early 1900’s, and later in the century the United States Department of Agriculture released several parasitoid species to help manage alfalfa weevil  (Hymenoptera: Ichneumonidae)  . Aphid dmonidae) . Longevi alfalfa . Dandelity as in  or 2) foty as in , we hypoty as in .Other in-field characteristics that can influence insect abundance and diversity in alfalfa include weed coverage , field aH. postica and the activity of its parasitoid B. curculionis, 2) determine if pest abundance was associated with alfalfa plant damage, and 3) examine the impact of past pesticide use and alfalfa stand age on H. postica density and parasitism rates by B. curculionis.The objectives of this work were to 1) determine how landscape factors and in-field characteristics affected both abundance of H. postica were reared to quantify parasitism using methods described below and parasitism rates of H. postica by B. curculionis averaged 42.8%, ranging from 13.2 to 74.9%. No other parasitoid species were detected or have been in our other efforts in Wyoming . Producers were asked if they had applied insecticide to control alfalfa weevil in the last five years. Insecticide had been applied 10 fields sometime in the last five years, five fields had not received insecticide application, and spray history was unknown for five fields. All fields were sampled in June 2015 prior to the first hay cutting or any insecticide application within that season.Producers were contacted via phone before sampling to determine stand age, variety grown, irrigation type, and general management practices, and to confirm permission to sample their fields. Not all producers were available to answer questions and in some cases producers were unsure and unable to provide information for all questions. Of these, 18 fields were pivot irrigated, and two fields were flood irrigated. Stand age ranged from one to eight years with an average of 3.25 years  to limit temporal differences in H. postica life stages and parasitism rates.Sampling dates were determined based on growing degree days accumulated above 48 degrees Fahrenheit after March 1 to target the third instar larval stage of  postica . Fields Insects were collected using sweep net sampling (38.1 cm in diameter) on warm, sunny, and low wind days. Six, 50-sweep samples were collected from each field . The first sweep transect began 20 m into the field and was conducted parallel to the field edge. Each subsequent transect began another 10 m into the field (to create a ladder pattern if seen from above). The contents of each 50-sweep sample were transferred into a gallon size bag with a dry paper towel to soak up excess moisture. Gallon bags were transported back to the lab in a cooler for further processing.H. postica larvae by parasitoids, a subsample of larvae was selected to rear to adulthood. Within twenty-four hours of sampling, one hundred third or fourth instar H. postica larvae were randomly removed for rearing. These H. postica larvae were divided into two brown paper bags  with two stems of fresh alfalfa per bag . The remainder of the insects from the sweep sample was frozen until sorted. H. postica larvae and adults were counted, as well as aphids (Hemiptera: Aphididae) to include in models to predict parasitism.To determine parasitism rates of  per bag . An addiEach field was characterized to account for differences in insect damage, alfalfa height, and floral resources through data collection at the starting location of each of the six sweep transects per field described above. Insect damage to alfalfa was scored by visually inspecting five plants in a 1 m × 1 m quadrat for 10 s and scoring damage on a scale from zero to five, with zero indicating no visible damage and five complete defoliation. Alfalfa height was measured in the middle of each of six the quadrats. Floral resources provided by weeds were quantified along two 50 m transects, each 50 m apart, beginning in-field, and moving towards the field edge. The number of open blooms for each blooming species  was recorded along the transect.Landscape composition surrounding field sites was analyzed to determine their relationship to insect abundances and parasitism rates. Latitude and longitude coordinates  were taken with a Global Positioning System (Garmin eTrex 10 Worldwide Handheld GPS Navigator) 20 m into each field site and imported into ArcMap . The area around each set of field coordinates was buffered at four radii: 500 m, 1,000 m, 2,000 m, and 3,000 m. We selected these scales based on the range of scale of response of 3,000 m and smaller for specialist enemies and specialist pests demonstrated in a meta-analysis by H. postica’s specialization on alfalfa. Edge density indicates the total length of edge over a given area. We calculated edge density . Also, at the 500,m scale, the proportion of alfalfa and other crops were negatively correlated  and at the 1,000 m scale, proportion of alfalfa was negatively correlated with natural/seminatural habitat . Due to these strong correlations, we used principal components analysis for these variables to create principal components (PCs) that were independent of one another . Linear regression was used to determine if stand age predicted densities of H. postica, aphids, or parasitism rates.Producers were interviewed to collect data on stand age and past insecticide use (Y/N). These variables were not included in the above modeling approach because these factors were not known for all fields and excluding fields for which these data were unknown would considerably reduce our sample size. We conducted H. postica ranged from 8 to 2,295 larvae per 50 sweeps with a mean (±SE) of 491 larvae (±153) per 50 sweeps. The ‘landscape composition’ model was the best model for predicting H. postica densities in alfalfa fields, with the 2,000 m scale the most predictive  and increased with the amount of alfalfa in the landscape (PC2). Lastly, H. postica densities were positively associated with alfalfa height. All other models tested for H. postica had an AICc that was comparable or greater than the null model . The ‘host density’ model had the lowest AICc value, more than 2 AICc lower than the null model . Aphid density was not a significant predictor of alfalfa damage . The overall model had an adjusted R2 of 0.43.H. postica densities were nearly 10 times higher in fields where insecticides had been applied in previous years than those fields that had never been sprayed (753 ± 266 weevils/50 sweeps vs 62 ± 18 weevils/50 sweeps) . Aphid densities  and parasitism rates  did not differ according to past insecticide use. Stand age reported by producers ranged from 1 to 8 years (average of 3.25 years). Stand age was not associated with H. postica densities , aphid densities , or parasitism rate .Producers reported insecticide use against alfalfa weevil in 10 out of 15 sampled fields in the previous five years, and insecticides were not used in five out of 15 fields. H. postica densities were negatively associated with landscape cover of nonalfalfa crops and positively associated with their food crop alfalfa. Specialist herbivores have been shown to be associated with the area of suitable host material in other cropping systems  around the border of alfalfa fields . WeevilsB. curculionis better than host density. Similarly, H. postica, although the relationship differed over the two years of her study. Parasitism rates may be lower in fields with higher H. postica densities if B. curculionis simply cannot keep up with heavy H. postica infestations (B. curculionis (In our work, no model predicted parasitism rate by stations , and thestations . Althougculionis . Rand anculionis . In addiculionis , suggestOur work supports the assertion from others  that varIn conclusion, our snapshot into alfalfa weevil-parasitoid interactions revealed that alfalfa weevil densities were best predicted by landscape composition at the 2,000 m scale and parasitism rates were best predicted by host density. Landscape configuration did not play a major role in predicting weevil density or parasitism rate. Future work that explores how weevil estivation relates to natural habitats, pairs fields with and without insecticide application history across landscape gradients, and studies the same fields over multiple years would be valuable next steps to understanding how to limit damage from this important pest.


The advanced glycation end products treated right ventricular outflow tract myocytes had a similar action potential duration as the controls, but exhibited a lower L‐type Ca2+ current, higher late sodium current and transient outward current. Moreover, the advanced glycation end products treated right ventricular outflow tract myocytes had more intracellular Na+, reverse mode Na+–Ca2+ exchanger currents, intracellular and mitochondrial reactive oxygen species, and less intracellular Ca2+ transient and sarcoplasmic reticulum Ca2+ content with upregulated calcium homeostasis proteins and advanced glycation end products related signaling pathway proteins. In conclusions, advanced glycation end products modulate right ventricular outflow tract electrophysiological characteristics with larger late sodium current, intracellular Na+, reverse mode Na+–Ca2+ exchanger currents, and disturbed Ca2+ homeostasis through increased oxidative stress mediated by the activation of the advanced glycation end products signaling pathway.Diabetes mellitus is associated with cardiovascular disease and cardiac arrhythmia. Accumulation of advanced glycation end products closely correlates with cardiovascular complications through mitochondrial dysfunction or oxidative stress and evoke proliferative, inflammatory, and fibrotic reactions, which might impair cardiac electrophysiological characteristics and increase the incidence of cardiac arrhythmia. This study examined the mechanisms how advanced glycation end products may contribute to arrhythmogenesis of right ventricular outflow tract—a unique arrhythmogenic substrate. A whole‐cell patch clamp, conventional electrophysiological study, fluorescence imaging, Western blot, and confocal microscope were used to study the electrical activity, and Ca 2+ homeostasis, which might play a role in diabetes related ventricular arrhythmogenesis and electrical remodeling.Diabetes mellitus is associated with cardiac arrhythmia. Advanced glycation end products (AGE) accumulation in diabetes mellitus may elicit mitochondrial dysfunction/oxidative stress generation and evoke proliferative, inflammatory and fibrotic reactions through AGE‐receptor for AGE (RAGE) signaling pathway, which might impair cardiac electrophysiological characteristics and increases the incidence of cardiac arrhythmias. Our result support that AGEs modulate RVOT electrophysiological characteristics through the activation of AGE‐RAGE signaling pathway with ionic currents modulation, increased intracellular and mitochondrial ROS, and disturbed Ca AGEs are a heterogeneous, complex group of compounds that are formed mainly through glycation   is an arrhythmogenic substrate with a unique electropharmacologic profile Maury, . PrematuHowever, knowledge of the role of AGEs in RVOT arrhythmogenesis remains limited. The cardiac effects of AGEs may contribute to the pathogenesis of RVOT arrhythmogenesis. Therefore, this study examined the role of AGEs in RVOT arrhythmogenesis, which may underlie DM‐related ventricular arrhythmogenesis.22.1Guide for the Care and Use of Laboratory Animals and the Guide for the Care and Use of Laboratory Animals, 8th Edition, published by the United States National Institutes of Health . Using a precision vaporizer, we anesthetized male rabbits (weighing approximately 1.5–2 kg) with an overdose of isoflurane (5% O2) while restraining them in a dorsal recumbent position. The lack of a corneal reflex and motor response to pain stimuli with a scalpel tip confirmed the adequacy of the anesthesia. The RVOT  and RVA (area within 5 mm of the RVA) were excised from all rabbits after euthanasia and prepared for analysis  and conformed to the institutional 2.23, 0.5 mM NaH2PO4, 0.5 mM MgCl2, 2.7 mM CaCl2, and 11 mM dextrose, at 37°C. The tissues were superfused at a constant rate (3 ml/min) with Tyrode's solution, which was saturated with a gas mixture of 97% O2 and 3% CO2. The transmembrane APs of the RVOT tissues were recorded using machine‐pulled glass capillary microelectrodes filled with 3 M KCl, and the tissue preparations were connected to a World Precision Instrument model WPI Duo 773 electrometer. The electrical and mechanical events were simultaneously displayed on a Gould 4072 oscilloscope and Gould TA11 recorder. Electrical stimuli were applied using a Grass S48 stimulator through a Grass SIU5B stimulus isolation unit. Burst firing was defined as the occurrence of an accelerated spontaneous AP with sudden onset and termination. After the control and AGE (100 μg/ml)‐treated RVOT or RVA tissues were analyzed and the effects of treatment during high‐frequency burst pacing (20 Hz) for 1 s. An inhibitor of late sodium current—ranolazine (10 μM) was added to observe burst firing in RVOT. Sustained VT and nonsustained VT were defined as ventricular bursting beating of more and less than 30 s, respectively. Spontaneous activity was defined as the constant occurrence of spontaneous APs in the absence of electrical stimuli. Burst firing was defined as accelerated spontaneous activity with a beat faster than the basal beating activity and sudden onset and termination. Triggered activity was defined as premature beats or delayed afterdepolarizations (DADs). EAD and DAD were defined as the presence of a spontaneous depolarization of the impulse after complete repolarization.The tissue preparations were bathed in Tyrode's solution (B5), which comprised 137 mM NaCl, 4 mM KCl, 15 mM NaHCO2.32+‐free Tyrode's solution containing 300 units/ml of collagenase type I (Sigma‐Aldrich) and 0.25 units/ml protease type XIV (Sigma‐Aldrich) for 8–12 min. The RVOT was excised and gently shaken in 50‐ml of Ca2+‐free oxygenated Tyrode's solution until single cardiomyocytes were obtained. The solution was then gradually replaced by normal oxygenated Tyrode's solution. The cardiomyocytes stabilized in a bath for at least 30 min before the experiments commenced. Isolated RVOT cardiomyocytes were treated in the BSA (100 μg/ml) or AGE (100 μg/ml) + BSA (100 μg/ml) for 4–6 h prior to ionic currents, Ca2+ transients, intracellular ROS, Na+, and Western blot analysis. The detailed contents of external (Tyrode's) and intracellular (pipette) solutions used in this study were summarized in Table Single cardiomyocytes from rabbit RVOTs and RVAs were enzymatically dissociated through a procedure described by Lu et al.  and were20, APD50, and APD90).Consistent with the procedure described by Lu et al. , a wholeICa‐L, NCX currents, INa, INa‐late, Ito and IK1 were recorded in the pipette solutions P1, P2, P3, P4 and P5, respectively.The APs was recorded in the pipette solution P5. INa was recorded during depolarization from a holding potential of −120 mV to the test potentials, which ranged from −90 to 0 mV in 5‐mV steps for 40 ms at a frequency of 3 Hz, at room temperature (25°C ± 1°C) in the bath solution B2.The INa‐late was recorded at room temperature in the bath solution B3 using a step‐ramp protocol . An equilibration period for dialysis was allowed for the adequate clamping of the cell currents. The INa‐late was measured as the tetrodotoxin (30 μM)‐sensitive portions of the current traces obtained when the voltage was ramped back to −100 mV, a process described by Lin et al.  was examined through a double‐pulse protocol. A 30‐ms prepulse from −80 to −40 mV was used to inactivate the Na+ channels; this was followed by a 300‐ms test pulse to 60 mV in 10‐mV steps at a frequency of 0.1 Hz. CdCl2 (200 μM) was added to the bath solution B1 to inhibit the ICa‐L. The Ito was measured as the difference between the peak outward current and the steady‐state current. The inward rectifier K+ current (IK1) was activated from −40 mV to the test potentials, ranging from −20 to 120 mV, in 10‐mV steps for 1 s at a frequency of 0.1 Hz under an infusion of 200 μM CdCl2 and 2 mM 4‐aminopyridine in the bath solution B1. The amplitudes of the IK1 were measured as 1 mM barium‐sensitive currents.The transient outward K+ current (IKr‐tail) was measured as the outward peak tail current density following a 3‐s prepulse from a holding potential of −40 mV to a voltage between −40 and 60 mV in 10‐mV steps at a frequency of 0.1 Hz in the presence of 30 μM chromanol 293B and 200 μM CdCl2 in the Ca2+‐free normal Tyrode's solution (the bath solution B1). Micropipettes were filled with the pipette solution P6.The rapid delayed rectifier K2.42+ (10 μmol/L) fluo‐3/AM was loaded onto the control and AGE‐treated RVOT cardiomyocytes for 30 min at room temperature. After intracellular hydrolysis of fluo‐3/AM was conducted for 30 min, excess extracellular dye was removed by changing the bath solution B1. Fluo‐3 fluorescence (F) was excited with the 488‐nm line of an argon ion laser and the emission was recorded at >515 nm. We repeatedly scanned cells at 2‐ms intervals for line scan imaging (8‐bit). F imaging was performed with a laser scanning confocal microscope  and an inverted microscope . To exclude variations in the F intensity caused by various volumes of injected dye and to correct for variations in dye concentrations, the fluorescent signals were calculated through the normalization of the F against the baseline F (F0), and reliable information about transient intracellular Ca2+ (Ca2+i) changes from baseline values [(F − F0)/F0] were thus obtained. The intracellular Ca2+ changes, including transient Ca2+i, peak systolic Ca2+i, and diastolic Ca2+i, were obtained during a 1‐Hz field stimulation with 10‐ms twice‐threshold‐strength square‐wave pulses. After the addition of 20 mM caffeine after electric stimulation at 1 Hz for at least 30 s, the estimated sarcoplasmic reticulum (SR) Ca2+ content, used as the total SR Ca2+ content, was measured from the peak amplitude of the caffeine‐induced Ca2+i transients .With reference to the experimental design developed by Suenari et al. , fluores2.5+ (Na+i) concentrations of control and AGE‐treated RVOT cardiomyocytes. Experiments were performed using a laser scanning confocal microscope (Zeiss LSM 510) and inverted microscope (Axiovert 100) with a 63 × 1.25 numerical aperture oil immersion objective, as described by Viatchenko‐Karpinski et al.  changes from baseline values [(F − F0)/F0].We used CellROX Green (Life Technologies) to assess cytosolic ROS production and Asante NaTRIUM Green‐2 AM (Teflabs) to evaluate cytosolic Nai et al. . Cardiomi et al. . Fluores2.62+ATPase 2a [SERCA2a]), Ca2+/calmodulin‐dependent protein kinase II (CaMKII), pCaMKII, ryanodine receptors (RYR), pRYR S2808, pRYR S2814, Cav1.2, NCX, and beta‐actin. All secondary antibodies were conjugated with horseradish peroxidase. Moreover, all bound antibodies were detected using an enhanced chemiluminescence detection system and analyzed using AlphaEaseFC software (Alpha Innotech). All targeted bands were normalized to beta‐actin to confirm equal protein loading.Control and AGE (100 μg/ml)‐treated RVOT cardiomyocytes were centrifuged and washed with cold phosphate‐buffered saline and lysed on ice for 30 min in a RIPA buffer comprising 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecylsulfate (SDS), and protease inhibitor cocktails (Sigma‐Aldrich). The protein concentration was determined with a Bio‐Rad protein assay reagent (Bio‐Rad). Proteins were separated in 4%–12% SDS‐polyacrylamide gel electrophoresis (PAGE) under reducing conditions and electrophoretically transferred into an equilibrated polyvinylidene difluoride membrane (Amersham Biosciences). All blots were probed with primary antibodies against RAGE, Sirtuin1 (SIRT1), pSIRT1, NFκB, inositol 1,4,5‐trisphosphate receptor (IP3R) and pIP3R, P38, protein kinase C , SR/endoplasmic reticulum (SR/ER)  with a post hoc Tukey's analysis by Sigmaplot version 12.0 for multiple comparisons. The ionic currents of RVOT cardiomyocytes without or with AGEs were compared using either the Wilcoxon rank‐sum test or unpaired 33.190, APD50, APD20, RMP, or APA, which was inconsistent with clinically observed QT interval prolongation in patients with DM. The reduction of the Ito following treatment with AGEs is depicted in Figure IK1 and IKr‐tail were not influenced by AGE treatment. The effect of AGEs on the sodium current is depicted in Figure INa remained unchanged regardless of the presence of AGEs  are presented in Figure s Figure . Howevers Figure . The chaA Figure . The AGEA Figure .3.22+i transients and SR Ca2+ content with a prolongation of decay time in RVOT cardiomyocytes  of RVOT cardiomyocytes , which was further sustained after the administration of 1 μM isoproterenol  and/or isoproterenol  was not performed in this study. The peaks of the caffeine transients with and without tetracaine are the same because the total cellular Ca2+ has not changed, however, baseline Ca2+i transient may decrease  or reverse (Ca2+‐influx) mode. In this study, reverse NCX was augmented with AGE 100 μg/ml, primarily because of the increased INa‐late and cytosolic Na+ concentration (Na+i). In the MERLIN‐TIMI 36 study, which enrolled patients, a third of whom had DM, during the first week after admission for acute coronary syndrome, treatment with ranolazine resulted in a significantly lower incidence of an episode of VT lasting ≥4 or ≥8 beats compared with the placebo  National Defense Medical Center.Supplemental Figure 1Click here for additional data file.


The peptide was labeled with 99mTc using a direct labeling method. We determined the in vitro binding fraction of 99mTc-pHLIP (Var7) to MDA-MB-231 cells. Serial biodistribution studies and small-animal SPECT/CT imaging in MDA-MB-231 TNBC-bearing mice were performed using 99mTc-pHLIP (Var7).The pHLIP (Var7) sequence was synthesized 99mTc-pHLIP (Var7) were 99.49 ± 0.17% and 99.63 ± 0.44%, respectively. The radiochemical purity was still more than 96% after 24 h in serum. The binding fraction of 99mTc-pHLIP (Var7) to MDA-MB-231 cells continuously increased in an acidic environment and was significantly higher than the cell-binding fraction (P < 0.01) at pH = 7.4 and the cell-binding fraction (P < 0.01) of 99mTc-kVar7 at different pH values  at each time point (P < 0.01). The distribution of 99mTc-pHLIP (Var7) in tumors at each time point was significantly greater than that of 99mTc-kVar7 (P < 0.01). SPECT/CT imaging was largely consistent with the biodistribution results; the tumor was clearly imaged at each time point after injection of 99mTc-pHLIP (Var7) but could not be imaged after injection of 99mTc-kVar7.The radiochemical yield and purity of 99mTc-pHLIP (Var7) showed a high radiochemical yield and stability and was highly concentrated in tumor tissues. Although there was strong radioactive background in the abdomen of tumor-bearing nude mice, it did not hinder early diagnosis of TNBC. In addition, a novel 99mTc labeling method was used: four amino acids, Gly-(D)-Ala-Gly-Gly, that formed an N4 structure were used as a chelating group and linked to the N-terminus of pHLIP (Var7) during peptide synthesis to realize direct 99mTc labeling. This method simplifies the labeling process and raises the radiochemical yield and purity (The tumor microenvironment (TME) of almost all solid tumors is acidic, with pH values as low as 6.0 , 2. The The main instruments employed in this study were a Chirascan plus ACD spectropolarimeter , CRC-55tR radiopharmaceutical dose calibrator , Wizard 1480 gamma counter , Scan-RAM Radio-HPLC/TLC scanner , and U-SPECT +/CT small-animal SPECT/CT imaging system .99mTc-pertechnetate , stannous chloride , isoflurane , and hydrochloric acid .The main reagents applied in this study were 99mTc labeling, four amino acids, Gly-(D)-Ala-Gly-Gly, were attached to the N-terminus of pHLIP (Var7) and kVar7 to form a strong chelating group containing an N4 structure. To prevent any spatial obstruction, gamma-aminobutyric acid (GABA) was introduced as a spacer between the N4 structure and pHLIP (Var7)/kVar7. The modified pHLIP (Var7) and kVar7 sequences were as follows:pHLIP (Var7), GaGG-GABA-ACEEQNPWARYLEWLFPTETLLLEL-NH2; kVar7, GaGG-GABA-ACEEQNPWARYLKWLFPTKTLLLKL-NH2.pHLIP Var7) and the control sequence kVar7 ( and the via high-performance liquid chromatography (HPLC) using a Shimadzu-LC2010 instrument  equipped with a Kromasil 100-5C18 chromatographic column  . The peptides were eluted with a gradient from 25 to 85% solvent A (0.1% trifluoroacetic in 100% acetonitrile) and 75 to 15% solvent B (0.1% trifluoroacetic in 100% water) at 1.0 ml/minute, with monitoring at 220 nm. The identities of pHLIP (Var7) and kVar7 were confirmed by mass spectrometric analysis using an Agilent 6125B mass spectrometer .pHLIP (Var7) and kVar7 were purified sn-glycero-3-phosphocholine  were dissolved in 10 mmol/L phosphate buffer (PB) at pH = 8 and equilibrated at room temperature for at least 10 h. The pH value of the solution was adjusted to 4.0 with 0.1 mol/L HCl.Next, 7 µmol/L PHLIP (Var7) and 2 mmol/L 1-palmitoyl-2-oleoyl-CD spectra of pHLIP (Var7) were recorded at pH = 8.0 and pH = 4.0 in a 1-mm-path-length quartz cuvette using a spectropolarimeter. The experimental settings were as follows: bandwidth, 1 nm; wavelength range, 190–260 nm; wavelength step size, 1 nm; time-per-point, 0.5 s; temperature, 25°C.99mTc-pertechnetate were added. The mixture was then placed in a 90°C water bath for 15 min. The product was purified in a Sephadex G25 column (1.0 × 25 cm) and eluted with PBS . The radiochemical yield and specific activity of 99mTc-pHLIP (Var7) and 99mTc-kVar7 were calculated.A total of 1 mg peptide powder was dissolved in 1 mL 0.02 M phosphate-buffered saline (PBS). Ten microliters of the above-prepared solution was added to 90 μL PBS. Then, 20 μL of 1 mg/mL stannous chloride (in 0.01 M HCl), 20 μL sodium phosphate solution (8.2 mg/mL), and 74 MBq fresh 99mTc-pHLIP (Var7) and 99mTc-kVar7 was determined by thin-layer chromatography (TLC). A thin polyamide layer was used as the stationary phase, and acetonitrile was used as the developing solvent. A sufficient amount of the labeled product was aspirated through a capillary pipette and spotted at the origin of the thin polyamide layer. The thin polyamide layer was air-dried and vertically placed in a test tube with developing solvent. The saturated thin polyamide layer was removed, dried, and placed on the TLC scanner for testing.The radiochemical purity of 99mTc-pHLIP (Var7) and 99mTc-kVar7 were placed in PBS at room temperature (25°C) and in fresh human serum at 37°C. Samples were taken to test the radiochemical purity at 1, 4, 6, and 24 h after labeling.2 in high-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and 1% antibodies (penicillin–streptomycin mixture).MDA-MB-231 human breast cancer cells (Chinese Academy of Sciences) were cultured in an incubator at 37°C with 5% CO6 MDA-MB-231 cells was subcutaneously inoculated into the right axilla of each mouse.Animal experiments were approved by the ethics committee of Qingdao University, and the management of animals followed ethical standards. We built an MDA-MB-231 tumor model using four- to- five-week-old female BALB/c nude mice . To do so, 0.1 mL of a cell suspension (50 µL PBS + 50 µL Matrigel) containing 1 × 105 cells/mL, and 1 mL of the cell solution was added to each well of a 12-well plate and cultured overnight in a 37°C cell incubator.a. When MDA-MB-231 cells grew to more than 90% confluence, they were digested. The cell concentration was adjusted to approximately 1.0 × 1099mTc-pHLIP (Var7) or 99mTc-kVar7 (20-30 μL) were added to each well. We adjusted the pH values of the wells to four levels .b. The medium was discarded, and the cells were washed with PBS twice. Then, 2 mL of fresh medium and 37 kBq 99mTc-pHLIP (Var7) or 99mTc-kVar7 = B/(B+F) × 100%.c. After culturing for 30, 60, 90, 120, and 180 min, the supernatant was aspirated into a supernatant tube, and each well was washed two times with PBS at the corresponding pH . The cleaning solution was also added into the supernatant tube. The cells were digested fully with 0.25% trypsin, and the cell suspension was aspirated into a cell tube and washed twice with PBS. The cleaning solution was also added to the cell tube. A γ counter was applied to measure the radioactivity count of the cell tube (B) and supernatant tube (F). The cell-binding fraction of in vivo biodistribution experiment. Each nude mouse was injected with approximately 1480 kBq/200 μL 99mTc-pHLIP (Var7) or 99mTc-kVar7 through the tail vein. The mice were sacrificed humanely 1 h, 2 h, 4 h, and 6 h after injection, and their tissues and organs, such as the brain, heart, lung, liver, kidney, stomach, small intestine, blood, muscle, bone, and tumor, were weighed and measured for their radioactivity count. The results are expressed as the percentage of injected dose per gram of tissue (%ID/g).When tumor diameters reached 0.8–1.0 cm, 24 tumor-bearing nude mice were selected for an 99mTc-pHLIP (Var7) or 99mTc-kVar7 through the tail vein. The tumor-bearing mice were anesthetized with 2% isoflurane at 1 h, 2 h, 4 h, and 6 h after injection and were fixed on the imaging bed of a small-animal SPECT/CT imaging system in the prone position. A pinhole collimator was applied. The main parameters were a matrix size of 256 × 256, magnification power of 1.45, and acquisition time at each time point of 10 min.When tumor diameters reached 0.8–1.0 cm, six tumor-bearing nude mice were selected for imaging. Each nude mouse was injected with approximately 11.1 MBq/200 μL via one-way analysis of variance. P < 0.05 was deemed significant.SPSS 24.0.0.0 (IBM) statistical software was applied for data processing. Quantitative data are expressed as the mean (M) ± standard deviation (SD). Variables were compared via solid-phase peptide synthesis. HPLC analysis of pHLIP (Var7) showed the formation of several compounds consisting of a major product  accompanied by two smaller peaks at retention times of 12.75 and 13.24 min. HPLC analysis of kVar7 showed the formation of several compounds consisting of a major product  accompanied by two smaller peaks at retention times of 11.47 and 12.33 min.pHLIP (Var7) and kVar7 were successfully synthesized MS analysis of pHLIP (Var7) showed two mass peaks with m/z values of 1131.70 [(M+3H)3H+] and 1697.13 [(M+2H)2H+]. MS analysis of kVar7 showed two mass peaks with m/z values of 1130.80 [(M+3H)3H+] and 1695.70 [(M+2H)2H+]. The measured molecular weights of pHLIP (Var7) and kVar7  were consistent with the theoretical molecular weights .The secondary structure of pHLIP (Var7) was determined by CD at pH 8.0 and 4.0. 99mTc-pHLIP (Var7) had a radiochemical yield of 99.49 ± 0.17% and purity of 99.63 ± 0.44%. 99mTc-kVar7 had a radiochemical yield of 99.46 ± 0.14% and purity of 99.63 ± 0.35%. The specific activities of 99mTc-pHLIP (Var7) and 99mTc-kVar7 were both 7.36 ± 0.01 MBq/μg.99mTc-pHLIP (Var7) and 99mTc-kVar7 was > 99% in PBS at room temperature at 24 h and > 96% in fresh human serum at 37°C at 24 h  to MDA-MB-231 cells steadily increased and reached the highest levels at 180 min, which were 3.33 ± 0.12%, 3.02 ± 0.04%, and 2.87 ± 0.03%, respectively. At pH = 7.4, the binding fractions of 99mTc-pHLIP (Var7) to MDA-MB-231 cells remained low at each time point . The binding fractions of 99mTc-kVar7 to MDA-MB-231 cells also remained low at each time point  at all tested pH values  at pH = 6.0, 6.5, and 7.0 at various time points  were significantly higher than the cell-binding fractions at pH = 7.4 (P < 0.01) and than the cell-binding fractions of 99mTc-kVar7 at pH = 6.0, 6.5, 7.0, and 7.4 (P < 0.01).The cell-binding fractions of 99mTc-pHLIP (Var7) in tumors at 1, 2, 4, and 6 h was 3.63 ± 0.92, 6.11 ± 0.66, 5.33 ± 0.40, and 3.72 ± 0.94% ID/g, respectively, all of which were higher than the distribution levels of 99mTc-kVar7 in tumors (P < 0.01). Of the measured off-target organs, 99mTc-pHLIP (Var7) and 99mTc-kVar7 showed high distribution levels in the liver and blood (The distribution of 99mTc-pHLIP (Var7) group, while no significant increasing trend was seen in the T/NT ratio in the 99mTc-kVar7 group. The T/NT ratio in the 99mTc-pHLIP (Var7) group was higher at each time point than in the 99mTc-kVar7 group  ratio increased over time in the 99mTc-pHLIP (Var7) or 99mTc-kVar7 (99mTc-pHLIP (Var7) but not after injection of 99mTc-kVar7. The liver was clearly imaged in both groups at each time point.SPECT/CT imaging was performed at 1, 2, 4, and 6 h after the injection of via mammography, magnetic resonance imaging, and ultrasound , which can target the TNBC TME (99mTc-pHLIP (Var7), which exhibits a typical pH-dependent transition from an unstructured conformation to an α-helix structure in an acidic environment (confirmed by CD spectroscopy), SPECT could be used for functional imaging of TNBC and is expected to be used for early diagnosis and screening of TNBC.TNBC can easily metastasize and has a poor prognosis. Early diagnosis of TNBC can effectively improve the prognosis. In daily clinical practice, the imaging diagnosis of breast cancer is typically performed trasound . Since Ttrasound , the relTNBC TME , 10, 11,99mTc labeling, indirect labeling is primarily used, which is relatively complex and leads to an insufficient radiochemical yield and poor stability in vitro. In this study, by referring to the labeling technology described by Zhao et al.  showed a high binding fraction to MDA-MB-231 cells in an acidic environment, significantly higher than the binding fraction of the control peptide 99mTc-kVar7, suggesting that the N-terminal labeling of pHLIP (Var7) with 99mTc did not change the ability of pHLIP (Var7) to insert itself into tumor cell membranes in acidic environments. The insertion of pHLIP family peptides into the lipid bilayer of cell membranes is triggered by low pH values. In acidic environments, the negatively charged amino acid residues (Asp or Glu) in pHLIP are protonated, and an α-helix forms for insertion into the cell membrane  and 99mTc-kVar7 had slow blood clearance, suggesting that they might bind strongly to plasma proteins and/or red blood cells. Complexes formed by binding are not conducive to probe elimination and lower the image quality. In future studies, we will consider changing the characteristics of probe molecules  to reduce their binding to plasma proteins and/or red blood cells. The high distribution of the probes in the kidney suggests that the probes might be excreted through the urinary system. One study found (Biodistribution assays showed that dy found  that thedy found  showed t99mTc-pHLIP (Var7), likely because 99mTc-labeled pHLIP (Var7) bound strongly to intrahepatic tissue proteins and 99mTc labeling changed the original elimination characteristics of pHLIP (Var7), thus, it was partially excreted in bile. Biodistribution data further showed that although there was a very strong radioactive background in the abdomen of mice, the distribution of 99mTc-pHLIP (Var7) in tumor tissue was significantly higher than that in muscle, bone, and other tissues. Based on the biodistribution characteristics of the probes, it could be predicted that primary TNBC would be easily identifiable in SPECT images, which would be valuable for the early diagnosis of TNBC.The distribution of both probes in the liver was high. The liver uptake of fluorescence-labeled pHLIP (Var7)  was muchin vivo biodistribution trend. The tumors in mice were imaged clearly at various time points after injection of 99mTc-pHLIP (Var7), while the tumors were not imaged after injection of 99mTc-kVar7, suggesting that 99mTc-pHLIP (Var7) was electrically neutral in the acidic TME due to protonation of the negatively charged Glu residue, and the peptide changed from a disordered structure to an α-helix structure and inserted itself into tumor cell membranes. 99mTc-kVar7 remained positively charged in the acidic TME and did not convert to an α-helix to insert itself into tumor cell membranes. Although the intense radioactive concentration in the livers of the tumor-bearing nude mice interfered with the detection of abdominal metastases, it did not hinder the imaging of primary TNBC lesions enriched in 99mTc-pHLIP (Var7); thus, 99mTc-pHLIP (Var7) can potentially be used for early diagnosis of TNBC and breast cancer screening in high-risk groups.The results of SPECT/CT imaging of tumor-bearing nude mice were largely consistent with the 99mTc-pHLIP (Var7) has a high radiochemical yield and high stability and shows high accumulation in TNBC. Although 99mTc-pHLIP (Var7) has a strong radioactive concentration in the abdomen, this does not prevent the imaging of primary TNBC. 99mTc-pHLIP (Var7) is expected to become a valuable radioactive molecular probe for early TNBC diagnosis and screening.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/The animal study was reviewed and approved by ethics committee of Qingdao University.YC, YS, XP, XS, and WZ performed the experiments and wrote the paper. MY designed and supervised the research. All authors read and approved the final manuscript.This study was funded by the Natural Science Foundation of Shandong Province (Grant No. ZR2021MH038), Shandong Provincial Medical and Health Science and Technology Development Program (Grant No. 202009040347) and the Affiliated Hospital of Qingdao University “Clinical Medicine+X” Scientific Research Project (Grant No. QDFY+X202101013).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SF3B1 mutations are recurrently found, most frequently in AML-MRC [SF3B1 mutations is considered as diagnostic criteria for AML-MR according to the 5th edition of the WHO classification , AML are classified into AML with recurrent genetic abnormalities, AML with myelodysplasia-related changes (AML-MRC) or AML, not otherwise specified (AML-NOS)  incorporSF3B1 is most frequently mutated in MDS or MDS/MPN and associated with a favorable prognosis and an indolent disease course [BCOR, BCORL1, NRAS, RUNX1, SRSF2, STAG2 and del(5q) [Within the last years, many prognostically relevant driver genes in AML have been identified including also spliceosome genes . In myele course –8. More  del(5q) .SF3B1mut cases were further classified according to WHO 2022 [For this analysis we selected 735 AML samples with material available to perform whole genome sequencing sent to the MLL Munich Leukemia Laboratory between 09/2005 and 01/2020. Therapy-related AML were excluded from this study. Within the cohort 89% (652/735) were de novo AML cases and 11% (83/735) s-AMLs. For further details on cohort and statistics see Supplementary Methods. All cases were classified into specific sub-groups according to the currently used WHO 2017 . SF3B1muWHO 2022 ). For abSF3B1 mutations in a small fraction  of AML patients , AML-MRC  and AML-NOS  , thereby confirming the association of SF3B1 mutations with GATA2::MECOM rearrangements as previously published [SF3B1 mutations were exclusively found in samples diagnosed with AML with maturation  and AML with GATA2::MECOM  were most frequent  per se are not prognostic in AML [We identified nts Fig.  in line 6%; 41/73 of AML p6%) Fig. . Within ublished . Notablyent Fig. . When stc in AML .Fig. 1DiSF3B1 mutations showed a mean variant allelic frequency (VAF) of 41% and those mutations affecting amino acids K666 and K700 were found most frequently  and NRAS . NPM1, TET2, or DNMT3A mutations or FLT3-ITD were detected in 15% (6/41) each. RUNX1 mutations were present besides within AML with RUNX1 mutation, also in AML-MRC (n = 3) and AML with GATA2::MECOM (n = 1). Interestingly, 37% (15/41) of SF3B1mut patients harbored at least one mutation in a DTA gene . Additional mutations were found in 5 to 21 different genes depending on the respective entity  with a different partner gene than GATA2. This resulted in 49% (20/41) of SF3B1mut patients harboring a MECOM-r . SF3B1 mutations were significantly associated with MECOM-r . In summary, the majority  of SF3B1mut AML were either AML with MECOM-r (n = 20) or AML-MR (n = 12), underpinning the strong association of SF3B1 mutations with these two entities . A prior history of MDS or MDS/MPN was documented in 20% (8/41) of SF3B1mut patients harboring on average 4.3 mutations at AML diagnosis , other mutations showed higher VAFs  remained detectable, despite complete hematologic remission and undetectable NPM1 mutation . However, it might be unidentified in others. Alternatively, SF3B1mut CHIP or CCUS  may represent relevant precursor lesions of SF3B1mut AML, in line with Venable et al., showing that SF3B1mut cases comprise the full pathologic spectrum of myeloid disorders from CCUS to AML [SF3B1mut s-AML patients, we frequently detected MECOM-r and RUNX1 mutations, both known AML driver genes [SF3B1mut AML patients, where no MDS or MDS/MPN history had been reported. In this line, we previously showed that SF3B1mut MDS patients harboring RUNX1 mutations frequently progressed to AML and that RUNX1 mutations and MECOM-r were gained during AML transformation [SF3B1mut AML without antecedent clinical documentation and further supports the guidelines of the WHO 2022 showing that SF3B1mut AML is diagnosed as AML-MR without knowing the patient’s clinical history.As shown, mutations in  studies , 14. Not studies  and the  studies . In our er genes . These tormation . Thus, oSF3B1 mutations are found in a small fraction of AML patients, are enriched in poor risk AML subtypes and are strongly associated with MECOM rearrangements and myelodysplasia-related changes. The persistently high VAF of SF3B1 mutations in AML patients suggests that SF3B1 mutations are acquired early in a pre-leukemic clone and may be indicative of an MDS pre-phase.In summary, Supplementary Material


An important strategy to support the professional development of mentors in health professions education is to encourage critical reflection on what they do, why they do it, and how they do it. Not only the ‘how’ of mentoring should be covered, but also the implicit knowledge and beliefs fundamental to the mentoring practice . This study analyzed the extent to which mentors perceive a difference between how they actually mentor and how they prefer to mentor.N = 228), was used to ask mentors about the how, what, and why of their mentoring in two response modes: (1) regarding their actual mentoring practice and (2) regarding their preferred mentoring practice. With an analysis of covariance, it was explored whether potential discrepancies between these responses were influenced by experience, profession of the mentor, and curriculum-bound assessment requirements.The MERIT (MEntor Reflection InstrumenT) survey (distributed in 2020, The averaged total MERIT score and averaged scores for the subscales ‘Supporting Personal Development’ and ‘Monitoring Performance’ were significantly higher for preferred than for actual mentoring. In addition, mentors’ experience interacted significantly with these scores, such that the difference between actual and preferred scores became smaller with more years of experience.Mentors can reflect on their actual and preferred approach to mentoring. This analysis and the potential discrepancy between actual and preferred mentoring can serve as input for individual professional development trajectories.The online version contains supplementary material available at 10.1186/s12909-022-03690-3. Mentors in health professions education are faculty who support their mentees’ personal and professional development –6. They To help mentors make the “what, why, and how” of their actual practice explicit, and explore the implicit system of knowledge and beliefs underpinning practice, we developed a survey called MERIT: MEntor Reflection InstrumenT . The intpersonal interpretative framework. Kelchtermans [professional self-understanding. This is the understanding mentors have of themselves as mentors at a certain point in time (the ‘what’ and ‘why’ of their mentoring). The second dimension, subjective educational theory, encompasses a mentor’s personal system of knowledge and beliefs about the way they mentor (the ‘how’) [The MERIT draws on research in the field of mentoring as well as our own empirical work on mentors’ htermans  describee ‘how’) . This mue ‘how’)  conceptuIn the current article, we report on additional data about ‘preferred mentoring’ gathered during the MERIT development study . With thWe invited mentors in health professions education to participate. In this article, our target population of mentors in health professions education is defined as faculty members who have a formal mentoring relationship with one or multiple (under)graduate students. The focus of this relationship is on supporting competence development and stimulating reflection (after Nicholls ). ResponAn extensive description of the design, distribution, and analysis of the psychometric properties of the survey can be found in Loosveld, Van Gerven, Driessen, Vanassche, Artino . The surA link to the online MERIT survey, which was hosted on Qualtrics , was distributed via Twitter accounts of the researchers (around 4000 cumulative followers), LinkedIn (around 800 connections), and via 128 e-mails to contact persons between November 2019 and March 2020. Because of this distribution via contact persons and social media, the exact overall denominator was unknown, as a result of which it was impossible to calculate the overall response rate. As this study did not intend to characterize a population, the lack of response rate was considered less problematic .actual mentoring practice and the second time, immediately after the first time, respondents were asked to envision their preferred mentoring. We included the following instruction to explain the two response modes  modelling professional development, with items about providing insight on how academia works (3) fostering autonomy about advice-seeking and problem-solving, and (4) monitoring performance, about accessing and understanding performance data. An extensive description of the design, distribution, and analysis of the psychometric properties of the survey can be found in Loosveld, Van Gerven, Driessen, Vanassche, Artino [Eight demographic questions and two open-ended questions about the content and design of the survey concluded the survey. The factor structure within the set of survey items was previously determined via Principal Access Factoring and the internal consistency reliability of the subscale scores evaluated using Cronbach’s alpha . Based oing, and  monitoriTo reach our current research objectives, we ran one-way analyses of covariance (ANCOVA) with Response Mode  as the within-groups independent variable. The dependent variables were the average score on the entire MERIT survey, as well as average scores for the four subscales, based on MERIT factors. We included three covariates in our model: (1) Experience, (2) Main Profession, and (3) Assessment. Experience was the amount of mentoring experience in years. Main Profession was defined as the profession that mentors primarily identified with . Assessment, finally, indicated whether mentors were required to assess their mentee or not . SPSS statistical software, version 25  and Microsoft Excel 2016  were used for data analysis and data management.This research was approved by the Maastricht University Ethics Review Committee (UM-REC), file number: FHML-REC/2019/033, October 1, 2019.After removing the data of four respondents mentoring outside the field of health professions education, 228 fully completed surveys remained for analysis.Our sample consisted of 77 (34%) mentors who identified as men and 148 (65%) who identified as women. One mentor indicated ‘other’ as their gender and two other mentors did not identify their gender (1%). The mean age of the respondents was 46.4 years . As can be seen from Table F = 15.20, p < .001, ηp2 = .064, indicating that the average total MERIT score was higher in the Preferred  than in the Actual  response mode  = 1.38, p = .241, ηp2 = .006, and neither did the other two covariates, Main Profession and Assessment (Fs < 1).The results of the ANCOVA yielded a significant main effect of Response Mode, F1, 22 = 15.20,F = 4.76, p = .030, ηp2 = .021, suggesting that the effect of Response Mode – that is, the discrepancy between Actual and Preferred MERIT scores – became smaller with more years of experience  = 13.75, p < .001, ηp2 = .058, indicating that mentors’ score on this factor was higher for Preferred than for Actual mentoring. For Monitoring Performance, mentors’ Preferred scores were again higher than Actual scores, F = 13.01, p < .001, ηp2 = .055  = 12.99, p < .001, ηp2 = .055. The other covariates did not show main effects on any of the four factors (Fs < 1) , suggesting that these covariates did not affect the discrepancy between Actual and Preferred MERIT scores  = 10.55, p = .001, ηp2 = .045, again suggesting that the effect of Response Mode, on the level of Supporting Personal Development, became smaller with more years of Experience  = 4.33 p = .039, ηp2 = .019, although this interaction did not survive Bonferroni correction. No further interactions between response mode and covariates were found. Table A significant Response mode × Experience interaction in that same direction was found for the subscale Monitoring Performance, Findings from this study suggest that the mentors in our sample perceive a discrepancy between their actual and preferred mentoring. Moreover, mentoring experience significantly moderated this discrepancy: The more years of experience as a mentor, the smaller the discrepancy became. This interaction effect appeared to be driven by responses on the subscale Supporting Personal Development.It is important to note that we did neither intend to make evaluative statements about mentoring capabilities, nor did we try to uncover the reason behind discrepancies between one’s actual and preferred mentoring. Moreover, identified discrepancies between actual and preferred mentoring do not imply that someone is not a good mentor. Rather these discrepancies may indicate conflicting narratives – for example, between professional self-understanding and curriculum requirements –, which could hamper mentors to put their personal knowledge and beliefs into practice –35. PrioBased on these findings, we believe that the merit of the MERIT survey for mentors lies in offering support during their professional development. Critical reflections on experiences from their daily practice can help mentors to identify and prioritize learning needs , 37, 38,A limitation to this study is that we had little means to control who filled out the survey. Even though we asked mentors to respond only when they met our inclusion criteria and we examined the responses for mentors who did not meet the inclusion criteria, we cannot be sure that all respondents indeed fit our description of mentors in health professions education. In addition, despite our efforts to distribute the survey globally, the majority of our respondents fulfilled their mentoring role in Europe (73.3%) or North America (18.9%). Therefore, our sample may not reflect a worldwide representation of mentors in health professions education and we cannot rule out the influence of, for example, local administrative rules and regulations. However, given the context specificity of the personal interpretative framework, we argue that an accurate representation of how individual mentors perceive their mentoring only exists within the specific professional working context of that mentor.Another limitation of this study is that we were not able to analyze how mentors interpreted or explained their reflections. Follow-up research could therefore take a more explanatory approach, where mentors are asked to reflect on their mentoring practice and then, together with an interviewer, explore their answers and discuss how those answers shape their personal interpretative framework. Because of our quantitative approach, we also do not know whether there are other factors that might influence mentors’ actual and preferred mentoring. Given the personalized and contextualized nature of mentoring, this is an avenue that warrants further exploration.The perceived discrepancy between actual and preferred mentoring of the mentors in our sample is influenced by their years of experience: More experienced mentors perceive a smaller discrepancy between their actual and preferred mentoring. This discrepancy could guide faculty development initiatives that involve active and collaborative formats to help mentors discuss, reinforce, and challenge their personal interpretative framework.Additional file 1.


However, the mechanisms that control the amplitude and termination of Ca2+ puffs are unresolved. To address these issues, we expressed SNAP-tagged IP3R3 in HEK cells without endogenous IP3Rs and used total internal reflection fluorescence microscopy to visualize the subcellular distribution of IP3Rs and the Ca2+ puffs that they evoke. We first confirmed that SNAP-IP3R3 were reliably identified and that they evoked normal Ca2+ puffs after photolysis of a caged analog of IP3. We show that increased IP3R expression caused cells to assemble more IP3R clusters, each of which contained more IP3Rs, but the mean amplitude of Ca2+ puffs (indicative of the number of open IP3Rs) was unaltered. We thus suggest that functional interactions between IP3Rs constrain the number of active IP3Rs within a cluster. Furthermore, Ca2+ puffs evoked by IP3R with reduced affinity for IP3 had undiminished amplitude, but the puffs decayed more quickly. The selective effect of reducing IP3 affinity on the decay times of Ca2+ puffs was not mimicked by exposing normal IP3R to a lower concentration of IP3. We conclude that distinct mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and closure of IP3Rs during the falling phase, and that only the latter is affected by the rate of IP3 dissociation.Ca Most Ca2+ signals are initiated by inositol 1,4,5-trisphosphate (IP3) produced when cell-surface receptors stimulate phospholipase C. IP3 binds to the four subunits of a tetrameric IP3 receptor (IP3R), priming it to bind Ca2+, and triggering the opening of an intrinsic Ca2+-permeable channel, through which Ca2+ flows rapidly from the lumen of the endoplasmic reticulum to the cytosol  that arise from the coordinated opening of a few channels within a cluster , which report the opening of a single IP3R   , 9. Ca2+resolved .2+-induced Ca2+-release between IP3Rs is potentially explosive, but the mechanisms that terminate the activity of Ca2+ puffs are incompletely understood. Delayed feedback inhibition of IP3Rs by substantial local increases in [Ca2+]c probably contributes  was expressed in human embryonic kidney (HEK) cells lacking endogenous IP3Rs (HEK-3KO cells)  . Our optimized methods, which required labeling of cells in suspension before plating for analyses using fluorescence microscopy (A–D), allowed SNAP-IP3R3 to be identified in cells, most of which expressed IP3R3 at modest levels  and with the punctate distribution of IP3R clusters typical of native IP3Rs   . Our useFig. 3A) .Figure 13R3 (Ab-IP3R3)  were immunostained with Ab-IP3R3 and imaged using total internal reflection fluorescence (TIRF) microscopy. The results demonstrate colocalization of immunostaining and SNAP-647 fluorescence , a tight linear correlation between the intensities of whole-cell immunostaining and SNAP-647 fluorescence (C), and a linear correlation for individual puncta between Ab-IP3R3 and SNAP-647 fluorescence intensities   in immunKO cells . HEK-SNArescence C, and a ensities F and S2.es F and  and 2 de2+ puffs evoked by photolysis of ci-IP3 in HEK-IP3R3 and HEK-SNAP-IP3R3 cells. Since the latter were transiently transfected (B), individual cells differed in their expression of SNAP-IP3R3 (E), we therefore selected cells in which the distribution of IP3R resembled that observed in HeLa cells with tagged endogenous IP3R . The results demonstrate that Ca2+ puffs occurred with indistinguishable frequencies and after similar latencies in the two cell types (B–D). Furthermore, the properties of individual Ca2+ puffs  were also indistinguishable for HEK-IP3R3 and HEK-SNAP-IP3R3 cells  to report SNAP-IP3R3 expression in individual HEK-SNAP-IP3R3 cells and compared it with the properties of the Ca2+ puffs evoked by photolysis of ci-IP3. The results demonstrate that over about a 60-fold range of expression there is a positive correlation between SNAP-IP3R3 expression and the frequency of Ca2+ puffs (A): Ca2+ puffs are more frequent in cells with more IP3Rs. The latency to the first detected Ca2+ puff after the photolysis flash decreased as SNAP-IP3R3 expression increased (B). However, the mean properties of individual Ca2+ puffs  were similar at all levels of SNAP-IP3R3 expression (C–F).We used SNAP-647 fluorescence intensity measured from the TIRF footprint of the region of interest (ROI) from which Ca2+ puffs A: Ca2+ pncreased B. Howevepression , C–F.Fig3R are distributed within cells expressing different numbers of IP3R. We restricted this analysis to HEK-SNAP-IP3R3 cells in which the SNAP-647 fluorescence intensity measured from ROIpuffs was <20,000 fluorescence units because at higher levels of expression, it was impossible to reliably distinguish individual puncta. The results demonstrate that both the number of SNAP-IP3R3 puncta (A and B) and their mean fluorescence intensity (A and C) were increased in cells expressing more SNAP-IP3R3. The rightward shifts in the fluorescence intensity distributions for individual puncta as IP3R expression increased indicates that when cells express more IP3Rs, most puncta contain more IP3Rs , the properties of individual Ca2+ puffs, including their mean amplitudes, are preserved, but they occur after shorter latencies and they are more frequent in cells with more IP3Rs. Since only a fraction of IP3R puncta are licensed to respond . This analysis demonstrates that in cells expressing many IP3Rs, puncta that contain more IP3Rs evoke Ca2+ puffs of unaltered amplitude.These results demonstrate that across a wide range of SNAP-IP respond , we had 2+ puffs  7) and  and 3R3 fs occur D. This a2+ puffs, indicating the number of open IP3Rs  within the conserved IP3-binding core (IBC) of IP3R1 causes a 9-fold reduction of its affinity for IP3 (3R3 and used it to explore the effects of increasing the rate of IP3 dissociation on Ca2+ puffs (A). We confirmed that SNAP-IP3R3RQ had the same subcellular distribution as SNAP-IP3R3 (B). Since the IBC is highly conserved in all IP3R subtypes and each IBC binds IP3 with the same affinity (3R3RQ mutation to exactly replicate the 9-fold decrease in affinity reported for IP3R1RQ (2+ puffs evoked by photolysis of ci-IP3 is reduced in cells expressing IP3R3RQ (C).Since the mechanisms that terminate Caresolved , 28, we  for IP3 . We intr2+ puffs A. We conAP-IP3R3 B. Since affinity , we expe IP3R1RQ . The ass IP3R3RQ C.Figure 3R for IP3 while maintaining the submaximal concentration of i-IP3 delivered after the UV flash to reduce the number of IP3R occupied by i-IP3 and so reduce the frequency of Ca2+ puffs and increase the latency before the first observed Ca2+ puff. Our results confirm these expectations. As with SNAP-IP3R3, the frequency of Ca2+ puffs increased with increased expression of SNAP-IP3R3RQ, the latencies shortened with increased expression, and the mean amplitude of individual Ca2+ puffs was unaffected by the overall level of SNAP-IP3R3RQ expression (C–E). However, across a wide range of IP3R3 expression levels, the frequencies of Ca2+ puffs were lower, and the latencies were longer for SNAP-IP3R3RQ than for SNAP-IP3R3 (C and D).We expected that reducing the affinity of an IPpression , C–E. HoAP-IP3R3 , C and D3R3 and SNAP-IP3R3RQ, we pooled data from cells in which the SNAP-647 fluorescence intensity measured from ROIpuffs fell within a defined range (1800–5900 FU), which coincided with cells in which the subcellular distribution of IP3R3 resembled that of native IP3Rs. The pooled data confirmed that the average level of IP3R3 expression (A) and mean puff amplitude (C) were indistinguishable for HEK-SNAP-IP3R3 and HEK-SNAP-IP3R3RQ cells, but the frequency of Ca2+ puffs was significantly reduced in the HEK-SNAP-IP3R3RQ cells (B).To allow direct comparisons of cells expressing SNAP-IPpression A and meamplitude C were inRQ cells B.Figure 2+ puffs in cells expressing SNAP-IP3R3 or SNAP-IP3R3RQ. Automated analysis of the rise times of Ca2+ puffs is distorted when Ca2+ puffs have a sustained plateau phase (‘square’ puffs) (A). We therefore excluded ‘square’ Ca2+ puffs from our analyses of the kinetics of individual Ca2+ puffs. The exclusion rate was similar (∼18%) for analyses of SNAP-IP3R3 and SNAP-IP3R3RQ (B), confirming that it did not bias our analyses. We note in passing that although we have not specifically addressed the mechanisms underlying ‘square’ Ca2+ puffs, their similar abundance in cells expressing IP3R3 and IP3R3RQ indicates that ‘square’ Ca2+ puffs are not selectively affected by changes in IP3R affinity. Rise times were indistinguishable in cells expressing SNAP-IP3R3 and SNAP-IP3R3RQ (D), but decay times and the duration of Ca2+ puffs were significantly shorter for SNAP-IP3R3RQ (E and F and C–E).We next compared the kinetic properties of individual Ca’ puffs)  because ’ puffs) A. We the-IP3R3RQ B, confir-IP3R3RQ D, but de3R for IP3 were solely attributable to reduced occupancy of IP3R by IP3, we would expect Ca2+ puffs evoked by a 9 to 10-fold lower concentration of i-IP3 through normal IP3R to have the same properties as Ca2+ puffs evoked by IP3RRQ. We therefore compared Ca2+ puffs evoked by photolysis of ci-IP3 in HEK-SNAP-IP3R3RQ cells with the usual UV flash duration (250 ms) to those evoked in HEK-SNAP-IP3R3 cells exposed to a 10-fold shorter flash (25 ms). The frequency of the Ca2+ puffs and the relationship between IP3R expression and frequency were similar under the two conditions (A and B). These results confirm that for the frequency of Ca2+ puffs, a 9 to 10-fold reduction in IP3-binding affinity can be compensated by a comparable increase in i-IP3 concentration, suggesting that steady-state occupancy of IP3R by IP3 may determine the frequency of Ca2+ puffs. As expected, the amplitudes of individual Ca2+ puffs recorded under the two conditions were indistinguishable (C). The more important result is that neither the shorter duration of Ca2+ puffs nor the faster decay times in cells expressing IP3R3RQ were reproduced by exposing IP3R3 to a 10-fold lower concentration of i-IP3 (D–F). Our results show that in cells expressing SNAP-IP3R3, all measured properties of individual Ca2+ puffs are indistinguishable whether evoked by low concentrations of i-IP3 (25-ms flash) or a 10-fold higher concentration (250-ms flash), but the decay times and durations are longer than for IP3R with reduced affinity for IP3 (SNAP-IP3R3RQ) (G).If the effects of reducing the affinity of the IPnditions , A and Buishable C. The moof i-IP3 , D–F. OuIP3R3RQ) G.Figure 3R3 to be expressed in HEK cells at levels comparable to expression of endogenous IP3R and, by attaching a SNAP-tag to the expressed IP3R3, we were able to reveal both the subcellular distribution of IP3R3 and the Ca2+ puffs they evoke in response to photolysis of ci-IP3  and to termination of Ca2+ puffs (through delayed inhibition of IP3R by Ca2+). It is, therefore, relevant that the biphasic effects of [Ca2+]c on IP3R is itself regulated by IP3. We, for example, have suggested that IP3 binding to IP3R primes them to open by facilitating the binding of Ca2+ to a stimulatory site; while in the absence of IP3, Ca2+ binds to an inhibitory site that prevents channel opening (3 regulating the effects of Ca2+ on IP3R gating (3 dissociation from an active IP3R may influence its susceptibility to Ca2+ inhibition. We therefore examined the effect of manipulating the rate of IP3R dissociation on the properties of Ca2+ puffs. We demonstrated that IP3R3 with about a 10-fold lower affinity for IP3 (SNAP-IP3R3RQ) evoked Ca2+ puffs in response to photolysis of ci-IP3. As expected, the Ca2+ puffs mediated by SNAP-IP3R3RQ were less frequent than those mediated by SNAP-IP3R3, consistent with the steady-state occupancy of IP3R by IP3 determining the number of active IP3Rs  was reduced for SNAP-IP3R3 cells to provide matched frequencies of Ca2+ puffs in the two cell lines (indicative of comparable IP3R occupancies by i-IP3) (3R for IP3 affects the rate at which Ca2+ puffs terminate: reducing the affinity for IP3 (increasing the rate of IP3 dissociation) causes Ca2+ puffs to terminate more quickly. We draw two important conclusions from these data. Firstly, the rate of IP3 dissociation from IP3R contributes to termination of Ca2+ puffs. Various mechanisms might align with this conclusion, but it is consistent with a local increase in [Ca2+]c contributing to termination by closing IP3R but only after IP3 has dissociated from active IP3R  . These a3Rs recruited within a cluster during the rising phase of a Ca2+ puff and the closure of active IP3Rs during the falling phase. The key observation here is that although Ca2+ puffs evoked by IP3R with reduced affinity for IP3 (SNAP-IP3R3RQ) terminate more quickly, their mean peak amplitude is indistinguishable from that of Ca2+ puffs evoked by normal IP3Rs  causes both channel closure and its immediate susceptibility to Ca2+ inhibition, which might then prevent reopening.Our second conclusion relates to whether the same mechanism might account for the constraint on the number of IPal IP3Rs  and 8. T3Rs, cells incorporate more of them into clusters, assemble more IP3R clusters, and generate more frequent Ca2+ puffs in response to IP3, but the properties of individual Ca2+ puffs, including their mean peak amplitudes, are unchanged. We suggest that different mechanisms constrain recruitment of IP3Rs during the rising phase of a Ca2+ puff and cause closure of IP3Rs during the falling phase; only the latter is affected by the rate of IP3 dissociation.We conclude that when presented with more IP3/PM benzyl-myo-inositol 1,4,5-trisphosphate hexakis(propionoxymethyl) ester) was from SiChem. 35-mm glass-bottom imaging dishes  were from Cellvis (IBL Baustoff+Labor GmbH). EGTA-AM, human plasma fibronectin, Pluronic F-127, and doxycycline hydrochloride were from Merck. SNAP-Cell 647-SiR, BsrGI-HF restriction enzyme, Q5 High-Fidelity Master Mix, and Gibson Assembly Master Mix were from New England Biolabs. pcDNA3.2/V5-DEST, pDONR221, pcDNA6/TR and pT-Rex-DEST30 vectors, LR and BP Clonase II enzyme mixes, Pfl23II (a.k.a. BsiWI) and CpoI (a.k.a. RsrII) restriction enzymes, and custom oligonucleotides were from Thermo Fisher Scientific.Cal520-AM and Calbryte590-AM were from AAT Bioquest. ci-IPSources of additional materials are provided in relevant sections of Experimental procedures.3R3 was obtained from Harvard PlasmID Database  and cloned from pENTR223 into pcDNA3.2/V5-DEST using LR Gateway cloning. We tagged human IP3R3 with a SNAP-tag, a self-labeling enzyme tag based on the human DNA repair protein, O6-alkylguanine-DNA-alkyltransferase, which reacts with O6-benzylguanine derivatives conjugated to fluorescent dyes (3R3 construct (SNAP-IP3R3), PCR was used to amplify an IP3R3 fragment , and BsrGI restriction digest was used to generate a backbone fragment. Gibson assembly was used to introduce a DNA string  encoding SNAPf (fast-labeling SNAP-tag) with a GGSGGGSG peptide linker between the backbone fragment and the IP3R3 fragment (A). For expression of SNAP-IP3R3 under control of a tetracycline-inducible promoter, SNAP-IP3R3 was transferred from pcDNA3.2 to pDONR221 and then to pT-REx-DEST30 using BP and LR Gateway cloning, respectively.A plasmid encoding human IPent dyes . To genefragment A. For ex3R3 with reduced affinity for IP3 , a DNA string was synthesized encoding a region of SNAP-IP3R3 between two restriction sites, BsiWI and RsrII, which contained an Arg to Gln mutation at residue 568 . A BsiWI/RsrII-restriction digest of SNAP-IP3R3 in pDONR221 was used to generate a fragment encoding the remainder of the construct. Gibson assembly was used to assemble the two fragments, and SNAP-IP3R3RQ was transferred to pT-REx-DEST30 using LR Gateway cloning. Sequences of all coding regions were confirmed using Sanger sequencing (Source BioScience). A silent point mutation was detected in bases (ATC to ATA) encoding Ile2512 in both constructs.To generate the construct encoding IP3R subtypes  or expressing only IP3R3  were generated by CRISPR/Cas9 (2 and passaged every 3 to 4 days using Gibco TrypLE Express (Thermo Fisher Scientific). Regular screening confirmed that cells were free of mycoplasma.HEK cells lacking all IP3R3 cells) expressing SNAP-IP3R under control of the Tet repressor (TetR), we used pcDNA6/TR, a plasmid encoding TetR, and the Invitrogen T-REx expression system. HEK-3KO cells were seeded in 25-cm2 flasks and after 24 h, when they were ∼70% confluent, they were transiently transfected using TransIT-LT1 Transfection Reagent  with SNAP-IP3R3 in pT-REx-DEST30 (2.6 μg/T25 flask) and pcDNA6/TR (15.8 μg). After 24 and 48 h, medium was exchanged for fresh complete medium containing doxycycline (1 μg/ml) to block activity of TetR and derepress the expression of SNAP-IP3R3 (B).To generate cells . To avoid background labeling, it was essential to label cells in suspension before reattaching them to imaging dishes for microscopy (C–E). Transfected cells were detached using TrypLE Express, resuspended in fresh complete medium (3 × 105 cells in 300 μl) containing SNAP-Cell 647-SiR (1 μM), and incubated for 15 min at 37 °C with 5% CO2. Cells were then washed three times , resuspended in fresh complete medium, plated into a 35-mm glass-bottomed imaging dish (#1 cover glass) coated with 10 μg/ml human fibronectin, and used after 12 to 14 h (B).HEK-SNAP-IPcroscopy , C–E. Tr to 14 h B.via a Cairn MultiLine LaserBank (488 and 647 nm) and an iLas2-targeted laser illumination system (Cairn Research), through which the excitation angle for TIRF was adjusted to achieve a theoretical penetration depth of 80 to 100 nm. Excitation light of 488 or 647 nm was passed through a quad-band filter set , and emitted light was passed through an appropriate band-pass filter within a high-speed filter wheel . A 395-nm LED  was used for flash photolysis of ci-IP3. Detection of emitted light was via an EMCCD camera . Image capture used MetaMorph Microscopy Automation and Image Analysis Software , through which image capture  was fully automated. Both live and fixed cells were imaged at 20 °C.Fluorescence microscopy used an inverted Olympus IX83 microscope equipped with a 100× oil-immersion TIRF objective  or for  a 60× oi3R3 cells in imaging dishes were fixed (20 min) with 4% paraformaldehyde (Alfa Aesar) in PBS (Thermo Fisher Scientific), washed twice in PBS, permeabilized (5 min) with Triton X-100 , washed twice in PBS, and blocked (1 h) in PBS with 5% bovine serum albumin . Cells were then incubated  with primary monoclonal mouse anti-IP3R3 antibody  in PBS with 3% BSA, washed three times in PBS, and incubated  with secondary rabbit anti-mouse antibody conjugated to Alexa Fluor 488  in PBS with 3% BSA for 1 h. Cells were then washed five times in PBS before imaging. All steps were performed under conditions that minimized exposure to light. We confirmed the specificity of Ab-IP3R3 by demonstrating the absence of immunostaining in HEK-3KO cells   was measured as the mean gray value from a region (19.2 × 19.2 μm) within which Ca2+ puffs were recorded. We refer to this measure of SNAP-IP3R3 expression as SNAP-647 fluorescence from ROIpuffs. To allow comparisons between experiments, fluorescence (FTetraSpeck) from TetraSpeck beads  was recorded and used to normalize measurements of SNAP-IP3R3 fluorescence (FSNAP). For experiments addressing the effects of varying SNAP-IP3R3 expression (SNAP/FTetraSpeck) were used for quantitative analyses.All TIRF images were first background-corrected by subtracting the gray value of an area from outside a cell using MetaMorph or FIJI (iji.sc/) . To quanpression , normali3R3 immunostaining (488 nm) and SNAP-IP3R3 labeling (647 nm) (C), the mean gray value from a ROI demarcating the entire TIRF footprint of the cell was measured. Pixel-based colocalization analysis used the FIJI plugin JACoP . M values of 1.0 indicate perfect colocalization. Costes’ randomizations  were generated for each image and used to assess the statistical significance of any colocalization (For comparisons between IP(647 nm) C, the mein JACoP . Images rescence B. M valulization .3R3 immunofluorescent puncta (F) or SNAP-IP3R3 puncta (B and C)  were background-subtracted  in FIJI to improve signal-to-noise. The FIJI plugin TrackMate (v 6.0.1) was used for automated detection of IPt puncta F or SNAP3 puncta , B and CB and C) .F), the coordinates of the pixel with the maximum fluorescence intensity for each detected spot in the IP3R3 immunofluorescence image were mapped onto the corresponding pixel of the SNAP-IP3R3 image. We used these point fluorescence intensity measurements to compare expression determined by immunostaining and SNAP-tag labeling. The approach is a valid measure of the fluorescence intensity of the entire punctum because the point intensity is linearly related to total intensity of the spot for the IP3R3 immunofluorescence image , EGTA to restrict Ca2+ diffusion  , incubated (1 h) with Cal520-AM (5 μM) and ci-IP3/PM (1 μM) in HBS with pluronic acid . Cells were then washed and incubated with EGTA-AM (5 μM) in HBS with pluronic acid (0.02%). After 45 min, cells were washed and incubated in HBS for a further 30 min before imaging. All steps were performed at 20 °C with minimal exposure to light.To allow imaging of Caiffusion , and ci-ites 200–00 nm  , 7. EGTA2+ puffs , 38. HEK2+ puffs, image streams were captured at 200 frames s-1  from a 19.2 × 19.2 μm ROI . Image stacks typically comprised 6000 to 10,000 frames (∼30–50 s). After 2 s of recording, a UV flash lasting 250 ms  within ∼100 nm of the plasma membrane. To achieve the high temporal resolution required to resolve the kinetics of Ca2+ puffs were performed using the detect_puffs plugin in FLIKA, an open-source Python-based image processing and analysis software (0 stack was generated by dividing the fluorescence intensity of each pixel from each frame (F) by the average intensity of that pixel over the first 390 frames before photorelease of i-IP3 (F0). Image stacks (F/F0) were then divided by the SD of the baseline F/F0 images, a Gaussian blur (σ = 2) was applied, and these stacks were then used to identify Ca2+ puffs and measure their properties.Raw image stacks were background-corrected in MetaMorph and frames containing the UV flash artefact were removed in FIJI. Automated detection and analysis of Casoftware , 40. An 2+ puffs, a Butterworth temporal bandpass filter (low frequency cut-off = 0.01) was applied to remove the slow increase in baseline fluorescence. In some cases, global increases in [Ca2+]c completely obscured Ca2+ puffs causing premature termination of the analysis; the shortest of these terminated recordings was ∼12 s. The recording interval was defined as the time from the first Ca2+ puff to either the end of the recording or the time when it became impracticable to resolve Ca2+ puffs against an elevated global [Ca2+]c.When a global increase in fluorescence obscured Ca2+ puff, to which a 2D Gaussian is fitted to identify its centroid. The centroid, defined with sub-pixel resolution, reports the center-of-mass of the clustered IP3Rs underlying the Ca2+ puff  centered on the centroid of the Ca2+ puff. Detected puffs were manually inspected to exclude noisy traces and to ensure the entire F/F0 trace was included in the analysis. In some cases, ‘square’ puffs (detected by eye) were manually removed from analyses of kinetic properties  of each other were considered to belong to the same Ca2+ puff site. FLIKA successfully identifies these sites when Ca2+ puffs are relatively infrequent and sparsely distributed. However, when Ca2+ puffs are more frequent and more densely packed, FLIKA merges large numbers of Ca2+ puffs into fewer sites, causing the number of sites to be underestimated (A and B). This occurs because FLIKA assigns Ca2+ puffs to the same site if each successively assessed Ca2+ puff is < 1 μm from the last; hence, a site can grow in a chain-like fashion as densely packed Ca2+ puff centroids become linked across considerable distances. To overcome this difficulty, we used a MATLAB script, ClusterXYpoints  (2+ puff sites. The XY coordinates of Ca2+ puff centroids generated by FLIKA were saved as .csv files and imported into MATLAB (version R2021a). ClusterXYpoints starts by sorting the XY coordinates of Ca2+ puffs (a.k.a. points) in ascending order. The first point is assigned as a cluster, and the cluster centroid is calculated. If the second point is < 1 μm from the first cluster centroid, it is added to the cluster and the centroid is recalculated. Subsequent points are added to the cluster if they are < 1 μm from the updated centroid of the cluster. If a point is > 1 μm from the current cluster centroid, a new cluster centroid is generated. The process continues until all Ca2+ puffs have been assigned to a site.Caof IP3Rs , 8. In ostimated , A and Bdinates) , to iden2+ puffs were infrequent, but it identified more sites than FLIKA when Ca2+ puffs were more frequent. At the highest frequencies of Ca2+ puffs, when FLIKA merged most Ca2+ puffs into a single site, ClusterXYpoints continued to identify discrete sites (A and B). We therefore used ClusterXYpoints to identify the number of Ca2+ puff sites. The problems encountered with ‘coalescing’ Ca2+ puff sites in FLIKA analyses arise only because the centroids of many temporally separated Ca2+ puffs are overlaid for analysis, they therefore have no impact on the analyses of individual Ca2+ puffs.ClusterXYpoints detected the same sites as FLIKA when Cate sites , A and B3R3 expression in cell populations (E), HEK-SNAP-IP3R3 or mock-transfected HEK-3KO cells were labeled with SNAP-Cell 647-SiR. Cells were washed with HBS and loaded with Calbryte590-AM (2 μM) in HBS with pluronic acid (0.02%) for 1 h at 20 °C in darkness. Here, Calbryte590 is used to define cell boundaries rather than as a Ca2+ indicator. Cells were washed and incubated in HBS for a further 1 h before imaging.For measuring SNAP-IPulations E, HEK-SN3R3 fluorescence intensity was measured from individual whole-cell ROIs.Epifluorescence imaging used an EVOS M7000 Cell Imaging System equipped with a 10× objective and a CMOS camera with a large field of view (Thermo Fisher Scientific). Calbryte590 and SNAP-Cell 647-SiR fluorescence were captured using EVOS LED cubes for Texas Red and Cy5, respectively. For image analysis, Calbryte590 fluorescence was used to demarcate whole cells because it is bright and uniformly distributed in the cytosol, making it ideal for automated segmentation. A custom-written ImageJ macro was used for segmentation (available from H.A.S.); the detected cell boundaries were then confirmed by manual inspection. Raw mean SNAP-IP2+ puffs, and independent analyses are reported in figure legends. Statistical comparisons used unpaired Student’s t tests; correlation analyses used Pearson’s correlation coefficient; comparisons of frequency distributions used χ2 test for trend . The statistical significance of colocalization analyses (Manders’ split coefficients) was determined after Costes’ randomization, where a Costes’ p value > 0.95 indicates that < 0.05 of the randomized images had a Pearson’s correlation coefficient value greater than the nonrandomized image   .All data required to support the conclusions presented are contained with the article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


By combining molecular, biochemical, proteomic, electrophysiological, and imaging techniques, we here show that MLC1 is a Ca2+/Calmodulin-dependent protein kinase II (CaMKII) target protein, whose phosphorylation, occurring in response to intracellular Ca2+ release, potentiates VRAC-mediated ICl,swell. Overall, these findings reveal that MLC1 is a Ca2+-regulated protein, linking volume regulation to Ca2+ signaling in astrocytes. This knowledge provides new insight into the MLC1 protein function and into the mechanisms controlling ion/water exchanges in the brain, which may help identify possible molecular targets for the treatment of MLC and other pathological conditions caused by astrocyte swelling and brain edema.Astrocytes, the main glial cells of the central nervous system, play a key role in brain volume control due to their intimate contacts with cerebral blood vessels and the expression of a distinctive equipment of proteins involved in solute/water transport. Among these is MLC1, a protein highly expressed in perivascular astrocytes and whose mutations cause megalencephalic leukoencephalopathy with subcortical cysts (MLC), an incurable leukodystrophy characterized by macrocephaly, chronic brain edema, cysts, myelin vacuolation, and astrocyte swelling. Although, in astrocytes, MLC1 mutations are known to affect the swelling-activated chloride currents (ICl, MLC1 gene encoding the MLC1 protein  ATP (>3000 Ci/mmol) for 10 min at 30 °C under gentle stirring. When indicated, the CaMKII inhibitor KN93  was used in the reaction mixture. The kinase reaction was stopped by adding 4X SDS loading buffer and the samples were resolved on 12% SDS-PAGE. The dried gels were exposed to X-ray film for autoradiography. When indicated, myelin basic protein (MBP) was added to the reaction mixture.The phosphorylated active enzyme CaMKII was immunoprecipitated from U251 cells using a specific anti-pCaMKII (Thr286) pAb . The enzyme was then used to phosphorylate in vitro the agarose-bound-GST N-terminal peptide and the His-fused MLC1 FL protein obtained as described above. The in vitro kinase assay was performed by incubating the immunoprecipitated pCaMKII or the commercial recombinant purified pCaMKII  with the recombinant proteins in kinase buffer . Proteins eluted from the beads were separated on a 1D-gel NuPAGE 4–12% . The bands were stained with Coomassie blue , cut, and digested with trypsin , Asp-N-protease (Promega), or Glu-C endoproteinase (Sigma-Aldrich). To identify phosphorylated sites, the peptide mixture was analyzed using LS-MS/MS as described : MS/MS a2+-free (0 Ca2+) solutions, as well as the internal solution, were used for electrophysiological recordings of the ICl,swell, as previously described  ATP, using the full-length MLC1 protein (FL) and the GST-MLC1 NH2-t (where the putative CaMKII phosphorylation motif had been observed) as substrates for the CaMKII enzyme precipitated from U251 cells. As shown in 2-t peptide and the reaction mixture was subjected to LC-MS/MS analysis. These experiments allowed the recognition of a phosphorylation site at Threonine (T), in position 17 of the MLC1-N-t peptide, within the supposed CaMKII consensus sequence RXXS/T  and cell volume regulation in several MLC disease models [swell potentiation. To this aim, patch-clamp recordings were performed on U251 cells expressing MLC1-WT and, as controls, on U251 cells transfected with an empty vector (Ø) or expressing the already characterized pathological mutant MLC1-S280L [swell, right), (swell density was significantly larger in MLC1-WT than in Ø and MLC1-S280L cells (swell inactivation (steady state/Ipeak)), was significantly larger in MLC1-WT (0.86 ± 0.03) compared to MLC1-Ø (0.71 ± 0.02) cells. Overall, these findings further support the ability of MLC1 to also act as a ICl,swell modulator when expressed in U251 cells. As MLC1 was involved in the activation of Cl-currents , A. The qu0L cells B,C. Notativation . Specifiswell, we investigated if the CaMKII-dependent phosphorylation of MLC1 represented a key step for its activation and the modulation of ICl,swell by MLC1. To this aim, we assessed the ICl,swell density in U251 expressing MLC1-WT or carrying the T17A or T17D mutations. Voltage ramps from −100 to +100 mV from a holding potential of −40 mV were applied. In ,swell activation in U251 cells with the hypotonic solution, together with representative current ramps (Inset: left) under hypotonic conditions and after application of 10 µM DCPIB, which selectively inhibits VRAC. The inset on the right of swell induced by step potentials during hyposmotic solution perfusion in the absence or presence of 10 µM DCPIB, which almost fully blocked the hyposmosis-activated currents. The results from these experiments revealed that the hyposmotic solution induced an inward current at −80 mV  in all cell models, as shown in the bar plot of swell from T17D and T17A mutants we observed that the T17D cells, exhibited an ICl,swell density similar to WT cells (~56 pA/pF), while the ICl,swell density observed in T17A cells (~32 pA/pF) was similar to MLC1-Ø cells , KN93 was not able to reduce the hyposmosis-induced ICl,swell.Once it was determined that the expression of functional MLC1 in U251 cells was required for the enhancement of the ICl,-Ø cells B. These WT cells C,D. Nota2+ binding protein Calmodulin upon increase of cytosolic Ca2+. Increased cytosolic Ca2+ due to cell swelling has also been shown to activate ICl,swell in astrocytes [2+ as a key player in the activation pathways of both MLC1 and ICl,swell. Since specific Ca2+ sources are known to be linked to specific functions, we aimed to distinguish the roles of external Ca2+ influx and Ca2+ release from internal stores in the CaMKII/MLC1-dependent enhancement of the ICl,swell. First, we carried out Fura-2-based Ca2+ recording experiments, to discriminate between the contribution of external Ca2+ influx and intracellular Ca2+ release in the hyposmosis-induced Ca2+ movements in U251. A hyposmotic challenge lasting 15 min was imposed on Fura-2-loaded MLC1-WT cells in the presence or absence of external Ca2+ , which, instead, was represented by the asynchronous Ca2+ transients predominant in the later phase. Finally, the slowly rising phase recorded in 0-Ca2+ depicts a homogeneous response among cells, due to release from unidentified intracellular stores, and is not attributable to a CICR mechanism. To offer a quantitative evaluation of the very variable response to hyposmotic challenge observed in the presence of Ca2+, we calculated the integral  of the Ca2+ signal at different time intervals, in both the presence and absence of extracellular Ca2+. The resulting graph  upon addition of external Ca2+, while in all the other cell types  the current density remained unchanged  was markedly greater than that elicited in the cell group probed in the absence of extracellular Ca2+ (~32 pA/pF)  in MLC1-WT cells and in the same cells pretreated for 5 min with 1 µM Thapsigargin (TG), a non-competitive inhibitor of the ER Ca2+ ATPase (SERCA), known to cause a massive Ca2+ release from the ER . A representative experiment carried out in WT cells pretreated with TG, with the hypotonic solution with 0-Ca2+ activated ICl,swell, is shown in 2+ influx (0-Ca2+ condition) the TG pretreatment did not significantly alter the ICl,swell density. As the influx of Ca2+ from extracellular space is able to further activate the ICl,swell in WT cells, we then asked whether Ca2+ entering into the cell could directly enhance the ICl,swell via the CaMKII/MLC1 pathway, or indirectly, by stimulating the release of Ca2+ from the intracellular stores via CICR. To this end, we assessed the ICl,swell density activated by hypotonic solution with 0-Ca2+, in MLC1-WT cells pretreated or not with TG, and then verified if ICl,swell density could be further increased upon switching to the hypotonic solution with 2 mM Ca2+ (swell activated by the Ca2+-free hypotonic solution further increased (by ~35%) upon addition of external Ca2+, while in TG-treated cells, the current density remained unchanged  C. The ab mM Ca2+ . The ananchanged F. Simila2+ increase in astrocytes. Since Ca2+ release is strongly linked to astrocyte activation in response to several pathological stimulations, these findings may have interesting implications for the comprehension of MLC1′s proper function and its central role in the control of volume changes and astrocyte activation processes in different brain pathological conditions, as discussed below. Pathological astrocyte swelling often occurs in response to CNS damage, leading to cellular (cytotoxic) edema. Cytotoxic edema represents a first step in the formation of tissue edema, as it generates the driving force for the vasogenic edema formation, causing tissue swelling . This pa2+ channel TRPV4 [2+ release in astrocytes  favored MLC1/CaMKII interaction, suggesting a possible functional outcome of this interaction. The identification by proteomic analysis of the T17 aa as the CaMKII target site and the characterization of the U251 cell lines expressing the MLC1 mutant insensitive to CaMKII phosphorylation (T17A) or mimicking constitutive phosphorylation (T17D), proved that CaMKII favors MLC1 protein assembly (dimer formation), an indication of MLC1 functional activation [2+ release. The functions of many proteins involved in volume regulation, such as AQP4 [We, here, have provided evidence that CaMKII binds MLC1 at its COOH terminal, similarly to what has been observed for the Cael TRPV4 . In additivation ,23,25,26 as AQP4  and calc as AQP4  and chlo as AQP4 ,47,48, aswell and volume regulation in several types of cell. Once activated, VRAC is permeable to osmolytes such as taurine, glutamate, and Cl– anions, whose efflux results in cell water outflow and restoration of the original cell volume [2+ dependence to this channel, which would be otherwise insensitive to Ca2+ ions in glioma cells [2+, ICl,swell is increased in MLC1-WT, but not in T17A or T17D mutants, is in accordance with the ability of Ca2+ influx to promote CaMKII-dependent MLC1 phosphorylation only in MLC1-WT expressing cells. Moreover, the abolition of a Ca2+ influx-mediated increase of ICl,swell after pretreatment with TG and 2-APB, confirmed the contribution of internal Ca2+ stores in this process, possibly via activation of the CICR mechanism. As summarized in 2+ influx by hypotonic cell swelling, most likely due to opening of stretch-activated Ca2+ channels (such as TRPV4); (2) triggering of Ca2+ release from intracellular stores through the CIRC mechanism; (3) Ca2+-induced CaMKII activation; (4) CaMKII-mediated MLC1 phosphorylation and consequent dimer stabilization of the newly activated protein and/or of that already present at the PM; (5) MLC1 potentiation of VRAC current. Besides the MLC1-mediated VRAC potentiation, the molecular mechanisms of VRAC activation is still an open question. The initial hypothesis involving changes in ion strength is now paralleled by other mechanisms, among which are membrane tension and signaling pathways. The heterogeneity of the activation mechanism may be attributed to cell-specific properties of VRAC and different interactions of the channel with Ca2+-sensitive effectors. In astrocytes, VRAC is also stimulated by neurotransmitters and neuromodulators such as ATP, through an increase of intracellular Ca2+, PKC, and CaM/CaMKII pathways activation [2+-dependent modulation of VRAC in astrocytes. This notion is further supported by the fact that in astrocytoma/glioblastoma cells, where MLC1 is virtually absent , changes in intracellular Ca2+ do not affect ICl,swell [2+-modulation of the ICl,swell was reported [2+-sensitivity to VRAC in other cell types where the Ca2+ dependence of ICl,swell has been demonstrated. To date, no direct interaction between MLC1 and the LRRC8A protein, the most generally recognized molecular component of the VRAC channels [swell in astrocytes [VRAC is the channel mainly responsible for both ICl,l volume ,50. Lackl volume ,15; convl volume . We herema cells . The obstivation . AlthougCl,swell . On the reported ,43. It wchannels ,55, has channels . In addichannels . In lightrocytes , it will2+ changes occurring in response to osmotic stress. Interestingly, in different in vitro and in vivo models, osmotic stress has been found to elicit Ca2+ responses in astrocyte end-feet, where MLC1 is preferentially expressed [2+-permeable gap-junctions, the CICR mechanism generates Ca2+ waves propagating through the astrocyte syncytium [2+ wave spreading is known to modulate several fundamental astrocyte activities  [2+ wave propagation via extracellular ATP release [2+ wave spreading. It is worth noting that alterations of Ca2+ diffusion at the perivascular end-feet can cause serious consequences for NVU and neuron functionality, since a Ca2+ increase in this compartment adjusts cerebral blood flow during synaptic activity [2+ in perivascular astrocyte end-feet, followed by vascular smooth muscle Ca2+ oscillations and arteriolar dilation [2+ dynamics in astrocyte end-feet due to MLC1 mutations might influence arteriolar contractility and BBB permeability, causing edema and NVU dysfunctions. In accordance with this hypothesis, a recent study provided evidence of alterations of vascular smooth muscle contractility and NVU coupling in MLC1 KO mice [Overall, the data here presented reveal that, in astrocytes, MLC1 protein function is regulated by Caxpressed ,22. We cxpressed . In addiyncytium . Ca2+ waonality) . By pote release , and by  release , MLC1 miactivity . Indeed,dilation . Thus, w KO mice . The eme KO mice , who gen2+ release from intracellular stores occurs in astrocytes in response to different damaging stimuli, leading to astrocyte swelling and activation [Dysfunction of NVU and of cerebral blood flow are also implicated in the pathogenesis of cerebral disorders, such as traumatic brain injury, Alzheimer’s (AD) and Parkinson’s (PD) diseases, multiple sclerosis (MS), and glioblastoma malignancy ,66. Intetivation .2+-sensitive protein linking VRAC-dependent volume regulation to Ca2+ dynamics in astrocytes. Although further analyses are needed to fully understand MLC1′s function and the details of MLC/VRAC cooperation, the results here presented open new perspectives for comprehension of the astrocyte-mediated molecular events governing brain water/ion exchanges in normal and pathological conditions. Considering the potential of astrocytes as pharmacological targets, this knowledge may be of interest for the identification of astrocyte-based therapeutic approaches for MLC and other brain diseases where astrocyte swelling and brain edema have a central role in the pathological process.In the present study, we revealed for the first time that MLC1 is a Ca