Background. Recent clinical trials suggest that nonoperativemanagement (NOM) of patients with acute, uncomplicated appendicitis is anacceptable alternative to surgery. However, limited data exist comparing thelong-term cost-effectiveness of nonoperative treatment strategies.Design. We constructed a Markov model comparing thecost-effectiveness of three treatment strategies for uncomplicated appendicitis:1) laparoscopic appendectomy, 2) inpatient NOM, and 3) outpatient NOM. The modelassessed lifetime costs and outcomes from a third-party payer perspective. Thepreferred strategy was the one yielding the greatest utility without exceeding a$50,000 willingness-to-pay threshold. Results. Outpatient NOM cost$233,700 over a lifetime; laparoscopic appendectomy cost $2500 more whileinpatient NOM cost $7300 more. Outpatient NOM generated 24.9270 quality-adjustedlife-years (QALYs), while laparoscopic appendectomy and inpatient NOM yielded0.0709 and 0.0005 additional QALYs, respectively. Laparoscopic appendectomy wascost-effective compared with outpatient NOM ; inpatient NOM was dominated by laparoscopicappendectomy. In one-way sensitivity analyses, the preferred strategy changedwhen varying perioperative mortality, probability of appendiceal malignancy orrecurrent appendicitis after NOM, probability of a complicated recurrence, andappendectomy cost. A two-way sensitivity analysis showed that the rates of NOMfailure and appendicitis recurrence described in randomized trials exceeded thevalues required for NOM to be preferred. Limitations. There arelimited NOM data to generate long-term model probabilities. Health stateutilities were often drawn from single studies and may significantly influencemodel outcomes. Conclusion. Laparoscopic appendectomy is acost-effective treatment for acute uncomplicated appendicitis over a lifetimetime horizon. Inpatient NOM was never the preferred strategy in the scenariosconsidered here. These results emphasize the importance of considering long-termcosts and outcomes when evaluating NOM. While usually safe and effective, appendectomy is not totally benign:surgery incurs perioperative risk and has been associated with short- and long-termcomplications, with reported rates ranging from 2% to 23%.13 The necessaryobservation period to assess these long-term outcomes is impractical for prospective trials,14 and certainly exceeds individual surgeons’ typical episodes of care. However,accurate assessment of the cost-effectiveness of NOM warrants consideration of thelong-term costs of appendicitis-related care, including the lifetime potential forrecurrence, ongoing risk for appendiceal malignancy, or potential postoperativecomplications such as adhesive bowel obstructions. Two prior cost-effectiveness analysescompared NOM with appendectomy in the adult and pediatric populations,16 but these analyses were completedover 1-year time horizons and did not model long-term costs.Proponents of nonoperative management (NOM) contend that it is less expensive thanappendectomy, as it minimizes operative costs and may reduce hospitalization days.Trials have estimated the costs of NOM versus surgical management for the indexhospitalization, but have not assessed long-term costs of recurrence or othercomplications.To determine the cost-effectiveness of NOM versus operative management of acuteuncomplicated appendicitis, we conducted an analysis comparing two NOM strategiesagainst laparoscopic appendectomy. A novel aspect of our analysis is the modeling ofboth short- and long-term costs over a reference patient’s lifetime to study theenduring effects of NOM and surgery. This analysis is intended to inform surgeons andthird-party payers regarding long-term economic implications when comparing NOM andlaparoscopic appendectomy as first-line therapies for uncomplicated appendicitis.This computer simulation modeling study was deemed exempt from human subject reviewbecause it uses only publicly available, deidentified, and aggregate datasources.We first defined the reference case as a healthy 20-year-old male in the UnitedStates presenting to the emergency room with imaging-confirmed uncomplicatedappendicitis. Uncomplicated appendicitis was defined as inflammation of theappendix in the absence of abscess, phlegmon, perforation, fecalith on imaging,or diffuse peritonitis on initial clinical exam.11 Two NOM arms were includedas we wished to consider both the most typical nonoperative treatment strategyas well as the potentially least resource-invasive, as recent pilot studies haveshown comparable outcomes between inpatient and outpatient management strategies.17 Appendectomy was considered the reference treatment, as it is the currentstandard of care.We constructed a decision model using decision analysis software . We used literature review and consultation with experts tobuild a decision tree comparing three management strategies: 1) laparoscopicappendectomy, 2) inpatient NOM with 3-day hospitalization for intravenousantibiotics (piperacillin-tazobactam) followed by completion course of 7 days ofoutpatient oral antibiotics (amoxicillin-clavulanate), and 3) outpatient NOMwith 7 days of oral outpatient antibiotics only (amoxicillin-clavulanate) . For eac19 and NOMmay result in a missed opportunity to treat an early, confined appendicealtumor. Recent data suggest that appendiceal tumors are incidentally discoveredin around 1% of appendectomies.2022 It is thus reasonable toconsider the potential downstream effects of leaving an unidentified tumor insitu following NOM over the rest of a patient’s life.Inpatient failure of NOM was defined as symptom progression during the indexhospital admission. Outpatient failure was defined as symptom progression orreturn of symptoms after discharge from the index hospitalization within 30 daysof initial presentation. Treatment failures occurring after 30 days wereconsidered to be recurrent appendicitis. Complicated failure or recurrence wasdefined as perforation with associated abscess. We considered short-termperioperative complications including ileus, surgical site infection, anestheticcomplications, and postoperative hemorrhage. We considered long-termcomplications including trocar-site hernia and adhesive small bowel obstruction.Finally, we accounted for the possibility that appendicitis may be the firstpresentation of an otherwise unrecognized appendiceal tumor,We also conducted additional scenario analyses to explore how much variousfactors would influence the assessment of NOM. These included the following: 1)if an older patient underwent NOM (if the base case was instead an average 40-or 65-year-old), 2) if patients who were managed nonoperatively never developedappendiceal cancer , 3) if patients who were managed nonoperatively neverfailed or recurred, and 4) if all failures or recurrences were uncomplicated.23 Age-appropriate background mortality and costs were considered in themodel.25 We used Markov modeling with a cycle length of 1 month toexamine probabilities of long-term events over the expected lifespan of thepatient. After initial chance nodes accounted for events and costs occurring inthe first 30 days, Markov nodes incorporating half-cycle correction were used.Incremental cost-effectiveness ratios were compared with a $50,000 per QALY gained willingness-to-pay threshold.26 For the strategies considered, we examined the incrementalcost-effectiveness ratios (ICERs)—defined as the incremental cost of a givenstrategy divided by its incremental benefit compared with the next bestalternative. Future costs and life-years were discounted at an annual rate of 3%.27The time horizon for the model was the expected lifetime of the reference case,derived from US life tables.28 and expert opinion from clinicians. Base case values were felt byclinicians to represent the best available estimates from the literature.Probabilities were obtained from literature review ; these w29 We summed the physician work, practice expense, and malpractice relativevalue units (RVUs) and multiplied by a conversion factor of 35.8887 (per CMSdata) for the Current Procedural Terminology (CPT) codes relevant to our study. Hospitalization costs were obtained from the CMS final rulefor fiscal year 2017,30 and were calculated by multiplying the conversion factor by the sum ofthe RVUs for 1 admit day (CPT 99222), 1 discharge day (CPT 99239), and anyintervening hospital days (CPT 99231). Average length of hospitalization perprocedure was obtained from literature review. We multiplied the sum of thelabor ($3420), non-labor ($2096), and capital cost rates ($447) by theappropriate DRG (diagnosis-related group) multiplier. The costs of emergencydepartment evaluation and follow-up clinic visits were obtained from the CMSHospital Outpatient Prospective Payment System.31 The sum of hospital charges and physician charges for a level 4 visit(CPT 99284) was added to the cost of imaging; assuming that half of the patientsreceived an abdominal computed tomography scan and half received an ultrasound.Medication costs were obtained from the Federal Supply Schedule.32 The average wholesale price per medication was multiplied by 0.64 toobtain the net final price paid by Medicaid for each medication . The Medicaid final price includes distribution anddispensing costs. Costs used in our analysis are summarized in Costs were considered from the perspective of a third-party payer. Proceduralcosts were calculated using the 2017 Centers for Medicare and Medicaid Services(CMS) physician fee schedule.33Utilities related to hospitalizations, surgical or procedural interventions, andrecovery from appendicitis or appendectomy were drawn from published literatureas available. Previously published studies assessing age-related backgrounddecline in health-related quality of life was used to estimate the baselineutility of the reference case.3437 We assumed that all NOMfailures or recurrences underwent laparoscopic appendectomy . All patients with complicated failures orrecurrences were assumed to undergo percutaneous drain placement byinterventional radiology followed by interval appendectomy. We assumed that 70%of patients returning with an appendiceal malignancy presented with a carcinoidtumor >2 cm and underwent right hemicolectomy, and the remaining 30%presented with Stage III appendiceal adenocarcinoma and underwent righthemicolectomy followed by 12 cycles of FOLFOX chemotherapy.3840 We purposefully assumedthat patients would present with advanced-stage malignancies due to a delay incancer diagnosis related to NOM. As a small percentage of patients undergoingappendectomy may be found to have malignancies at that time, we assumed thatthese patients would be cured by appendectomy alone and would not requirefurther treatment. We felt this assumption was reasonable, as we defined ourreference case as having confirmed appendicitis without concerning imagingfindings correlated with diagnosis of later-stage malignancy duringappendectomy.We assumed that all appendectomies were completed laparoscopically, in line withmodern case series describing very low rates of conversion to open appendectomyfor uncomplicated appendicitis.We first conducted one-way deterministic sensitivity analyses to evaluate how thedecision might change across the plausible range of each model parameter givenits uncertainty while holding all others at their base case values. We usedsensitivity analyses to assess the importance in changes of utilities for healthstates individually and in combination, while preserving the preference orderingof health states. Baseline values were varied over the ranges displayed in 41 The uncertainty for normal distributions was calculated using apreviously defined method.42 Values were repeatedly sampled from their appropriate uncertaintydistributions 10,000 times.We then conducted Monte Carlo probabilistic sensitivity analyses using betadistributions for input probabilities and utility weights and normaldistributions for defined procedural costs. Univariate utility distributionswere then correlated to establish joint uncertainty distributions exploitingordinal preferences over health states, in order to avoid bias and improveprobabilistic sensitivity analyses.Authors’ funding sources had no role in the study.When considered over the expected lifetime of the base case patient, outpatientNOM proved the least costly and generated the fewest QALYs and life-years . BothlaWe first assessed whether the cost-effectiveness of NOM was sensitive to the ageof the reference patient considered. When the age of the reference case wasincreased to either age 40 or age 65, laparoscopic appendectomy remained themost effective strategy and was cost-effective at age 40 but not at age 65.When we considered the situation where the risk of developing appendiceal cancerafter NOM was zero , appendectomy’s healthbenefits relative to NOM gained by removing the appendix were attenuated . Hence,When we considered the scenario where all NOM was successful , the costs of both NOM strategiesdecreased compared with the base case . OutpatiFinally, we considered the situation where all failures and recurrences wereuncomplicated and proceeded directly to appendectomy instead of requiringinitial percutaneous drainage followed by interval appendectomy. The cost ofboth NOM strategies decreased relative to the base case and outpatient NOMremained the least costly. QALYs and life-years increased for both NOMstrategies; appendectomy continued to generate more life-years and QALYs. Inthis scenario, laparoscopic appendectomy was no longer cost-effective comparedwith outpatient NOM . Inpatient NOM was dominated bylaparoscopic appendectomy.Model results were sensitive to changes in the following parameters in one-waysensitivity analyses: perioperative mortality rates during appendectomy, theprobability of developing recurrent appendicitis and the probability that therecurrence be complicated, the probability of developing an appendicealmalignancy after NOM, and the cost of laparoscopic appendectomy. When theprobability of perioperative mortality during laparoscopic appendectomy was>0.26%, outpatient NOM dominated laparoscopic appendectomy. When theprobability of developing recurrent appendicitis in the 5 years following NOMwas >26%, laparoscopic appendectomy dominated. Laparoscopic appendectomy alsodominated when the probability of a complicated appendicitis recurrence was>4.6%. Laparoscopic appendectomy dominated outpatient NOM when the long-termrisk of developing an appendiceal malignancy after NOM was <0.48%. Varyingthe cost of laparoscopic appendectomy also changed the preferred strategy: whengreater than $10,173 , outpatient NOM was preferred overlaparoscopic appendectomy. In no sensitivity analysis that we considered wasinpatient NOM the cost-effective strategy.We conducted a two-way sensitivity analysis when the probabilities of short-termNOM failure and long-term appendicitis recurrence were varied concurrently.In probabilistic sensitivity analyses, laparoscopic appendectomy was thepreferred strategy in 62% of 10,000 simulated cases when the willingness-to-paythreshold was set at $50,000 per QALY, while outpatient NOM was preferred in theother 38%. A cost-effectiveness acceptability curve is included as Our analysis finds that laparoscopic appendectomy is the most effective managementstrategy for acute uncomplicated appendicitis when the lifetime effects ofappendicitis-related treatment are considered. It is cost-effective compared withoutpatient NOM with an ICER of $32,300 per QALY. Inpatient NOM is the costlieststrategy and was less effective than laparoscopic appendectomy.16 A costanalysis of the APPAC trial comparing appendectomy to NOM concluded that NOMincurred fewer costs over the duration of the trial.13 In contrast, our model suggests that laparoscopic appendectomy is acost-effective treatment option despite the additional costs associated with surgerywhen the lifetime implications of appendicitis-related treatment are considered.Conversely, inpatient NOM as conventionally defined by prospective trials was notcost-effective over a lifetime horizon. This demonstrates the importance ofcollecting long-term data from registries and prospective trials assessing NOM.Prospective trials of short duration and individual surgeons’ personal experienceswill not account for long-term outcomes and may make NOM appear overlyfavorable.These findings are important as few other studies have modeled or assessed thelong-term health and economic consequences of NOM for uncomplicated appendicitis.Prior cost-effectiveness analyses assessing NOM were limited to a 1-year timehorizon and suggested that NOM dominated laparoscopic appendectomy.When considered over a patient’s lifetime, the differences in costs and QALYs betweenappendectomy and NOM were relatively small. This likely reflects the overallexcellent outcomes following appendicitis and low rates of long-term complicationsfollowing either management strategy. However, the sensitivity and scenario analysespresented here are informative in determining important factors in the long-termcost-effectiveness of appendicitis treatment. Appendectomy cost and perioperativemortality proved influential in one-way sensitivity analyses, suggesting thatminimizing operative costs and mortality are important targets in real-lifepractice. Model outcomes were insensitive to changes in age until age 65, suggestingthat these results are applicable to a broad adult population.4 Although rare, it is thus important to consider the possibility of futuremalignancy when considering NOM.2022The probabilities of developing and dying from appendiceal malignancies were stronglyinfluential on treatment preferences in this analysis, despite their relativelyrarity. Appendectomy was preferred over outpatient NOM when the probability offinding an incidental appendiceal malignancy at index appendicitis presentation wasgreater than 0.59%. Recent data suggest a 1.1% probability of incidentallydiscovering an appendiceal malignancy at index appendicitis presentation forpatients aged 18 to 65, and a 2.7% probability for ages >65.512 The thresholds of NOM failureand appendicitis recurrence resulting in dominance by laparoscopic appendectomy arenotably lower than those reported in prior cost-effectiveness analyses conductedover a 1-year time horizon, which found that NOM was not the preferred strategy whenshort- and long-term failure rates ranged from 32% to 45%.16 These results suggest thattreatment failure rates of large-scale pending prospective trials should be analyzedclosely as they bear significant financial implications for the cost-effectivenessof NOM, and that careful tracking of long-term recurrence rates is important.As expected, rates of NOM treatment failure and appendicitis recurrence wereinfluential on model outcomes and the preferred strategy. Two-way sensitivityanalyses revealed that the threshold rates of treatment failure and appendicitisrecurrence resulting in dominance of outpatient NOM over laparoscopic appendectomywere at the low end of recurrence rates seen in randomized trials, where treatmentfailure rates have reached as high as 38%.6 Although not yet common clinical practice, recent pilot studies have movedtoward assessing fully outpatient treatment strategies, consistent with the leastresource-invasive strategy presented here.17 Clinicians must be aware that NOM protocols that result in hospitalizationslonger than hospitalizations typical for laparoscopic appendectomies  are unlikely to prove cost-effective on a population level.Notably, inpatient NOM as defined in this analysis was never cost-effective or thepreferred strategy compared with outpatient NOM or laparoscopic appendectomy. Thedefinition of inpatient NOM was modeled to be consistent with the largest randomizedtrial to date: a 3-day admission for intravenous antibiotics followed by 7 days ofcompletion oral antibiotics.44; thus, cost estimates and their effects on the model may not begeneralizable to other settings. Parameters used to model outpatient NOM were drawnfrom prospective trials that included an all-oral antibiotic regimen, as no majorprospective trials to date have tested a fully outpatient NOM strategy. For thepurposes of studying the costs associated with outpatient treatment, we assumed thatoral antibiotics given in hospital would yield similar results to oral antibioticstaken at home. However, this may be unrealistic as patients may derive additionalbenefit from hospitalization alone, making outpatient management appear overlyeffective in this analysis.We recognize the limitations associated with this study, particularly the relativelylimited prospective data used to generate model parameters. This is especially truefor parameters involving long-term probabilities, including the long-term risk ofappendicitis recurrence after NOM and long-term risks of postoperativecomplications. Every effort was made to preferentially use model parameters drawnfrom high-quality randomized data, only supplementing with retrospective data andexpert clinician opinion if no other parameter estimates were available. Healthstate utilities used as model parameters were limited in availability and drawn fromsingle studies, and thus may significantly influence model outcomes. Health carecosts in the United States are known to exceed costs in other developednations4547 These results may not beapplicable to low-resource settings where preoperative diagnostic imaging is lessfrequently utilized and rates of negative appendectomy are higher.Finally, the model is built on the assumption that patients have imaging-confirmedappendicitis, consistent with modern surgical practice. Rates of negativeappendectomy have decreased significantly secondary to the high diagnostic accuracyof modern diagnostic imaging.In this cost-effectiveness analysis of treatment strategies for acute uncomplicatedappendicitis, laparoscopic appendectomy is the most effective management strategyfor acute uncomplicated appendicitis when considered over a lifetime horizon and iscost-effective compared with outpatient NOM. In cases where there is a desire not toperform surgery, outpatient NOM should be considered, as inpatient NOM as previouslydefined in randomized trials is costlier and is not cost-effective.


Improving outcomes in acute myeloid leukemia (AML) remains a major clinical challenge. Overexpression of pro-survival BCL-2 family members rendering transformed cells resistant to cytotoxic drugs is a common theme in cancer. Targeting BCL-2 with the BH3-mimetic venetoclax is active in AML when combined with low-dose chemotherapy or hypomethylating agents. We now report the pre-clinical anti-leukemic efficacy of a novel BCL-2 inhibitor S55746, which demonstrates synergistic pro-apoptotic activity in combination with the MCL1 inhibitor S63845. Activity of the combination was caspase and BAX/BAK dependent, superior to combination with standard cytotoxic AML drugs and active against a broad spectrum of poor risk genotypes, including primary samples from patients with chemoresistant AML. Co-targeting BCL-2 and MCL1 was more effective against leukemic, compared to normal hematopoietic progenitors, suggesting a therapeutic window of activity. Finally, S55746 combined with S63845 prolonged survival in xenograft models of AML and suppressed patient-derived leukemia but not normal hematopoietic cells in bone marrow of engrafted mice. In conclusion, a dual BH3-mimetic approach is feasible, highly synergistic, and active in diverse models of human AML. This approach has strong clinical potential to rapidly suppress leukemia, with reduced toxicity to normal hematopoietic precursors compared to chemotherapy. L and the recently described MCL1 inhibitor S63845, which our group has shown to be well tolerated in mice and active against a number of malignancies, including a subset of AML at low nanomolar concentrations [Acute myeloid leukemia (AML) is a hematopoietic malignancy arising from the transformation of myeloid progenitor cells. Recurrent mutations in over 70 genes characterize the genomic landscape of AML, with each patient harboring a unique spectrum of ancestral, clonal, and sub-clonal populations that evolve over time and in relation to selective pressures exerted by chemotherapy , 2. Althtrations .L or MCL1, supporting the rationale to target multiple pro-survival proteins simultaneously [Although single agent clinical activity with venetoclax has been modest in AML, clinical responses are increased in combination with either hypomethylating agents or low-dose cytarabine for treatment-naive elderly patients ineligible for intensive chemotherapy –12. In aaneously , 16, 17.aneously . Althouganeously .The potential to clinically target MCL1 was realized by development of the potent and selective small molecule inhibitor S63845 . We now MV4;11, MOLM-13, PL-21, ML-2, Nomo-1, THP-1, EOL-1, Kasumi-1 (from ATCC) were cultured in RPMI (GIBCO) supplemented with FBS 10% (v/v); HL-60, KG1, KG1a were cultured in IMDM (GIBCO) supplemented with FBS 20% (v/v); and OCI-AML3 in MEM alpha (GIBCO) supplemented with FBS 20% (v/v). In addition, all media contained penicillin (100 IU/ml), streptomycin (100 µg/ml), and L-glutamine (2 mM). All cell lines were determined to be free of mycoplasma contamination.5 cells/mL and drugs tested over a 5-log concentration range. After 48 h, cell viability was determined by FACS analysis of cellular exclusion of SYTOX Blue Dead Cell Stain (Life Technologies Cat No S34857) using an LSR-Fortessa (BD) [50).S55746 and S63845 were provided by Servier Laboratories. A1331852 was synthesized as previously described . Venetocssa (BD) , 20. FAC50’s were calculated using standard four-parametric curve fitting. IC50 was defined as the compound concentration at which the Cell Titer Glo (CTG) signal was reduced to 50% of that measured for the vehicle (DMSO) control. In order to analyze the activity of the compounds in combination, the cells were seeded and treated with seven or eight 3.16-fold serial dilutions of each compound dispensed, either individually or in all possible permutations in a checkerboard fashion. Effects of the single agents, as well as their checkerboard combinations on cell viability were assessed after three days of incubation at 37 °C/5% CO2 using CTG at 75 μL reagent/well. Two independent experiments, each one performed in duplicate, were performed. Luminescence was quantified on a multipurpose plate reader. Potential synergistic interactions between compound combinations were assessed using the Loewe additivity model  and reported as the Synergy Score [50 determination and colony forming unit (CFU) assays have been previously described [For the larger scale drug synergy assays, cells were seeded and treated with nine 2-fold serial dilutions of each compound dispensed into cell assay plates. Single agent ICgy Score . Cell viescribed .Genomic DNA libraries were prepared using either a custom Haloplex (Agilent) panel, as previously described , or the 5 cells into 6-week old female and male irradiated (100 Rad) non-obese diabetic/severe combined immunodeficient (NOD/SCID/IL2rγnull) mice as previously described [20 at a ratio of 40:10:50 or S63845 (200 µL 25 mg/kg) twice weekly or weekly IV dissolved in 50% 2-hydroxypropyl)-β-cyclodextrin (Sigma) and 50% 50 mM HCL or the drug combination, or vehicle. Blood counts were determined using a hematology analyzer . Bioluminescent imaging was performed using the Caliper IVIS Lumina III XR imaging system. Mice were anesthetized with isoflurane, injected intraperitoneally with 100 µL of 125 mg/kg luciferin , and BLI performed 5 minutes later.All studies in mice were performed under the institutional guidelines approved by the Alfred Medical Research and Education Precinct Animal Ethics Committee. MV4;11 cells transduced with the luciferase reporter (pLUC2 promega) were intravenously injected at 1 × 10escribed . Engraft6 leukemic blasts were injected into 6-week old female NOD-IL2Rcγnull (NRG-SG3) mice  via tail-vein injection and animals monitored for leukemia progression using flow cytometric analysis of peripheral blood for hCD45 + cells. hCD45 + cell counts in the bone marrow from the femurs of euthanized animals were used to determine the extent of leukemia infiltration. Bone marrow cells were extracted by flushing femurs in PBS supplemented with 2% fetal bovine serum. To determine the efficacy of S55746 and S63845, mice were gavaged daily with S55746 (100 mg/kg) for 5 days or received S63845 25 mg/kg twice weekly IV. Drug efficacy was determined by flow cytometric analysis of hCD45 + cells in bone marrow isolated from flushed femurs. Sternums and spleens were fixed in formalin, sectioned, and stained with hematoxylin and eosin or anti-hCD45 to assess leukemic burden and cellularity.To establish mouse models of primary patient AML, 1 × 10For additional methods refer to L (A1331852). Comparison was made with idarubicin and cytarabine, drugs commonly used in the treatment of AML. Freshly derived primary AML samples were studied if the viability of the cells in control media plus DMSO after 48 h was at least 70%. Primary samples were derived from patients with treatment naive , BCL-2 (ABT-199/venetoclax or S55746/BCL201), or BCL-Xive Fig.  or chemoive Fig.  AML.Fig.50 < 100 nM) to BCL-2 (venetoclax or S55746), or MCL1 (S63845) targeting alone [L inhibitor A1331852  was apparent in > 50% of the primary AML samples when treated simultaneously with equimolar concentrations of BH3-mimetics targeting BCL-2 and MCL1  in a subset of primary AML samples resistant to the anthracycline drug idarubicin , or the hypomethylating agent decitabine Fig. . SynergiTP53 or the RNA splicing/chromatin machinery  [50 < 100 nM) was observed across a broad spectrum of AML cases, including those with recognized poor risk genomic features, such as mutated RUNX1 (11/15 cases sensitive), DNMT3A (10/16 cases sensitive) or ASXL1 (7/14 cases sensitive) among cases harboring mutant TP53 (7/8 resistant). As overall numbers were limited, an expanded sample set will be required to confirm this preliminary association.Poor survival outcomes in AML are associated with adverse-risk chromosomal lesions , inv(3), MLL-fusions), mutations involving  MLLPTD) . Therefo MLLPTD) . SensitiL inhibitor A1331852 was toxic to both leukemic and normal progenitors in clonogenic assays -SG3 mice, which have been transgenically modified to express human SCF, GM-CSF, and IL3 in vivo, to enhance engraftment of human AML blasts [To examine the potential for dual BCL-2 and MCL1 targeted therapy to suppress patient-derived AML in vivo, primary AML samples were xenografted into immunodeficient NOD.L blasts , 26. StrMcl1 gene knockout studies suggested that long-term Mcl1 ablation was poorly tolerated by mouse hematopoietic cells [Having demonstrated the potential for dual BH3-mimetic targeting of BCL-2 and MCL1 to rapidly induce remissions and extend the survival of mice xenografted with AML, we next examined the tolerance of normal hematopoietic cells to combined S55746/S63845 therapy. Prior published conditional ic cells . The MCLL targeting. This avoids undesirable on-target thrombocytopenia that may result from BCL-XL inhibition [Since its inception in the 1970’s, combined cytarabine/anthracycline or “7 + 3” chemotherapy has been an enduring constant in the therapy of AML. Our current work highlights several new findings of translational importance for patients with AML. We demonstrate for the first time that small molecule BH3-mimetics directly targeting BCL-2 and MCL1 can potently suppress human AML, with limited toxicity to normal human hematopoietic progenitors, potentially overcoming one of the major limitations from the use of cytotoxic drugs. Furthermore, primary AML blasts were more sensitive to combined MCL1/BCL-2 than to combined MCL1/BCL-Xhibition . Our stuTP53 mutation in this sub-group. The mechanistic basis of this resistance remains to be determined. Possible causes include expression of non-targeted pro-survival factors, such as BFL-1 or BCL-XL, the requirement to target multiple pro-survival proteins simultaneously  or defective expression of downstream BAX or BAK. Examination of these possibilities will be the subject of future research.Although venetoclax has previously been shown to synergistically enhance the activity of the MCL1 inhibitor A-1210477, the latter was only been shown to be effective in vitro and against AML cell lines at micromolar concentrations . In contL, and MCL1 enables determination of which pro-survival proteins are important for the survival of normal, as well as malignant tissues, both in vitro and in vivo. Importantly, the sensitivity of intact cells to BH3-mimetics can be measured quantitatively, utilizing compounds intended for clinical use.Prior to this study, the feasibility of targeting both BCL-2 and MCL1, even with pharmacological inhibitors, remained uncertain. Previous lineage-specific deletion models indicated potential risk to cardiac , 31, graIn conclusion, our studies using S55746 and S63845 provide proof-of-concept demonstration that targeting BCL-2 and MCL1 simultaneously can lead to rapid suppression of diverse AML subtypes, with limited toxicity to normal human bone marrow cells, thereby providing strong rationale for further clinical development.Moujalled et al_Supplementary information


Among them, the Ca2+ sensor recoverin undergoes a pronounced Ca2+-dependent conformational change, a prototypical so-called Ca2+-myristoyl switch, which allows selective targeting of G protein-coupled receptor kinase. Zebrafish (Danio rerio) has gained attention as a model organism in vision research. It expresses four different recoverin isoforms  that are orthologs to the one known mammalian variant. The expression pattern of the four isoforms cover both rod and cone cells, but the differential distribution in cones points to versatile functions of recoverin in these cell types. Initial functional studies on zebrafish larvae indicate different Ca2+-sensitive working modes for zebrafish recoverins, but experimental evidence is lacking so far. The aims of the present study are (1) to measure specific Ca2+-sensing properties of the different recoverin isoforms, (2) to ask whether switch mechanisms triggered by Ca2+ resemble that one observed with mammalian recoverin, and (3) to investigate a possible impact of an attached myristoyl moiety. For addressing these questions, we employ fluorescence spectroscopy, surface plasmon resonance (SPR), dynamic light scattering, and equilibrium centrifugation. Exposure of hydrophobic amino acids, due to the myristoyl switch, differed among isoforms and depended also on the myristoylation state of the particular recoverin. Ca2+-induced rearrangement of the protein-water shell was for all variants less pronounced than for the bovine ortholog indicating either a modified Ca2+-myristoyl switch or no switch. Our results have implications for a step-by-step response of recoverin isoforms to changing intracellular Ca2+ during illumination.Primary steps in vertebrate vision occur in rod and cone cells of the retina and require precise molecular switches in excitation, recovery, and adaptation. In particular, recovery of the photoresponse and light adaptation processes are under control of neuronal Ca Recoverin is a NCS protein and inhibits G protein-coupled receptor kinase GRK1, also known as rhodopsin kinase, at high levels of free Ca2+ concentration. Biochemical in vitro data and results on recoverin knockout mice support such a role of recoverin  retina expresses four recoverin genes, rcv1a, rcv1b, rcv2a, and rcv2b coding for proteins zRec1a, zRec1b, zRec2a, and zRec2b, respectively . Target binding and regulation is suggested to occur with orthologs of mammalian GRK1 and GRK7  yielding pairs GRK1a-Rec1a (in rods) and GRK7a-Rec2a (in UV cones), but regulation of GRK1b and GRK7b by Rec2b is feasible as well  are related to recoverin and are activator/inhibitor proteins that regulate membrane bound sensory guanylate cyclases . The vargulation . We suggEscherichia coli as described previously for bovine recoverin (bRec) . The soluble and insoluble fractions of all recoverin forms were purified by a hydrophobic interaction chromatography except of zRec1b, which was purified by an ammonium sulfate precipitation followed by a size exclusion chromatography. After purification, recoverin containing fractions were combined and dialyzed against 50 mM ammonium hydrogen carbonate to remove residual EGTA that is present from the hydrophobic interaction chromatography step, followed by a buffer exchange against decalcified 50 mM ammonium hydrogen carbonate . Degree of myristoylation was determined by reversed phase high-performance liquid chromatography as previously described  were heterologously expressed in n (bRec) . The zReescribed  yieldingvia western blot .Recombinant non-myristoylated zrecoverin isoforms were used as antigens by a company  for producing polyclonal antibodies in rabbits. The obtained sera were further purified with an affinity chromatography column. For this purpose, each recombinant non-myristoylated recoverin  was immobilized on a CNBr-activated sepharose column and the corresponding serum was passed over the column to remove unspecific antibodies. Due to the elution, all antibodies were diluted 1:10. Anti-zRec2a crossreacted also with zRec2b; therefore, two purification steps were necessary. The first step was catching cross-reactive antibodies by immobilizing zRec2b to the column and collecting the non-bound fraction, which was passed over the zRec2a affinity column in the second step. The specificities of all antibodies were tested 2+-dependent manner  were incubated with 2 mg/ml urea-washed rod outer segment (ROS) membranes and 2 mM CaCl2 or 2 mM EGTA. After 30 min of incubation , the samples were centrifuged , supernatants were discarded, and pellets were resuspended with 2 mM CaCl2 or 2 mM EGTA in HEPES buffer to remove unbound recoverin. After additional 30 min of centrifugation, supernatants were discarded and pellets were resolved in sample buffer. The samples were separated by SDS-PAGE, followed by a protein transfer to a nitrocellulose membrane by a semi-dry blotting using Towbin buffer . After the transfer, all blots were blocked with 1% milk powder  in TBS-T . After blocking, incubation followed with primary specific antibodies against zRec isoforms (see below) and against bRec  was used instead of Dibromo-BAPTA as the competing chelator. It has a KD value of 20 μM in MOPS buffer  . Recoverin concentrations of stock solutions were determined by using a recoverin-specific Bradford calibration curve. Titrations were performed with 15 μM recoverin and 0.5 μM BAPTA-5N in decalcified MOPS buffer. To decalcify the buffer, the buffer passed several times over a Chelex column . The initial [Ca2+] in the titration was determined by BAPTA and ranged between 15 and 60 nM. Chelator fluorescence spectra were measured with a fluorescence spectrophotometer from Photon Technology International using excitation at 494 nm and recording emission spectra between 500 and 560 nm. The Ca2+ titration was performed by using calcium stocks of 3 mM and 30 mM CaCl2 in decalcified MOPS-buffer. In a total volume of 500 μl protein solution, 15 injections of 0.5 μl of 3 mM CaCl2, followed by seven injections of 0.5 μl of 30 mM CaCl2 were done. For each [Ca2+], the peak amplitude at 524 nm was determined, normalized, plotted as a function of free calcium concentration and fitted by the CaLigator software (A chelator assay was employed for the determination of macroscopic Caonstants  with the pH 7.2) , thus masoftware .2+-induced conformational changes of recoverin variants as described before for other NCS proteins . After immobilization, increasing [Ca2+] in a decalcified Tris buffer  were injected and flushed over the protein surface. Control injections over an empty flow cell were performed and subtracted from the sample flow cell  instrument  was used for detecting Caproteins . Immobilproteins . All myrlow cell . By plot2+] in HEPES buffer were incubated for 20 min on ice. For every [Ca2+] above 1 μM, a CaCl2 stock solution in HEPES buffer was prepared, every [Ca2+] under 1 μM was adjusted by a mixture of K2H2EGTA and K2CaEGTA  of each experiment, followed by averaging all K1/2 values and determining the standard deviation.Changes in fluorescence emission of 8-anilinonaphthalene-1-sulfonic acid (ANS) caused by interaction with exposed protein regions  were recK2CaEGTA . ANS flu®syringe filter . Lyophilized protein was dissolved in decalcified Tris-buffer, protein concentration was determined by a Bradford assay and adjusted to a final concentration of 10 μM. After adjusting either a calcium concentration or EGTA concentration of 1 mM, the protein solution was filtered by an AnotopTM 10 filter  and the measurement was started. After recording, mean and standard deviation for each recoverin variant was determined.Dynamic light scattering (DLS) measurements were performed with a Zetasizer Nano-S . For the experiment a polystyrene, disposable, semi-micro cuvette (Ratiolab) was used. Refractive index and viscosity were set to 1.330 and 0.8872 cP , and temperature was set to 25°C with 2 min equilibration time. The measurement angle was 173° backscatter, and the analysis model was set to multiple narrow models (high resolution). For each measurement, a minimum of 11 runs with 30 repetitions were performed. The used Tris buffer  was filtered through a Rotilabo2+ concentration. This translocation process is connected to the functional role of mammalian recoverin controlling the activity of mammalian GRK1 in a Ca2+-dependent manner, inhibiting GRK1 at high Ca2+, and relieving the inhibition at low Ca2+. All zRec forms contain a consensus sequence for myristoylation and could in principle undergo a Ca2+-myristoyl switch and membrane translocation process. Therefore, we tested purified myristoylated and non-myristoylated zRec variants in a membrane binding assay using an equilibrium centrifugation assay. Surprisingly, only zRec1a, and to a lesser extent zRec2a, interacted with membranes in a Ca2+-dependent way like it is known from bRec . bRec served as a benchmark control, since its Ca2+-dependent association with membranes or lipid mixtures is well described in test tube experiments that show 20-25% of total applied recoverin  as all isoforms except zRec2b bound stronger to the membrane in their Ca2+-free state. By contrast, zRec2b showed no difference between its Ca2+-free and Ca2+-bound state.Bovine recoverin shows a reversible binding to membranes triggered by changes in free Caecoverin . The oth2+-binding affinities of zRec forms by measuring macroscopic Ca2+-binding constants with a modified chelator method . A representative example of a titration is displayed in Figure 2 for myristoylated zRec2a revealing an apparent KD of 11.9 μM (Table 1), which is close to the apparent KD values of 14-17 μM obtained previously with bRec . Titrations with non-myristoylated zRec isoforms allowed the determination of two apparent KD values. Fitting of binding curves gave for all zRec isoforms one apparent KD of higher affinity and one of lower affinity (Table 1) resembling the Ca2+-binding studies with non-myristoylated bRec , which allow the investigation of conformational changes from different perspectives.Although all myristoylated zRec forms bound Ca2+-sensor proteins such as recoverin are well suited for monitoring conformational changes by SPR devices under precisely defined experimental conditions . All titrations with immobilized myristoylated zRec variants showed an increase in the maximal amplitudes at defined Ca2+ concentrations resembling those reversible changes in resonance units (RU) that were previously reported for bRec. Table 2 gives a summary of the determined K1/2 values . K1/2 values of zRec forms are around 9-10 μM and are thus slightly higher than 6.1 μM obtained for bRec and those reported in the literature  and therefore, can monitor conformational changes of the whole protein by increasing (or decreasing) fluorescence emission. ANS fluorescence emission of myristoylated zRec2a was half-maximal at a K1/2 of 22.6 μM [Ca2+]  similar to the bovine control variant . All other zRec forms showed significantly different K1/2 values being either fivefold lower (myristoylated zRec1b) or two- to-threefold higher (Supplementary Table S1). However, more unexpected differences became visible except for zRec1a, when we compared myristoylated and non-myristoylated variants (Supplementary Table S1) indicating a more specialized role of the myristoyl moiety for each zRec (see Section “Discussion” below).ANS interacts non-covalently with hydrophobic regions in proteins. Ca2+-saturating and the Ca2+-free state . Most similar to the myristoylated bRec control was zRec2a, all other zRec variants exhibited lower net changes leading to the following sequence of ΔF: bRec > zRec2a > zRec1a > zRec2b > zRec1b. This pattern changed completely, when the myristoyl group was absent leading to a large net change in zRec1b, a lower change for other zRec forms that was still higher than the comparison with bRec . Maximal amplitudes of ANS fluorescence emission in the presence of Ca2+ gave an identical sequence (Supplementary Table S2).We further analyzed difference fluorescence spectra providing information about the net fluorescence change between the Ca2+ to determine a potential difference in the conformation based on Ca2+ binding. Every variant was tested at least 40 times in a myristoylated and non-myristoylated form. To determine the hydrodynamic radius, an intensity plot of DLS data was used. The intensity plot showed one higher peak and one lower peak . Concerning the absence of the second peak in a number plot , the second peak was negligible and only the first peak was used for the further analysis. This peak reflected the size of the investigated protein revealing the hydrodynamic radius . Myristoylated bRec showed the largest difference of the hydrodynamic radius between the Ca2+-bound and Ca2+-free state among all recoverin forms (Table 3) revealing for the Ca2+-bound state, a larger hydrodynamic radius of 7.54 ± 0.17 nm than for the Ca2+-free state, 6.15 ± 0.14 nm (Table 3). Nearly all other myristoylated zRec forms also showed a larger hydrodynamic radius in the Ca2+-bound state but the difference was not as prominent as for the bovine form. Only myristoylated zRec2a had a slightly larger hydrodynamic radius in the Ca2+-free state (Table 3).For dynamic light scattering, all recoverin isoforms were investigated in the absence and presence of Ca2+-free than in the Ca2+-bound state (Table 3), which was opposite to non-myristoylated bRec, whose hydrodynamic radius was nearly the same in the Ca2+-bound (6.4 ± 0.11 nm) than in the Ca2+-free state (6.34 ± 0.13 nm).All non-myristoylated zRec forms except zRec2b have a larger hydrodynamic radius in the CaSupplementary Figure S2). However, our comparative analysis of the molecular properties of zebrafish recoverin forms demonstrate a pattern of significant differences, which we will discuss for each of the experimental approaches.Zebrafish recoverin isoforms share a high sequence homology to the bovine  ortholog  amino acids at the corresponding positions  indicating that all Ca2+-free zRec isoforms keep the myristoyl group buried in a hydrophobic pocket. Critical residues for the Ca2+-triggered transition (and extrusion of the myristoyl group) are Gly42 and Gly96, around which rotational movements occur, and His68 and Leu108, which are involved in the changing domain interactions of EF-hand 2 and 3 (2+-switch mechanisms as we see with zRec1b and zRec2b must therefore have other causes (see Section “Discussion” further below).Mammalian recoverin, in particular bRec, is the prototype of those NCS proteins that undergo a Caembranes . Other N 1 and 3 , 2002. I 2 and 3 . All the2+-free non-myristoylated zRec1a, 1b, and 2a to membranes than binding of their Ca2+-saturated forms to membranes. Binding of proteins to membranes can be the result of hydrophobic and electrostatic interactions. Amino acid sequences of zRec isoforms differ in a few segments from those of bRec. Most of these short stretches of two to three amino acids show no consensus to corresponding amino acids in bRec. Furthermore, they are located on the protein surface  and could account for the observed differences.Unexpected was the higher binding of Ca2+ affinities of zRec isoforms should determine Ca2+sensitive properties such as the Ca2+-dependent membrane association. In mammalian and zebrafish recoverin forms, the two functional EF hands 2 and 3 are highly conserved in all critical positions with oxygen containing side chains . However, in EF-hand 3 of zRec2b, a Pro is at the N-terminal part of the exiting helix and located next to the Glu that is essential for Ca2+ coordination. Figure 6B illustrates the position of Glu next to Pro that is substituted for Asn. Pro interfering with the helix structure could slightly disturb or shift the position of the complexing Glu leading to the lower Ca2+ affinity observed with zRec2b. Furthermore, the C-terminus in bRec was previously identified as an internal modulator of Ca2+ sensitivity. Truncation of C-terminal amino acids causes a shift to lower Ca2+ affinity in bRec  and could partially account for the lower ΔF value determined for zRec1a and even more for zRec1b, where the surface of the patch decreases.ANS fluorescence emission is a tool to estimate the increase of solvent accessible hydrophobic surfaces depending on experimental conditions, for example, Ca-binding . It has Figure 7). This hydrophobic groove might be another target of ANS. The Gly in zRec2b decreases the degree of hydrophobicity thereby lowering binding of ANS, whereas Ala in zRec2a has a side chain of lower hydrophobicity than Ile, but probably sufficient for ANS binding . In non-myristoylated bRec, the large hydrophobic patch is solvent accessible in both forms of bRec, with one or two Ca2+ bound . However, other zRec variants did not exhibit similar correlations.Two other hydrophobic patches of lower size are visible on the surface of bRec, one made of Leu167, Phe172, Ile173, Leu187, and Ala188 and the second one made of Phe158, Phe159, Ile182, Leu183, Leu185, and Ile186. The latter one forms a kind of canyon-like structure, in which Ile182 in bRec is replaced by Ala and Gly in zRec2a and zRec2b, respectively . Zang et al. further showed that cone photoresponse recovery differs in zRec2a and zRec2b morphants depending on illumination that triggers transient changes in cytoplasmic Ca2+ concentration (2+-binding constants that we determined for zRec2a and zRec2b (Table 1). Membrane binding and Ca2+-binding constants of zRec1a were the most similar to those of bRec. Due to the expression of zRec1a in rods (and UV cones) and the relationship of their targets zGRK1a and bovine GRK1, our results are in agreement with a functional pair of zRec1a and zGRK1a. Finally, we cannot exclude that one or more zRec forms target a different protein as this was observed for bRec interacting with caldendrin (2+ concentration in combination with the DLS data could point to conformational differences that are suitable for interacting with different targets.We conclude from these observations that all zRec isoforms undergo Cantration . These fldendrin  and suggldendrin . For exaDE, AS, and K-WK designed the study. DE and AS performed experiments. DE and K-WK analyzed data. K-WK wrote the first draft of the manuscript. All authors corrected and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer PG and handling editor declared their shared affiliation at the time of the review.


The CA2 region of the hippocampus is a somewhat obscure area lacking in an understanding of its form and function. Until recently, the CA2 has been thought of as merely an extension of the CA3, with some referring to it as the CA3a region. Recent investigations into this area have not only delineated the CA2, but also defined it as a region distinct from both CA1 and CA3, with a unique set of cortical inputs and outputs and contributions to cognitive processes. One such process that has been shown to depend on the CA2 is the ability to recognize a novel or familiar conspecific, known as social recognition memory. Here, we review these findings and discuss the parallels between CA2 dysfunction and social impairments. Social cognitive neuroscience is a burgeoning interdisciplinary field of research that aims to delineate the “why” and “how” of interactions between individuals. It aims to explore the relationship between cognitive processes and behavior in a social context. First described by One of the processes that is paramount to the structure and stability of relationship networks that define societies is that of social memory . Social The ability to recognize a familiar versus novel individual is the foundation on which social relationships are built. Memory for individual conspecifics is not only necessary to engage in meaningful relationships but is also required to express appropriate behavioral responses based on previous encounters . Social While social recognition is a common process across a variety of mammals, there are species and sex differences which may influence how it is expressed . StudiesSex-differences have also been reported to exist in social recognition processes. In a social recognition task, female rats were reported to spend less time investigating a conspecific juvenile compared to males, yet females displayed stronger remote social recognition than males . Based oEvidence suggests that the hippocampus is required for proper processing of social memories as data have shown that hippocampal lesions disrupt social recognition . Rats wiThe two major divisions of the hippocampus are the dorsal and the ventral hippocampus , each wiThe ventral hippocampus has been shown to be involved in emotional memory processes, like fear, and influences the fear memory processing of the amygdala, given that the ventral, but not the dorsal, hippocampus projects directly to the amygdala . The dorThe dentate gyrus  receivesThe ability to organize and separate spatial events from one another, allowing an organism to temporally remember one place as distinct from another, is largely mediated by the dentate gyrus . This spstratum pyramidale, the stratum lacunosum-moleculare, the stratum lucidum, the stratum oriens, and the stratum radiatum. The stratum pyramidale contains the cell bodies of pyramidal cells and various interneurons  has been widely accepted to be the cellular underpinning of synaptic plasticity, and memory acquisition, storage, and retrieval in the central nervous system . High-frno avail .A inhibitory currents were blocked using picrotoxin, yet all of these protocols consistently induced LTP in the CA1  imaging to compare cell ensembles in CA1, CA2, and CA3 subregions in mutant mouse lines lacking either NMDA receptors at the CA3 synapse (CA3-NR1 KO) or at the perforant path inputs from the entorhinal cortex to the dentate gyrus (DG-NR1 KO). This allowed the investigators to manipulate the quality of incoming information to CA2. CA3-NR1 KO leads to deficits in rapid encoding information and associative memory storage, making a familiar representation more novel, while DG-NR1 KO leads to deficits in context discrimination, making a novel representation more familiar  or the environment in which the conspecific was initially encountered could ostensibly create errors in recognition. Together with the temporal processing of information, the CA2 can be viewed as processing the general ‘when, where, and what’ aspects of social recognition.stratum pyramidale of CA2 in schizophrenic and manic-depressive patients compared to controls  light chain in CA2 pyramidal neurons, which blocked neurotransmission. To test the efficacy of this method, they co-expressed channelrhodopsin-2 (ChR2) with either TeNT or yellow fluorescent protein (YFP) using Cre-dependent AAVs. From there, they used whole-cell current clamp recordings to determine the strength of synaptic transmission from CA2 to CA1 pyramidal neurons. Focal photostimulation over a wide range of intensities delivered to CA1 stratum oriens and stratum radiatum regions evoked robust monosynaptic potentials in the CA1 pyramidal neurons in slices in which ChR2 was co-expressed with YFP in CA2 pyramidal neurons. Slices in which ChR2 was co-expressed with TeNT in CA2 pyramidal neurons produced little to no synaptic response in CA1 neurons when CA2 neurons were illuminated (Recent advancements in genetic manipulation techniques have allowed for investigations into the role of CA2 in social cognition in a more precise and sensitive manner. uminated .Given the efficacy of TeNT lesions, they then investigated the behavioral consequences of CA2 inactivation. A three-chamber social novelty test was used to compare performance between control mice, expressing YFP in CA2 pyramidal neurons, and CA2-TeNT mice. In this test, the amount of time that the experimental mouse spent interacting with a novel, unrelated mouse was compared to the time spent interacting with a familiar co-housed littermate in order to measure social recognition . While tA five-trial habituation/dishabituation social memory was used to confirm that CA2-inactivation resulted in impaired social memory. In this test, the experimental mouse was exposed to a stimulus mouse for four consecutive trials then introduced to a novel stimulus mouse on the fifth trial. The CA2-TeNT mice showed no significant habituation during the first four trials as evidenced by a lack of decreased exploration and no significant dishabituation to the novel mouse as shown by a lack of increased exploration . The CA2A three-chamber test of sociability was used to compare performance between CA2-YFP mice and CA2-TeNT mice. This test examined the normal preference of the experimental mouse for a chamber containing a littermate versus an empty chamber. Both the CA2-YFP and CA2-TeNT groups displayed a significant preference for the compartment containing their littermate, suggesting that CA2 inactivation did not alter sociability . In theiFollowing up to the work by The CA2 is a dynamic region with a composition and connectivity unique to the hippocampus, and not just an extension of the CA3, as was previously believed. Given that research into this region is a relatively new endeavor, the precise role of the CA2 is not fully understood. Nevertheless, burgeoning research into this area has established the role of CA2 in social memory processes, with a particular focus on social recognition, and as such, dysfunction in this area has been implicated in the social impairments common to a number of psychiatric diseases. Beyond these preliminary findings, little is known concerning the cellular pathways that regulate this brain region and how it mediates social recognition representations, while its contribution to neuropathologies is somewhat vague and inferential, at best. Further research into the cellular mechanisms that govern the CA2 may provide insight into the social deficits underlying certain neuropathologies, such as autism and schizophrenia, and potentially yield novel treatment strategies for these disorders.NT wrote the initial and revised versions of the manuscript. MH commented and edited the manuscript. Both authors approved the final manuscript for submission.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.


In particular, the quantitative and kinetic properties of purinergic-induced Ca2+ release from intracellular stores and Ca2+ entry from extracellular medium were investigated. The main conclusions are: (1) stimulation of TECs with high eATP triggers [Ca2+]c signals which include Ca2+ mobilization from intracellular stores (mainly ER) and Ca2+ entry through the plasma membrane; (2) the long-lasting Ca2+ influx phase requires both store-operated Ca2+ entry (SOCE) and non-SOCE components; (3) SOCE is not significantly involved in the antimigratory effect of high ATP stimulation; (4) ER is the main source for intracellular Ca2+ release by eATP: it is required for the constitutive migratory potential of TECs but is not the only determinant for the inhibitory effect of high eATP; (5) a complex interplay occurs among ER, mitochondria and lysosomes upon purinergic stimulation; (6) high eUTP is unable to inhibit TEC migration and evokes [Ca2+]c signals very similar to those described for eATP. The potential role played by store-independent Ca2+ entry and Ca2+-independent events in the regulation of TEC migration by high purinergic stimula deserves future investigation.Tumor microenvironment is particularly enriched with extracellular ATP (eATP), but conflicting evidence has been provided on its functional effects on tumor growth and vascular remodeling. We have previously shown that high eATP concentrations exert a strong anti-migratory, antiangiogenic and normalizing activity on human tumor-derived endothelial cells (TECs). Since both metabotropic and ionotropic purinergic receptors trigger cytosolic calcium increase ([Ca Stromal TRPC1-7 . SOCE iser cells ,19. It ier cells , includir medium .Importantly, SOCE mechanisms are involved in the regulation of cellular proliferation, migration, differentiation and apoptosis ,23, playNowadays, growing interest has been focused on the therapeutic potential of purinergic signaling since it plays a fundamental role in both physiological and physio-pathological processes. In particular, eATP controls several acute vascular functions ,26,27,28Rapaport and colleagues have demonstrated for the first time an anti-neoplastic activity of eATP. Consistently, recent studies have revealed an antitumor activity of extracellular nucleotides in different types of cancer ,32. P2RsFurthermore, very few studies on the effects of purinergic stimulation in the tumor endothelium are currently available. The tumor vasculature strongly differs from the vasculature of healthy tissues from both a morphological and functional point of view. A deeper knowledge of the mechanisms underlying the sensitivity of tumor endothelium to high eATP concentrations  could be helpful in this context.We have recently shown that high non-physiological eATP doses (>20 µM) strongly inhibit migration of ECs isolated from human breast carcinoma (BTEC) and enhance attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization . This pr2+ signaling, some P2YRs (mainly P2Y11) and P2XRs promote intracellular cAMP increase that can in turn regulate endothelial barrier stabilization and inhibit cell migration c increased in the great majority of cells examined (2+ transient (peak) is usually followed by a sustained long-lasting phase (plateau). Based on the dose-dependent amplitude and area of peak and plateau phases (out), only a dose-dependent transient spike was detectable, due to Ca2+ release from intracellular stores, while the plateau phase, mainly related to Ca2+ entry from the extracellular medium, was completely abolished  evoked c increase induced by TG and Baf-A1 in cells pretreated with ATP or UTP, globally indicating an interplay among ER, mitochondria and lysosomes in the Ca2+ release triggered by both the purinergic agonists , 20 µM Bafilomycin A1  and 1 µM FCCP (to uncouple mitochondria). All the experiments were performed in 0 Caagonists J–L.2+ signals in TECs.We have recently shown that high non-physiological eATP doses (>20 µM) strongly inhibit migration of ECs from human breast carcinoma (BTEC) and enhances in vitro endothelial normalization . These e2+ signals and their role on migration in both breast and renal-derived ECs.For these reasons, we investigated ATP-mediated Ca2+ signals in BTEC and RTEC; these data are in accordance with the expression of both metabotropic and ionotropic receptors previously reported [2+]c rise due to Ca2+ mobilization from intracellular stores, followed by a long-lasting plateau due to Ca2+ entry through the plasma membrane. Considering the recruitment of both metabotropic and ionotropic ATP receptors, the long-lasting phase could be due to both store-operated calcium entry (SOCE) and/or non-SOCE mechanisms. SOCE is clearly observed both in BTEC and RTEC when stimulated with 30 µM CPA or TG, two widely used ER depletors. On the other hand, the pharmacological inhibition of SOCE with 20 µM BTP-2 significantly prevented ATP-mediated Ca2+ entry in BTEC but did not completely abolish the plateau phase, revealing a non-SOCE component of Ca2+ entry induced by 100 μM ATP, presumably related to P2RX-ionotropic receptors.Here we show that high ATP stimulation (100 μM) triggers biphasic Careported . In part2+ signals with BTEC migration. The ability of 100 µM ATP to decrease BTEC migration even in the presence of BTP-2 unveils a SOCE-independent component in the anti-migratory effect of ATP, possibly related to the recruitment of P2XR-ionotropic receptors that act as calcium-permeable channels c measurements. Fluorescence was acquired by Nikon Eclipse TE-2000S  inverted microscope. [Ca2+]c was expressed as a ratio (R) of emitted fluorescence at 510 nm corresponding to excitation wavelengths of 340 nm and 380 nm.Cells were grown on glass gelatin-coated coverslips  was used for image acquisition (3 s frequency). For each experiment, several regions of interest (ROIs) have been selected corresponding to single cells in the chosen image field. Real time background subtraction was applied in order to limit noise. Average traces shown in figures include at least 20 ROIs.Calcium imaging analysis and quantification  were performed using Clampfit software  and analysed with GraphPad Prism 7 .2+ was evaluated at 300 s after the onset of the response. Total area under calcium spikes in 0 Caout was measured by the use of Event Detection protocol in Clampfit software.Area underlying calcium influx during the sustained phase in the presence of extracellular Ca2+ store depletion and store-operated Ca2+ entry (SOCE) were evaluated by exploiting the Ca2+ add-back protocol. Briefly, the cells were treated with the agonist to induce depletion of Ca2+ stores in Ca2+-free medium  and, subsequently, replaced with Ca2+-containing solution (2 mM Ca2+) so that SOCE could be measured.CaScratch Wound Healing Assay. Cells were grown to confluence on 24-well culture plates. Cell monolayers were allowed to rest for 12 h in EndoGro-MV and a wound was made by scraping the middle of the cell monolayer with a P10 pipette tip. Floating cells were removed by washing twice with Phosphate-Buffered Saline, PBS, and the cell monolayer was put in a serum-free RPMI medium for 1 h and then in RPMI 10% FBS.2 and pictures were taken every 2 h using Metamorph software  [Cells did not undergo any significant degree of mitosis during the experiments. Images were acquired using a Nikon Eclipse Ti-E microscope with a 4X objective. Cells were kept at 37 °C and 5% COCA, USA) ,44. MigrCA, USA) ,46. At l® RNA Mini Kit . For quantitative real-time polymerase chain reaction (qRT-PCR), 0.5–1 μg total RNA was transcribed into complementary DNA (cDNA) by High-Capacity cDNA Reverse Transcription Kit  upon treatment with RQ1 RNase-Free DNase . qRT-PCR was performed using the QuantStudio™ 6 Flex Real-Time PCR System (Applied Biosystems). Primers and probes were designed using the Universal ProbeLibrary Assay Design Center software (www.lifescience.roche.com). Transcript abundance, normalized to 18 s mRNA expression, is expressed as a fold increase over a calibrator sample.Total RNA was extracted from cell samples using PureLinkp-value of <0.05 was considered significant.Data were analyzed with GraphPad Prism 7 . Each experiment was repeated at least three times. Preliminary Shapiro-Wilk test was performed to check the normal distributions of each dataset: accordingly, statistical analysis was performed by using either the non-parametric Mann-Whitney test or the Student’s t-test. A 2+ signals according to the agonist and its concentration. High eATP doses strongly inhibit BTEC [2+ signals due to Ca2+ release from intracellular stores (mainly ER) and Ca2+ entry from extracellular medium. Similar calcium signals and antimigratory effects are triggered by ADP and Adenosine, while P2RY-selective agonist UTP promotes calcium signals without any detectable modulation of TEC migration. Calcium release from ER is not required to account for the entire antimigratory activity of high ATP, while SOCE is not significantly involved. Non-SOCE mechanisms, presumably related to P2RXs recruitment, could be good candidates as negative regulators of TEC migration, but calcium-independent pathways cannot be excluded.Purinergic stimulation in BTEC evokes different Cabit BTEC  and RTEC


Our aim was to determine the purinergic pathway in isolated human olfactory neuronal precursor cells (hONPC) that exhibit MpSC features. Cloning by limiting dilution from a hOE heterogeneous primary culture was performed to obtain a culture predominantly constituted by hONPC. Effectiveness of cloning to isolate MpSC-like precursors was corroborated through immunodetection of specific protein markers and by functional criteria such as self-renewal, proliferation capability, and excitability of differentiated progeny. P2 receptor expression in hONPC was determined by Western blot, and the role of these purinoceptors in the ATP-induced exocytosis and changes in cytosolic Ca2+ ([Ca2+]i) were evaluated using the fluorescent indicators FM1-43 and Fura-2 AM, respectively. The clonal culture was enriched with SOX2 and OCT3/4 transcription factors; additionally, the proportion of nestin-immunopositive cells, the proliferation capability, and functionality of differentiated progeny remained unaltered through the long-term clonal culture. hONPC expressed P2X receptor subtypes 1, 3-5, and 7, as well as P2Y2, 4, 6, and 11; ATP induced both exocytosis and a transient [Ca2+]i increase predominantly by activation of metabotropic P2Y receptors. Results demonstrated for the first time that ex vivo-expressed functional P2 receptors in MpSC-like hONPC regulate exocytosis and Ca2+ signaling. This purinergic-triggered release of biochemical messengers to the extracellular milieu might be involved in the paracrine signaling among hOE cells.Extracellular ATP and trophic factors released by exocytosis modulate  In many species including humans, neurogenic process in the central nervous system (CNS) and the olfactory epithelium (OE) prevails in adulthood , 2. ThisStem cells can be dissociated from neurogenic niches to develop primary cultures in a monolayer . However2+ signaling in neuronal cells, for instance, the olfactory transduction in sensory neurons i was calculated according to the Grynkiewicz formula i was calculated from 5 cells by treatment and compared through a Student t test between stimuli.To evaluate i. Therefore, we corroborated the functionality of the expressed P2 receptors by microfluorometry, detecting cytosolic free Ca2+ with the indicator Fura-2 AM. hONPC perfusion with ATP induced a transient increase in [Ca2+]i  (2+]i increase was induced by P2 receptor activation.It is well-known that activation of purinoceptors is followed by an increase in [Ca [Ca2+]i . When tw10.4 nM) . In addi10.4 nM) , indicatα-β-methylene ATP) were used. The former induced a [Ca2+]i increase equivalent to 91% of the ATP-evoked response    or P2X ( 6.9 nM) , whereas 6.4 nM) . Additio 6.4 nM) , while P 6.4 nM) .2+]i by two pathways, by depletion of intracellular Ca2+ stores through activation of metabotropic P2Y receptors, or by influx of extracellular Ca2+ upon activation of ionotropic P2X receptors. To explore the participation of these mechanisms, hONPC were perfused with ATP in a Ca2+-free solution to discard Ca2+ influx. In this case, the response to ATP reached 84%    ; conceiv12.3 nM) . These rex vivo cultured hONPC that exhibit MpSC-like features expressed functional purinergic P2 receptors coupled to Ca2+ signaling. This purinergic pathway induced exocytosis and a transient increase in [Ca2+]i, predominantly by the activation of metabotropic P2Y receptors. In CNS neurogenic niches, trophic factors released by exocytosis play a key role in the regulation of proliferation, migration, or differentiation of MpSC. Hence, the ATP-mediated exocytosis in hONPC might be involved in paracrine signals to modulate these processes in the hOE.In this study, we found for the first time that in vivo, SOX2 expression is restricted to MpSC and this transcription factor is absent in committed progenitors and immature or mature OSN i i augmentation was induced by UTP. As mentioned, metabotropic P2Y2 and P2Y4 receptors (both detected in this work by Western blot) are activated by purine and pyrimidine nucleotides [2+]i increase; nevertheless, its participation in exocytosis remains to be elucidated. These ionotropic receptors could be involved in other cellular functions; for instance, the P2X7 receptor has been associated with apoptosis regulation in cells from different tissues [Another well-studied function of ATP in stem cells is the induction of changes in [Ca2+]i . Our datleotides . Particuleotides  and humaleotides . In thisleotides , 73. On  tissues . This su tissues . AlthougEvidence concerning physiology of neuronal precursors obtained from healthy humans could constitute the basis to perform future comparison studies with hONPC isolated from patients diagnosed with neuropsychiatric disorders such as schizophrenia. In this regard, anomalies in the purinergic signaling pathway have been detected in patients diagnosed with this illness , 77. In 2+]i mainly through activation of metabotropic P2Y receptors. Hence, in the human olfactory neuroepithelium, ATP might function as a chemical messenger modulating the paracrine signaling among the different cell types that need to be coordinated in this neurogenic structure. Our results indicate that the culture of hOE-derived neuronal precursor cells is a suitable model to study the Ca2+-dependent purinergic signaling pathway and the involvement of P2 receptors in proliferation, migration, or differentiation in human multipotent precursor cells.The long-term cell culture obtained by cloning was composed predominantly by hONPC that showed proteomic and functional traits closely related to MpSC. These stem cells were confirmed to have originated from the olfactory neuroepithelium because they expressed vimentin and OMP. The preparation of ex vivo hONPC expressed a broad variety of P2 receptors. This purinergic pathway induced both an exocytotic process and a transient increase in [Ca


Ceramides, a pleiotropic class of sphingolipids, are important mediators of cellular senescence, but their role in neuronal aging is still largely unexplored. In this study, we investigated the effects of L-cycloserine (L-CS), an inhibitor of thede novoceramide biosynthesis, on the aging phenotype of cortical neurons cultured for 22 days, a setting employed as anin vitromodel of senescence. Our findings indicate that, compared to control cultures, ‘aged’ neurons display dysregulation of [Ca2+]ilevels, mitochondrial dysfunction, increased generation of reactive oxygen species (ROS), altered synaptic activity as well as the activation of neuronal death-related molecules. Treatment with L-CS positively affected the senescent phenotype, a result associated with recovery of neuronal [Ca2+]isignaling and reduction of mitochondrial dysfunction and ROS generation. The results suggest that thede novoceramide biosynthesis represents a critical intermediate in the molecular and functional cascade leading to neuronal senescence and identify ceramide biosynthesis inhibitors as promising pharmacological tools to decrease age-related neuronal dysfunctions.Although aging is considered to be an unavoidable event, recent experimental evidence suggests that the process can be counteracted. Intracellular calcium (Ca Aging is the time-dependent process characterized by the loss of the physiological integrity of living organisms . AlthougCeramides are a class of sphingolipids involved in a wide array of biological functions including cell growth, proliferation, differentiation, and programmed cell death as well as cellular senescence . The appde novobiosynthesis from palmitoyl-CoA and serine, or cleavage of sphingolipid precursors in membranes ihandling, mitochondrial functioning, spontaneous neuronal Ca2+signaling, and activation of age-related and ceramide-driven molecular pathways.To gain further insights on the role played by ceramides in neuronal aging, we used L-cycloserine (L-CS), an amino acid that inhibits SPT activity both safely and selectively, and has been employed bothynthesis ,17–20. Win vitromodel of neuronal senescence iwere measured by using the high-affinity (Kd≈140nM) ratiometric dye fura-2. The analysis of resting fura-2 signals showed that no regional differences in [Ca2+]ilevels were detectable in proximal or distal dendrites of aged or control neurons treated with either vehicle or L-CS  or 10 µM cyclopiazonic acid , two agent employed to promote cation release from mitochondria or the ER, respectively. Analysis of the cytosolic [Ca2+]ichanges showed that exposure to L-CS did not affect mitochondrial Ca2+release (2+release was instead found in aged neurons (2+release (data not shown) and to facilitate the evaluation of this pathway, ER was pre-loaded with Ca2+derived from the opening of the voltage-gated Ca2+channels (VGCCs) as result of a 5-min depolarization induced by exposing cultures to 60 mM K+. Compared to vehicle-treated and age-matched neurons, Ca2+released from the ER was found to be lower in L-CS-treated control cultures (Mitochondria and the endoplasmic reticulum (ER) constitute the main intracellular Ca+release . A signi neurons . As our cultures . No diffcultures .+-Ca2+exchanger (NCX) [2+]i. We, therefore, investigated the effects of ceramide on NCX functioning in L-CS or vehicle-treated cultures. Data obtained from this set of experiments were compared with the ones originated from age-matched and vehicle-treated sister cultures. In the experiments, fura-2 loaded neurons were exposed to a Ca2+free medium while in the presence of the Na+/K+-ATPase pump blocker, ouabain (100 µM), a maneuver set to promote the intracellular accumulation of Na+. Ouabain-treated cultures were then switched to a Na+-free medium to force the NCX to operate in the reverse mode to promote Ca2+influx. Analysis of the cytosolic [Ca2+]irises showed that L-CS does not affect NCX activity in control and aged cultures treated with either vehicle- or L-CS  , a low c or L-CS . Changescultures .Mitochondrial functioning is a key target of the aging process ,21. In oAberrant ROS generation and accumulation of ROS-driven by-products are key features of aging . To asse2+]itransients, an index of neurodegenerative hyperexcitability, we eployed high-speed real-time microfluorimetric Ca2+imaging [d≈ 335 nM) and Ca2+sensitive fluorescent probe fluo-4 and spontaneous changes in somatic [Ca2+]iwere evaluated in terms of spike frequency and amplitude. In line with previous studies .,32.31,32in vitromodel to characterize the effects of pharmacological blockade ofde novoceramide biosynthesis on neuronal senescence. The results indicate that exposure to the SPT inhibitor, L-CS, concurrently lowers ceramide levels in aged neurons and attenuates critical molecular and functional changes induced by aging. In particular, we found that L-CS actively counteracts the age-related mitochondrial dysfunction and neuronal Ca2+dyshomeostasis.In this study, we used an2+]ilevels  which in turn contribute to the increase in ceramide by-products (i.e.: sphingosine), a likely hypothesis suggested by our lipidomic analysis  of the mitochondrial Δp and a reduction of the ROS production in L-CS-treated neurons when compared to vehicle-treated aged-matched cultures. These findings are also in line with previous reports showing that ceramides contribute to mitochondrial dysfunction by impairing the organelle electron transport chain, generating ROS, and promoting the permeabilization of the mitochondrial outer membrane ,56. ROS de novoceramide biosynthesis, prevents the mitochondria-driven activation of senescent-related pathways.To investigate the downstream effectors of the mitochondrial pathways involved in neuronal dysfunction, we evaluated changes in levels of p53, pJNK, and pP38. The selection of these proteins was driven by the fact that 1) these factors are altered in and participate to senescence-related processes; 2) their activity is intertwined with mitochondrial dysfunction; 3) their levels are modulated by ceramides ,48,58–60in vitroneuronal development imeasurements HCSS was supplemented with 200 - 500 nM TPEN (Merck) to avoid interferences due to other metal ions (i.e. zinc) with [Ca2 +]i-dependent fluorescence signals itransients. Images were acquired at full-frame resolution  at 1 Hz sampling rate for up to 5 min. Conversely, to evaluate dendritic Ca2+transients, the camera frame was cropped (with a 1x binning) around proximal primary dendritic branches and images acquired at a 10 Hz sampling rate for up to 60 s.Fluo-4 AM  was employed to measure spontaneous [Ca2+]itransients from vehicle- and L-CS-treated cultures were analyzed with a custom made MATLAB code, as previously described [2+transients when the threshold was set at 25% of the baseline were excluded from the analysis.Fluorescence changes of each cell/dendrite were expressed as a percentage of baseline fluorescence . Spontaneous [Caescribed ,41. For x) were normalized to basal fluorescence intensity (FCCCP) obtained by exposing the cells to the mitochondrial uncoupler CCCP (10 µM). Experiments were halted when, after CCCP application, the fluorescence signal was stable for up to 30 s.Measurements of the mitochondrial Δp were performed as previously described , with miNeurons plated on glass coverslips were washed and loaded for 30 min at room temperature with MitoTracker Green FM (100 nM in HCSS), a mitochondrially-targeted and Δp-insensitive dye. Cells were imaged on the stage of an inverted Zeiss LSM800 Super-resolution confocal microscope equipped with a 488 nm LED-based laser line, a 63x oil immersion objective (N.A.: 1.4), and an Airyscan imaging module. Appropriate emission filter-sets were selected with the built-in Smart Setup function. Laser power was maintained at the minimum (0.65%) to achieve an optimum signal-to-noise ratio and avoid photobleaching. Acquisition parameters  were constant among experimental sessions. Single-plane images were acquired with the Zeiss ZEN proprietary software and stored for offline analysis.Confocal micrographs were Airyscanned to obtain super-resolution images that were further analyzed with the ImageJ-based MiNA toolkit, following the developer workflow . BrieflyEvaluation of basal ROS production was performed by employing hydroethidine , a fluorescent probe sensitive to ROS. Basal ROS production levels were measured after loading vehicle- or L-CS-treated cultures with HEt (5 µM). Cells were loaded for 2 h at 37 °C to allow dye oxidation by endogenously released ROS. Resting fluorescence signals were recorded and normalized to that of vehicle-treated control cultured neurons.Western blot analysis was performed as previously described ,78. BrieSptlc-2relative expression was corrected forGAPDHhere employed as endogenous control. Samples were run in triplicate.Sptlc-2relative fold change was calculated with the ΔΔCt method.RNA extraction and qRT-PCR were performed as previously described . Brieflythand 75thpercentiles, and whiskers extend 1.5 times the interquartile range from the 25thand 75thpercentiles [2+transients were analyzed by unpaired Student’s t-test. Experimenters were not blinded to treatment allocation. By conventional criteria, P values are represented as * for P ≤ 0.05 and ** for P ≤ 0.01.Data are represented as box plots. Center lines and boxes represent medians and means, respectively. Box limits indicate 25centiles . No statSupplementary FiguresSupplementary Table 1