Extracellular acidity is a marker of highly aggressive breast cancer (BC). pH-low insertion peptides (pHLIPs) target the acidic tumor microenvironment. This study evaluates the distribution and therapeutic efficacy of radioiodine-labeled pHLIP variant 3 (Var3) in a mouse model of BC. 125I) labeled Var3-pHLIP to MDA-MB-231, 4T1, and SK-BR-3 BC cell lines under different pH values was evaluated in vitro. The distribution of 125I-labeled Var3-pHLIP and wild-type- (WT-) pHLIP in tumor-bearing mice was analyzed in vivo using micro-SPECT/CT imaging. The therapeutic efficacy of radioiodine-131 (131I)-labeled Var3-pHLIP in MDA-MB-231 xenografts was evaluated by relative tumor volume measurement and immunohistochemical analysis.  The binding of fluorescein isothiocyanate (FITC)- or radioiodine-125 . The relative tumor volume in MDA-MB-231 xenografts was significantly lower in the 131I-Var3-pHLIP-treated group than in the groups treated with Var3-pHLIP (p = 0.027), 131I (p = 0.001), and saline (p < 0.001). The 131I-Var 3-pHLIP group presented a lower expression of Ki67 and a higher expression of caspase 3.  The binding ability of FITC- or  Radioiodine-labeled Var3-pHLIP effectively targeted BC cells in an acidic environment and inhibited the growth of MDA-MB-231 xenografts by ionizing radiation. Breast cancer (BC) is the leading cause of cancer death among women . Triple-+ production and excretion, whereas extracellular pH in healthy tissue under physiologic conditions is 7.2–7.4. In addition, the acidic microenvironment is not affected by clonal selection and thus is promising for diagnostic imaging and targeted therapy [Acidosis in the tumor microenvironment promoted by the Warburg effect is a hallmark of cancer. The extracellular pH in this environment is below 6.5 because of Hα-helix that crosses the cell membrane lipid bilayer [99mTc), iodine-125 (125I), and iodine-131 (131I) are simpler for in vivo imaging than labeling with positron-emitting nuclides. Furthermore, 131I can emit beta radiation for tumor therapy. 99mTc-labeled wild-type- (WT-) pHLIP [125I-labeled pHLIP variant 7 [The 37-amino-acid pH-low insertion peptide (pHLIP) is water-soluble at pH ≥ 7.4 and has a low affinity for cell membranes. However, pHLIP is lipophilic under acidic conditions and forms an  bilayer . Therefo bilayer –8, radio bilayer , 10, and bilayer , 12. Var bilayer , 13, 14.-) pHLIP  and 125Iariant 7  are prom125I-WT-pHLIP and 125I-Var3-pHLIP in mice-bearing human BC xenografts and investigated the therapeutic efficacy of 131I-Var3-pHLIP in MDA-MB-231 xenografts.This study compared the in vivo distribution of 2.The human BC cell lines MDA-MB-231 (triple-negative BC), 4T1 (stage IV BC), and SK-BR-3 (HER2-positive BC) were purchased from The Cell Bank of the Chinese Academy of Sciences  and were cultured in DMEM  containing 4.5 g/L glucose, 10% fetal bovine serum , and 1% penicillin-streptomycin . Cells were incubated at 37°C in a humidified incubator with 5% COpHLIP was synthesized by GL Biochem Ltd.  through solid-phase chemical synthesis. The amino acid sequences of WT- and Var3-pHLIP are ACEQNPIYWARYADWLFTTPLLLLDLALLVDADEGTK and ACDDQNPWRAYLDLLFPTDTLLLDLLWK. The N-terminal of pHLIP was conjugated with fluorescein isothiocyanate (FITC), and fluorescence in vitro was measured at 495 nm excitation and 519 nm emission.125I for in vivo imaging experiments, and Var3-pHLIP was labeled with 131I for therapeutic efficacy experiments. Radioactive iodine binds to tyrosine residues in pHLIPs. Peptides were dissolved in 200 μL of 0.02 M PBS (pH 7.4) to reach a concentration of 1 mg/mL and mixed with 10 μL of Na125I (74 MBq) or 20 μL of Na131I (925 MBq). Chloramine-T  was added to the test tubes and mixed for 70 s at room temperature. The reaction was stopped by adding 200 μL of sodium metabisulfite (5 mg/mL). The peptides were eluted in ethanol through a C18 column equilibrated with 20 mL of 100% ethanol and 20 mL of distilled water. The radioactive purity of 125I-WT-pHLIP, 125I-Var3-pHLIP, and 131I-Var3-pHLIP was determined using thin-layer chromatography. Furthermore, the radiochemical purity of 125I-WT-pHLIP, 125I-Var3-pHLIP, and 131I-Var3-pHLIP was measured, and chemical stability was determined at 24 and 48 h at room temperature (25°C) in PBS and at 37°C in serum.WT-pHLIP and Var3-pHLIP were labeled with 4. After cell attachment, FITC-WT-pHLIP and FITC-Var3-pHLIP (50 μg/mL) were added to the wells in triplicate. Cultures were incubated on a rotary shaker for 30 min. The culture medium was aspirated, and cells were washed three times with a solution of the respective pH without FITC-labeled peptides. Subsequently, 250 μL of DAPI staining solution (100×)  diluted with DMEM medium at the respective pH was added to each well. The cells were incubated at 37°C in a humidified incubator with 5% CO2 for 10 min, washed three times with PBS at the respective pH, and imaged on a fluorescence microscope (×100).DMEM medium and MES buffer were mixed at different volume ratios  to simulate extracellular environments with a pH of 7.8, 7.4, 7.0, 6.6, and 6.2, respectively. MDA-MB-231, 4T1, and SK-BR-3 cells were seeded in 24-well plates at a density of 5 × 10125I-WT-pHLIP or 125I-Var3-pHLIP for 1 h at 37°C in the medium at different pH values ranging from 6.2 to 7.8 . At the same time, cells in the medium at pH 6.2 were further incubated with 125I-labeled peptide with an excess of unlabeled peptide (25 μg/well). The supernatant was aspirated into a tube. The cells were then washed twice with PBS of the corresponding pH to remove unbound radioactivity. The wash solution was also added to the supernatant tube containing free radioactivity (F). The cells with bound radioactivity (B) were completely lysed with 300 μl sodium hydroxide and aspirated into a cell tube. The radioactive counts of the cell and supernatant tube were measured by a γ counter. The cell binding fraction of the 125I-labeled peptides was calculated as B/(B + F) × 100%. All experiments were performed in triplicate.Cells were incubated with 3 cells per well) and cultured for 12 hours. Cultures were divided into a treatment group  and control group (100 μL of culture medium at the respective pH). The assays were performed in triplicate and repeated independently three times. The cells were incubated at 37°C in an incubator for 30 min, and the culture medium was aspirated. The CCK-8 solution was mixed with DMEM at a ratio of 1 : 9, and 100 μL of the solution was added to each well. Absorbance was measured at 450 nm in a microplate reader. Absorbance in wells containing medium at pH 7.8 without pHLIP was defined as 100% cell viability, and the percentage of cell viability was calculated.MDA-MB-231, 4T1, and SK-BR-3 cells were seeded in 96-well plates (5 × 106 cells (suspended in 150 μL PBS) into the right axilla of each mouse. The mice were used in subsequent experiments when tumor diameter reached 0.5-0.9 cm. The study protocol was approved by the institutional review board and by the Experimental Animal Center of Ruijin Hospital affiliated to the School of Medicine of Shanghai Jiao Tong University.Five-week-old female athymic BALB/c nude mice  were used in our study. Mouse xenograft models of MDA-MB-231, 4T1, and SK-BR-3 cells were produced by the subcutaneous injection of 5 × 10μL of 1% Kl solution was administered to mice by oral gavage once a day, and 0.1% Kl was added into the drinking water to block thyroid gland activity. Mice-bearing MDA-MB-231, 4T1, or SK-BR-3 tumors were intravenously injected with 3.7 MBq of 125I-WT-pHLIP or 125I-Var3-pHLIP. Each group contained four mice. The mice were anesthetized by isoflurane inhalation, placed in the prone position, and scanned using a four-head SPECT/CT system  and dedicated multipinhole apertures with a diameter of 1.5 mm at 1, 2, 4, 24, and 48 h after injection of 125I-labeled peptides. CT images were acquired after static SPECT imaging (10 min/frame). The images were reconstructed using U-SPECT REC software and analyzed using PMOD software version 3.9 (Switzerland). Regions of interest (ROIs) were drawn in the tumors and organs, including the brain, heart, liver, lungs, kidneys, intestine, and bladder, at various time points. The radioactivity per gram (ID%/g) in the ROIs was measured to obtain the dynamic tumor-to-background ratio  and the in vivo distribution and kinetics of 125I-WT-pHLIP and 125I-Var3-pHLIP in the xenograft models of BC.Three days before micro-SPECT/CT imaging, 300 131I-Var3-pHLIP in the tail vein on days 0 and 3 (29.6 MBq each day). Groups 2, 3, and 4 were injected with the same volume of Var3-pHLIP, 131I (29.6 MBq), and saline solution, respectively. Tumor size was measured every 2 to 4 days postinjection (pi) for 26 days using a caliper. Tumor volumes /2, where a and b are tumor length and width, respectively. Relative tumor volumes were calculated as V/V0 . The body weight of mice was also determined.Twenty mice-bearing MDA-MB-231 human BC xenografts were divided into four groups of five animals. Group 1 was injected with The animals were sacrificed by cervical dislocation. The tumors were excised, fixed in paraformaldehyde, embedded in paraffin, and sectioned. The sections were mounted on glass slides, dewaxed in xylene, dehydrated through a graded ethanol series, and incubated in 3% hydrogen peroxide for 25 min. The samples were incubated with rabbit anti-human caspase 3  and rabbit anti-human Ki67  antibodies for 1 h at room temperature. Slices were washed with PBS thoroughly and incubated with HRP-labeled secondary antibody for 1 h at room temperature. Then, DAB kit was used for visualization and examined by light microscopy, and cells containing brown pigment were considered positively stained.p < 0.05.Data were analyzed using the SPSS version 20.0  and GraphPad PRISM version 7.0  and represented as means ± SD. Statistical significance was analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction and was established at High-performance liquid chromatography analysis showed that the concentration of the main constituent of WT-pHLIP was 91.4% with a retention time of 14.9 min, whereas the concentration of the main component of Var 3-pHLIP was 95.7% with a retention time of 9.5 min. Mass spectrometry analysis showed that WT-pHLIP had a peak at m/z 1135.4 and a molecular weight of 4536.1 Da, and Var3-pHLIP had a peak at m/z 1240.3 and a molecular weight of 3724.2 Da.125I-WT-pHLIP, 125I-Var3-pHLIP, and 131I-Var3-pHLIP was 99.6%, 98.9%, and 99.5%, respectively. At room temperature in PBS buffer, 125I-WT-pHLIP, 125I-Var3-pHLIP, and 131I-Var3-pHLIP had a stability of >95% at 24 h and >90% at 48 h. After 24 and 48 h at 37°C in serum, the stabilities were 86.8% and 86.6% for 125I-WT-pHLIP and 83.2% and 82.6% for 125I-Var3-pHLIP, respectively.The radioactive purity of Fluorescence imaging showed that the in vitro distribution of FITC-Var3-pHLIP and FITC-WT-pHLIP in the cell membrane decreased from pH 6.2 to pH 7.8 in the cell lines MDA-MB-231 Figures , 4T1, an125I-WT-pHLIP and 1.30 ± 0.10%, 12.3 ± 0.80%, 23.40 ± 0.44%, 23.88 ± 0.55%, and 24.90 ± 0.96% for 125I-Var3-pHLIP, respectively, (p < 0.0001) and 7.8 (p < 0.0001) suggesting pH-dependent binding of peptides to cells. Furthermore, with an excess of unlabeled peptide, the binding fractions of 125I-WT-pHLIP or 125I-Var3-pHLIP to MDA-MB-231 cells at pH 6.2, respectively, decrease to 3.11 ± 1.35% and 1.50 ± 0.11%, indicating binding specificity of 125I-labeled pHLIPs. Similar results were found for the trend of binding fraction of 125I-labeled pHLIPs to 4T1 or SK-BR-3 cells under pH gradient  but was not significantly different in 4T1  and SK-BR-3  xenografts.In vivo micro-SPECT/CT imaging at 1, 2, 4, 24, and 48 h after injection of  Figures . The TBR125I-Var3-pHLIP in tumors was the highest at 2 h pi  and decreased from 4 h to 48 h pi  (125I-WT-pHLIP (2.87 ± 0.60%ID/g), the maximum uptake of 125I-Var3-pHLIP was significantly higher in MDA-MB-231 xenografts at 2 h pi (p = 0.046). However, there was no statistical uptake difference of these two pHLIPs at the same time in 4T1  or SK-BR-3  xenografts.The distribution of  in 4T1) . Compare125I-Var3-pHLIP and 125I-WT-pHLIP was high in the heart, liver, lungs, bladder, and kidneys.The radioactive count in major organs or tissues of MDA-MB-231 tumor-bearing nude mice Tables  decrease131I-Var3-pHLIP-treated group (2.06 ± 0.47) than that in the groups treated with Var-pHLIP , 131I , and saline  at the end of treatment  and lower than that of 64Cu-Var3-pHLIP. Similarly, the maximum uptake of 125I-Var7-pHLIP was at 1 h pi [125I or 131I during the iodination of Var3-pHLIP using the chloramine-T method has little effect on peptide hydrophobicity.WT-pHLIP, Var3-pHLIP, and Var7-pHLIP are currently the most promising peptides of the pHLIP family. Positron emission tomography and fluorescence studies showed that, compared with WT-pHLIP, the distribution of Var3-pHLIP and Var7-pHLIP in the liver was lower probably because of their lower hydrophobicity at physiological pH. Var7-pHLIP is shorter and less hydrophobic than Var3-pHLIP, increasing clearance from the blood and liver but potentially decreasing its uptake by tumors , 14. The131I labeling is simpler than conjugating the above drugs to pHLIP. The present study showed that 131I-Var3-pHLIP inhibited tumor growth in MDA-MB-231 xenografts through ionizing radiation damage. However, treatment had some limitations. First, 131I-Var3-pHLIP was widely distributed in blood-rich organs such as the liver, heart, and lungs, increasing radiation damage to these normal organs. Second, 131I-Var3-pHLIP was cleared quickly from tumors, and its distribution in tumor tissue at 24 h pi was 12.5% of that at 2 h pi; thus, repeated injections are necessary to achieve the radiation dose required for effective treatment. Therefore, developing pHLIP variants with higher retention in tumor and faster blood clearance is necessary to improve the therapeutic efficacy and safety of 131I-labeled pHLIPs. Third, previous studies have shown that local inflammation was closely associated with increased acidity of extracellular microenvironment in the involved organs and tissues, which makes pHLIPs accumulate in the inflammatory diseases such as arthritis [The ability of pHLIP to target the acidic microenvironment of tumors allows its use as drug carriers. Studies have covalently bound therapeutic drugs that cannot cross the plasma membrane (phallacidin , amanitirthritis , pneumonrthritis , and actrthritis . Thus, iRadioiodine-labeled Var3-pHLIP effectively targeted breast cancer cells in an acidic environment and inhibited the growth of MDA-MB-231 xenografts by ionizing radiation.


Acute myeloid leukemia (AML) is an extremely aggressive form of blood cancer with high rates of treatment failure. AML arises from the stepwise acquisition of genetic aberrations and is a highly heterogeneous disorder. Recent research has shown that individual AML samples often contain several clones that are defined by a distinct combination of genetic lesions, epigenetic patterns and cell surface marker expression profiles. A better understanding of the clonal dynamics of AML is required to develop novel treatment strategies against this disease. In this review, we discuss the recent developments that have further deepened our understanding of clonal evolution and heterogeneity in AML.Acute myeloid leukemia (AML) is an extremely aggressive and heterogeneous disorder that results from the transformation of hematopoietic stem cells. Although our understanding of the molecular pathology of AML has greatly improved in the last few decades, the overall and relapse free survival rates among AML patients remain quite poor. This is largely due to evolution of the disease and selection of the fittest, treatment-resistant leukemic clones. There is increasing evidence that most AMLs possess a highly complex clonal architecture and individual leukemias are comprised of genetically, phenotypically and epigenetically distinct clones, which are continually evolving. Advances in sequencing technologies as well as studies using murine AML models have provided further insights into the heterogeneity of leukemias. We will review recent advances in the field of genetic and non-genetic heterogeneity in AML. More than a century ago Charles Darwin proposed the theory of evolution by natural selection of the fittest. In recent years, cancer researchers have accumulated evidence that this theory also applies to the ‘evolution’ of cancers ,2,3. DurAnimal models have often been used to study the genetic features of human cancers, to characterize mutations as well as to study their contribution to tumor formation, maintenance and progression ,19,20. AAML1/ETO, MYH11/CBFB, PML/RARA and MLL fusion genes, to name a few. However, nearly half of all AML patients have a normal karyotype and lack chromosomal abnormalities [RUNX1, CEBPA); those encoding signal transduction proteins such as FLT3, KIT and the RAS family of genes; genes involved in chromatin modification and epigenetic regulation including DNMT3A, IDH1/2, TET2 and ASXL1; genes of the spliceosome machinery  and genes encoding members of the cohesin complex , as well as genes, such as NPM1, which is the most frequently mutated gene in AML, that cannot easily be assigned to any of these rather broad functional categories [AML is a genetically heterogeneous disorder that arises from the malignant transformation of hematopoietic stem cells (HSCs) . Cytogenmalities ,7. In thmalities ,26. The tegories .FLT3, NPM1, IDH1/2, and the RAS genes, and to study the role of chromosomal translocations in AML [Although NGS studies have greatly contributed to elucidating the molecular pathogenesis of leukemias, not all mutations that are identified in a patient sample contribute to malignant transformation. Mutations that provide a proliferative and fitness advantage to a cell and contribute to the disease phenotype are called “driver” mutations, while those that have no effect on the growth and/or fitness of a cell are so-called “passengers” . The “pas in AML ,19. NPM1 or FLT3, alone have been shown to lead to myeloproliferative disorders (MPD) but not to AML, in vivo [FLT3 and NPM1 or in combination with other known driver mutations develop AML [MLL fusions or the CALM/AF10 fusion gene show incomplete penetrance and very long latencies suggesting that cooperating mutations are required for complete malignant transformation [AML1/ETO9a (a truncated isoform of the AML1/ETO fusion) and CALM/AF10 driven leukemias and shown that the mice acquire additional somatic mutations during the latency period [AML1/ETO was expressed in primitive hematopoietic stem cells (HSCs) of t positive patients who had achieved complete remission, these cells were able to differentiate normally and give rise to mature cells of the various blood lineages, thus indicating that the presence of the fusion alone is not sufficient for malignant transformation [AML1/ETO when expressed together with mutations in tyrosine kinases such as FLT3 and c-KIT leads to the development of AML [Murine models have also been an invaluable tool in understanding the contribution of a given genetic change to the development of leukemias. Studies in these models have provided evidence that a single genetic alteration is not sufficient to induce a full-blown leukemic phenotype. Some of the most common mutations detected in patient samples, including  in vivo ,31,32  tend to cooperate with each other, while mutations in genes with similar biological roles  are often mutually exclusive [From the studies mentioned above and other similar studies, it is now well established that AML is caused by the cooperation of several genetic aberrations that are acquired in a stepwise fashion. According to a hypothesis proposed by Gilliland and Griffin, as few as two cooperating mutations, each belonging to a different class, might be sufficient to induce AML ,43. Clasg others ,44,45. Rxclusive ,46.The number and kind of somatic mutations found in AML not only differs from patient to patient but can also vary considerably within the same tumor (intra-tumor heterogeneity), which is frequently very obvious when matched samples at diagnosis and relapse are studied. This heterogeneity within a sample is a result of evolutionary processes during the course of the disease. Like most cancers, AML is a clonal disorder, in which an initial transforming mutation leads to a transformed or malignant cell and the descendants of this cell form the founding clone. Individual cells of the founding clone will acquire additional genetic changes leading to the outgrowth of subclones ,27,47. DDNA sequencing and mutational profiling of AML samples has revealed two main types of clonal evolution patterns in AML, linear and branching evolution . In lineThe clonal complexity and the evolutionary history of a given AML sample is often determined from variant allele fraction (VAF) data obtained from massively parallel DNA sequencing of bulk tumor samples . The VAFAlthough studies using bulk patient samples have improved our understanding of the evolutionary histories of leukemias, these studies have their limitations as the data from bulk sequencing studies are often not sufficient to accurately reconstruct the clonal architecture and evolutionary patterns of a tumor. In the recent past, advances in single cell sequencing (sc-seq) technologies have enabled the study of mutations in AML with single cell resolution, which has facilitated a better understanding of the clonal architecture of AML ,50. In a+ BM cells were then analyzed using sc-seq to study clonal diversity. The study showed that the PDX models closely mimicked the clonal architecture observed in the patient samples. In one of the xenotransplanted samples, 11 of the 12 subclones detected in the original patient sample were also detected in the PDX samples. In the same study, two PDX models were established using samples from another patient who was found to have two AML subclones of similar clonal size. While both subclones had mutations in DNMT3A, ASXL1, STAG2, BCOR and U2AF1, they had acquired different RAS mutations. One subclone had an NRAS mutation, while the other had acquired a mutation in KRAS. In both the PDX models, the clone with the NRAS mutation was found to expand, while regression of the KRAS clone was observed, suggesting that the subclones had different proliferation potentials. Interestingly, similar clonal dynamics were observed in the actual patient on relapse [n = 57) and secondary (n = 27) PDXs using leukemic cells from 26 AML patients. They allowed engraftment and clonal evolution to proceed in the transplanted mice until the first signs of disease were observed. They then performed whole exome sequencing (WES) on the 26 patient samples (collected at diagnosis) and the 84 xenografts to study clonal dynamics of the leukemias. The authors identified five distinct patterns of clonal evolution in the PDXs. These included: “monoclonal leukemias”, which were defined as those samples in which the PDXs harbored the same individual clone detected in the corresponding patient at diagnosis; a “stable” leukemia wherein a dominant clone and a minor subclone were identified in both the patient and the PDX with similar frequencies; the “loss” pattern of evolution where the major clone at diagnosis was lost or detected at a much lower variant allele frequency (VAF) in the PDX models, and the parental clone which contained all but one or two of the AML mutations was found to be retained and was responsible for propagation of the leukemia; the “expansion” pattern wherein a minor subclone detected at diagnosis in the patient sample (average VAFs of 5% at diagnosis) was found to expand and become the dominant clone in the xenografts; and the “burst” pattern where a rare clone at diagnosis expanded in the primary xenografts but was lost in the secondary ones. The “burst” pattern of evolution was detected in 9% of the samples and revealed that the AML cells undergo continuous clonal competition in vivo, and some mutations may confer an initial proliferative advantage leading to expansion of a clone at the expense of long-term self-renewal [In addition to sequencing of patient samples at diagnosis and relapse, patient derived xenograft (PDX) murine models are often used to study clonal dynamics of leukemias. In the study by Morita et al., single cells isolated from three AML patients with branching evolutionary patterns were xenotransplanted into immunodeficient mice. Engrafted human CD45 relapse . Sandén -renewal . More imCALM/AF10  and AML/ETO9a, not only were the primary leukemic mice found to acquire several cooperating mutations that were identified via WES, but analysis of successive cohorts of serially transplanted leukemias revealed complex clonal evolution patterns. While in some murine leukemias a founding clone was detected that was found to further evolve on serial transplantation, analysis of other samples revealed a complicated pattern of losses and gains of mutations suggesting the expansion and contractions of leukemic subclones [Murine models established using other techniques, such as the MBMTLMs, have also been shown to mimic the clonal evolution patterns detected in AML patient samples. For instance, in MBMTLMs established using two fusion genes, the ubclones ,52. Inteubclones ,52. ThesDNMT3A, IDH1/2, TET2 and ASXL1 are predominantly acquired early on, during initiation of the disease. These mutations are frequently found at high VAFs indicating that they are present in virtually all tumor cells in a sample and represent the “founding clone” [FLT3 and RAS are found to occur later during disease progression. Consistent with these findings, sequencing of matched sample pairs at diagnosis and relapse has revealed that mutations in epigenetic modifiers are often retained after therapy and have also been found to persist in patients in complete remission, while mutations in FLT3 and RAS tend to be unstable and either expand or are lost in the relapse sample indicating that these mutations are subclonal [NPM1, one of the most frequently mutated genes in AML, are often thought to be disease initiating mutations, Shlush et al. demonstrated that in AML samples containing DNMT3A and NPM1c mutations, the DNMT3A mutation preceded the mutation in NPM1 [Despite the complex evolutionary patterns that have been identified in AML samples, recent studies suggest that mutations are most likely acquired in a specific order and the order in which mutations are acquired impacts the phenotype and clinical response of AML. Several studies have shown that mutations in genes encoding epigenetic regulators such as g clone” ,49,53,54ubclonal ,56. In aDNMT3A, TET2 and ASXL1 are not only detected in patients with hematopoietic malignancies, but have also been recurrently identified in samples from healthy people, especially in those obtained from elderly individuals [Intriguingly, recurring mutations in genes involved in epigenetic regulation, particularly in ividuals ,58,59. Tividuals . This phividuals ,59. The Dnmt3a mutation (corresponding to the R882H hotspot mutation in humans) and a Npm1 mutation (corresponding to the NPM1c mutation), respectively. The study showed that mice that were first induced for the Dnmt3A mutation followed by the Npm1c mutation developed MPD, which progressed to AML on subsequent transplantations. Notably, increasing the time between induction of the Dnmt3a mutation and that of the Npmc1 mutation resulted in a more aggressive MPD and a decreased overall survival in the mice suggesting that when the Dnmt3a mutant clone was allowed to expand for a longer duration, it increased the risk of progression to malignancy [To gain a better understanding of CHIP, Loberg et al. developed an in vivo dual recombinase system by combining interferon-inducible Cre and tamoxifen-inducible flippase recombinases, which enabled the sequential induction of a lignancy . This stSRSF2 gene (SRSF2P95), which encodes a splicing factor, and other driver genes can be correlated with distinct clinical phenotypes. When the SRSF2P95 mutation co-occurs with mutations in JAK2 or MPL myelofibrosis is observed. On the other hand, co-occurring mutations in TET2 and the RAS pathway genes lead to monocytosis and leukocytosis, respectively, while co-occurring mutations in STAG2, RUNX1 or IDH1/2 correlate with a blastic phenotype. Therefore, the diverse phenotypes observed in different patients with SRSF2P95-mutated neoplasms can be attributed to inter-patient mutational heterogeneity [+CD117dim” population that was positive for the HSPC marker CD34 and had low expression of CD117 , a “CD34+CD117bright (bri)” population which had high expression of CD117, and a population of “abnormal myeloid cells with partial maturation (AM)”. These compartments showed heterogeneous biological features such as variability in gene expression, morphology, proliferation ability and response to therapy. Interestingly, the CD34+CD117bri cells were less susceptible to chemotherapy than the other two populations. In addition, patients with a higher level of the CD34+CD117bri cell population showed worse overall and relapse-free survival than patients with fewer CD34+CD117bri cells. This suggested that a higher proportion of CD34+CD117bri cells in t AML patients might be associated with a poor prognosis and inferior outcomes [Leukemic cells are not only characterized by heterogeneity of their genetic lesions, but also show distinct phenotypes, including heterogeneous cell morphology and surface marker expression. A number of studies have demonstrated that phenotypic heterogeneity arises as a result of heterogeneity at the genomic level. For example, Todisco et al. have shown that the co-occurrence of a mutation in the ogeneity . In anotoutcomes . Overalln = 22) and relapsed AML (n = 25), the immunophenotype of the leukemic cells was found to change in the majority of the patients during the disease course. The most frequent changes observed were in the expression of CD13, CD33, CD56, CD7 (an early T-cell marker), CD4 (mainly expressed on helper T-cells) and CD11b (a myeloid marker) [Not only inter-patient but also intra-patient phenotypic heterogeneity is frequently observed in leukemia. In some patient samples, the pattern of the cell surface antigen expression profile of the leukemic cells has been shown to evolve from diagnosis to relapse ,66,67,68 marker) . The aut marker) .de novo AML revealed that different subclones of the same tumor gave rise to leukemias with distinct morphologies and phenotypes [The phenotypic shift in leukemia might result from the acquisition of new mutations and evolution of a pre-existing (sub)clone, from the emergence of a new clone at relapse, or the outgrowth of a minor (sub)clone that was present in the sample at diagnosis, which was resistant to therapy and expanded to drive relapse . Althougenotypes . For exaenotypes . Similarenotypes . These rNRAS and WT1, respectively, both of which evolved from a founding clone with a mutation in the CEBPA gene. The two subclones showed different cell surface marker expression profiles. The NRAS subclone was IL1RAP+ , whereas the WT1 subclone was IL1RAP−. In another patient, they found a subclone with a FLT3-ITD mutation, which had evolved from a founding clone with mutations in DNMT3A and RUNX1. Interestingly, the FLT3-ITD mutation was found to correlate with expression of CD25 (an activated T-cell marker) [FLT3-ITD mutations and CD25 expression has also been reported in other studies [Additionally, in a comprehensive study of 50 AML patients, de Boer et al. demonstrated that subclones sorted based on plasma membrane protein expression from each individual patient were genetically distinct. For example, in one patient, they identified two subclones with a mutation in  marker) . The cor studies . NPM1 or IDH1/2 genes correlated with reduced expression of the HLA-DR (human leukocyte antigen–DR isotype) and CD34 markers on leukemic cells. In contrast, TP53 mutations were linked to CD34 overexpression. Using these data, they also found a correlation between genetic and phenotypic evolution in their AML samples. For example, in one of the samples with a linear evolutionary pattern, they found that the stepwise acquisition of mutations in TET2, U2AF1, DNMT3A and NRAS was associated with phenotypic alterations. In this patient, a single mutation in TET2 correlated with the expression of both lymphoid  and myeloid (CD11b) markers, indicating that the TET2 mutation was most likely acquired in a pre-leukemic HSC. TET2-U2AF1 double-mutated cells had the expression of the same markers but at a lower level, as well as expression of early myeloid markers (CD123 and CD13). Then, on acquiring a DNMT3A mutation (TET2-U2AF1-DNMT3A triple-mutated cells), the cells were found to express the hematopoietic stem cell markers CD34 and CD117. Finally, after acquisition of a mutation in NRAS, the quadruple-mutated cells showed a myeloblastic phenotype , similar to the phenotype of the leukemic blasts [Furthermore, using simultaneous profiling of single-cell DNA and cell surface protein expression (scDNA + protein-seq), Morita et al. showed an interesting association between the mutational pattern and immunophenotypes in 26 AML patients. Their results indicated that mutations in the c blasts . CALM/AF10 driven MBMTLM established by our group, limiting dilution assays (LDAs) were performed and leukemias established from single leukemia stem cells  which correlated with the acquisition of a new subclone (subclone 3) and loss of subclones 1 and 2. The remaining two tertiary leukemias that were sequenced, each of which had originated from a single LSC, had high B220 expression and a much longer latency compared to the other leukemias, which correlated with an expansion in subclone 2 and loss of subclone 1. Surprisingly, an expansion of subclone 2 was observed only in these two samples, while in all other tertiary samples subclone 2 was lost [Similar observations with regard to genotypic-phenotypic evolution have also been made in murine models of AML. In a iewed in ) were chiewed in . The priwas lost . These rIn addition to providing insights on the complex evolutionary patterns, a probable mutational order and an extremely dynamic genetic and phenotypic landscape in AML, sequencing studies have also revealed that the overall mutational burden is relatively low in AML compared to other cancers ,71. FurtEpigenetic modifications are chemical changes to the DNA molecule or the chromatin proteins, which do not change the DNA sequence but influence gene expression, and include DNA methylation, histone modifications such as methylation, acetylation, phosphorylation and ubiquitinylation affecting chromatin remodeling. Epigenetic modifications can be stably transmitted through mitosis. However, unlike genetic aberrations, epigenetic marks show great plasticity and can change during normal development, during the course of a disease and in response to external stimuli. This often leads to epigenetic heterogeneity, which can be defined as genetically identical cells exhibiting distinct epigenetic states, leading to transcriptional diversity within a clonal population of cells ,75. AlonDNMT3A, TET2 and IDH1/2, as well those involved in histone modifications such as ASXL1 and EZH2. The dysregulation of these genes leads to aberrant methylation or other epigenetic patterns in tumor cells. Somatic mutations in TET2 or in the isocitrate dehydrogenase enzymes, IDH1/2, are often associated with hypermethylation signatures and subsequent inactivation of target genes that result in impaired hematopoietic differentiation. On the other hand, AML cases with mutations in the DNA methyltransferase enzyme, DNMT3A, have been found to exhibit hypomethylation and thereby activation of target genes. Mouse models of mutant IDH1/2, TET2 and DNMT3A recapitulate the epigenetic features observed in AML patients with these mutations, supporting their functional significance [CEBPA mutations, as well as those with the PML/RARA, AML1/ETO and MYH11/CBFB fusions, show specific patterns of methylation losses and gains compared to normal CD34+ BM cells [FLT3 and TET2 or NPM1 leads to an increase in epigenetic diversity in AML cells, and an enhanced epigenetic diversity has been associated with inferior patient outcomes [As mentioned in previous sections, mutations in genes encoding epigenetic modifiers are frequently found in AML and are also detected in pre-leukemic HSCs. The most common epigenetic mutations have been detected in genes involved in DNA methylation, which include ificance ,78. In aificance . SamplesBM cells ,77. Studoutcomes ,76,79.PU.1 and CEBPA transcription factor motifs and an increase in accessibility of the GATA and RUNX motifs [Interestingly, there is a growing body of evidence that suggests that epigenetic changes may also drive disease progression and relapse in AML, independent of underlying mutations C. This hX motifs ,80. PU.1CEBPA, that were aberrantly methylated at the pre-leukemic stage, indicating that the altered epigenetic state of these genes may play a role in the onset of AML [CEBPA locus has also been reported in human AML and has been proposed as a useful prognostic marker for AML patients [Alterations in DNA methylation patterns have also been associated with leukemic progression in AML murine models. Sonnet et al. examined the epigenetic profile of a mouse leukemia model driven by the hypomorphic expression of the PU.1 transcription factor . The stut of AML . Hypermepatients . The studies described above strongly suggest that epigenetic heterogeneity and plasticity are key drivers of leukemic transformation and disease progression, particularly during relapse. However, the mechanisms that mediate epigenetic heterogeneity and the complex interplay between genetic and epigenetic signals are not completely understood yet. The study by Sonnet and colleagues showed that mouse models of leukemogenesis can also be utilized to identify altered epigenetic states and study epigenetic evolution during leukemia progression. With advances in technologies such as ATAC-seq, it has now become possible to perform epigenetic analysis combined with genetic and transcriptomic analysis not only on patient samples but also in murine models. Such multi-omic studies of murine leukemia models at different stages of leukemia will further help us to understand the role of epigenetic reprogramming in the initiation and progression of AML. AML is a highly heterogeneous and aggressive malignant disease originating from hematopoietic stem cells. Although our understanding of the molecular basis of AML has improved greatly in the last four decades, overall survival rates, especially for older patients (>60 years of age), remain quite poor. Even though young patients initially respond to therapy and achieve complete remission, a majority of these patients eventually relapse ,83. In oAs technologies  of patient samples as well as murine leukemia models. Well characterized murine models will not only provide us with a deeper understanding on the underlying molecular pathogenesis of AML but can also be utilized to test novel drugs and treatment regimens in pre-clinical settings.


The patient was eventually diagnosed with acute myeloid leukaemia (AML) with mutated NPM1.An 86‐year‐old woman presented with fever and pneumonia. Her laboratory investigations showed leucocytosis 15 × 10⁹/L) with 92% blasts, anaemia (haemoglobin 5.5 g/dl), thrombocytopenia (platelet 31 × 10⁹/L), and elevation of fibrinogen degradation products (FDPs > 120 μg/ml). The patient refused bone marrow examination. Hypogranular blasts without Auer rods containing nuclear invagination, the so‐called “cup‐like” or “fish‐mouth” appearance, accounted for approximately 17% of the blasts in peripheral blood. Blasts were strongly positive for myeloperoxidase and showed normal female karyotype  [Figure 5 × 10⁹/LNPM‐1 and FLT3‐ITD mutations have been detected in more than 60% of CLB‐AML cases with normal karyotype. NPM1‐mutatd AML showing APL‐like immunophenotype, negative for both CD34 and HLA‐DR, often harbours co‐mutations in IDH1/2 or TET2 [NPM1 harbouring FLT3 or IDH1/2 co‐mutations [FLT3 mutation is less than 5% and much lower compared to that of NPM1 and IDH2, which means that FLT3 mutation may not affect the cup‐like morphology and clinical presentation in our case. NPM1‐mutated AML carrying concurrent FLT3‐ITD mutation especially with high (≥0.5) allelic ratio is well known to be associated with poor prognosis. Molecular analysis is very important in CLB‐AML with APL‐like phenotype in terms of predicting prognosis and determining the optimal treatment strategy including allogeneic stem cell transplantation or molecular targeted therapies such as FLT3 or IDH1/2 inhibitors.AML with cup‐like blasts (CLB‐AML) is defined as the presence of more than 10% of blasts with nuclear invaginations that span at least 25% of the nuclear diameter, which are commonly associated with AML M1/2 (myeloid) and M4/5 (myelomonocytic/monocytic) in the FAB classification. Y. Sugimoto has received honoraria from Novartis, funding from Takara Bio, and research support from Astellas, Kyowa Kirin, and Ono, and is an accepted researcher from Shojunkai Takeuchi Hospital. All other authors do not have any conflicts of interest to declare.


The intracellular Ca2+ concentration ([Ca2+]i) is about three orders of magnitude lower than the extracellular concentration, resulting in a steep transmembrane concentration gradient. Thus, the spatial and the temporal dynamics of [Ca2+]i are ideally suited to modulate Ca2+-mediated cellular responses to external signals. A variety of highly sophisticated methods have been developed to gain insight into cellular Ca2+ dynamics. In addition to electrophysiological measurements and the application of synthetic dyes that change their fluorescent properties upon interaction with Ca2+, the introduction and the ongoing development of genetically encoded Ca2+ indicators (GECI) opened a new era to study Ca2+-driven processes in living cells and organisms. Here, we have focused on one well-established GECI, i.e., GCaMP3.0. We have systematically modified the protein with sequence motifs, allowing localization of the sensor in the nucleus, in the mitochondrial matrix, at the mitochondrial outer membrane, and at the plasma membrane. The individual variants and a cytosolic version of GCaMP3.0 were overexpressed and purified from E. coli cells to study their biophysical properties in solution. All versions were examined to monitor Ca2+ signaling in stably transfected cell lines and in primary cortical neurons transduced with recombinant Adeno-associated viruses (rAAV). In this comparative study, we provide evidence for a robust approach to reliably trace Ca2+ signals at the (sub)-cellular level with pronounced temporal resolution.Calcium (Ca This is achieved by efficient transport mechanisms extruding Ca2+ ions from the cytosol to the extracellular side or to internal stores such as the endoplasmic reticulum or mitochondria i to low values since sustained increased [Ca2+]i may have detrimental effects causing cell damage or even cell death i. This interest triggered the development of a variety of strategies. The introduction of Ca2+-sensitive fluorescent dyes, such as Fura-2 or Fluo-4 [2+ signals in living cells and tissue [2+ transients with synthetic fluorescent dyes. Another important issue is that Ca2+-sensing fluorescent dyes allow imaging of [Ca2+]i only for a couple of hours after loading because cells continuously extrude or degrade them. These problems were overcome with the introduction of genetically-encoded Ca2+ indicators (GECI) i. Mitochondria are known to play a key role in controlling [Ca2+]i due to efficient sequestration and release mechanisms that have been intensively studied, e.g., with a variety of synthetic or genetically encoded Ca2+ indicators  . The finThe constructs were cloned into eukaryotic expression vectors and used for transient transfection of human embryonic kidney cells (HEK293) to monitor their subcellular distribution . In ordeAs expected, non-transfected cells did not emit fluorescent signals of GCaMP3.0, but the Golgi apparatus, ER, and mitochondria were specifically stained  to the N-terminus of the protein. The GCaMP3.0 variants were purified via Ni-NTA affinity chromatography and then applied to in vitro spectroscopic characterization.The constructs were cloned into pRSET A vector  that allows expression of the protein in 2+ affinity of the sensors was determined. Briefly, 2 µM protein were incubated with increasing concentrations of Ca2+ ([Ca2+]free). For each [Ca2+] the sample was excited at 470 nm and the fluorescence emission spectrum was registered. A representative example is depicted in In a series of experiments, the Ca2+]free. Fitting of the data resulted in the concentration–response curve displayed in 50 of 297 nM for GCaMP3.0nuc. Measurements for all other GCaMP3.0 variants followed the same regime. Calculation of mean EC50 ± SD are summarized in p = 0.06). Additionally, the sensors dynamic ranges, i.e., the ratio of (fluorescence (F) Fmax/Fmin) were different up to 1.2-fold  measured in each sample. The data show that at a higher pH, as it is present in mitochondria [2+]free. Measurements at pH 7.2 or 7.6 have almost no or only minor effects on the sensor’s response profile. The changed values for EC50 and the dynamic range of GCaMP3.0mito at a higher pH are considerably below 1 µM [Ca2+], and they will not prevent the detection of physiologically relevant Ca2+ signals in mitochondria  (right panel).Activation of GPCRs, however, does not always result in single transient Cahenomena ,55. In F2+] signals similarly well as the established GCaMP3.0cyto sensor or non-transfected cells loaded with Fluo-4. The mitochondrial construct, GCaMP3.0mito, showed a completely different response profile. Upon histamine application, the fluorescence signal increased, remained at an elevated value and then slowly returned to the basal level after histamine application was stopped  and 340 nm (Ca2+-bound form)  will lead to a decrease of fluorescence, opposite to increasing GCaMP3.0 signals. Similar to the previous experiments, Fura-2 loaded cells expressing either GCaMP3.0mom or GCaMP3.0mito were exposed to increasing concentrations of histamine  for 60 s intermitted by washing steps with histamine-free extracellular solution for 90 s. The application of different histamine concentrations was performed twice. During the first experiment, excitation at 960 nm was used to record GCaMP3.0mom and GCaMP3.0mito signals. During the second application with excitation at 760 nm, Fura-2 signals were registered. Representative experiments are shown in 2+-dependent fluorescence signals of both the GCaMP3.0 variants and Fura-2. The signals, however, differed with respect to their shape and their duration.We extended our comparative analyses and measured Cat 510 nm . Their and form) . With ou2+-dependent sensor fluorescence was detected with 0.5 µM histamine. Application of higher histamine concentrations caused oscillatory Ca2+-dependent signals. Binding of Ca2+ ions to Fura-2 causes a decrease of the fluorescence signal, when excited at 760 nm , fluorescence signals almost returned to base line values during the washing step see . At high2+-signals. Located at the outer membrane of mitochondria, the GCaMP3.0mom sensor registers changing [Ca2+]i in the cytosol as well. Thus, it was not surprising that both Fura-2 and GCaMP3.0mom were able to monitor the oscillatory Ca2+ responses initiated by histamine and caused by Ca2+ release from intracellular stores. In contrast, GCaMP3.0mito located within mitochondria responded with transient yet increasing Ca2+ signals, most likely reflecting influx of Ca2+ ions into the organelle serving as an alternative route in cellular Ca2+-homeostasis and nicely demonstrates the potential of targeted Ca2+-sensors.Since Fura-2 is homogeneously distributed in the cell, it primarily allows detection of cytosolic Ca2+ ions are intimately linked to neuronal signaling, we examined the suitability of all targeted GCaMP3.0 variants to report Ca2+ signals in primary cortical neurons. To deliver and express the sensors in primary neuronal cultures, we generated recombinant Adeno-associated viruses (rAAVs). In a previous study i, both in cell-based pharmacological assays as well as in individual cells and neurons. Basic biophysical and biochemical properties of the variants were determined on bacterially expressed and affinity purified proteins.Here, we have generated and characterized subcellular targeted versions of the genetically encoded Ca2+ signals induced by, e.g., GPCR activation, we decided to employ GCaMP3.0 known as a robust Ca2+ detector with pronounced sensitivity and a dynamic range i as long as the ligand was present. Other cells showed single transient responses or oscillations on top of elevated Ca2+ levels. These response repertoires were reminiscent of GPCR-induced signaling reported previously i than, e.g., GCaMP6 or GCaMP7 versions that have been developed for detecting even single Ca2+ sparks in neurons free) were registered. Purified protein was diluted in Ca2+ free buffer (30 mM 3-(N-Morpholino)propansulfonic acid (MOPS), 100 mM KCl, 10 mM K2EGTA, pH 7.2) to a final concentration of 2 µM. By adding a Ca2+-containing buffer  the [Ca2+]free was successively increased. Values of [Ca2+]free were calculated with WEBMAXC ). Samples were excited at 470 nm and emission spectra were recorded between 480 and 600 nm with a QM-4 spectrometer . Data analysis was performed with Graph Pad Prism 5.04 . Determination of GCaMP3.0 fluorescence at pH 7.6 and 8.0 was according to a protocol established by i in single cells, two-photon-laser scanning fluorescence microscopy measurements were performed. Details of the setup are described in [2+]i were registered either in Fluo-4 or Fura-2 loaded cells or in cells expressing GCaMP3.0 variants. Loading with Fluo-4 was in extracellular solution ) supplemented with 1.7 µM Fluo-4 AM or 2 µM Fura-2 AM , 3 mM probenecid, and 0.02% (w/v) pluronic F-127 (Sigma-Aldrich) for 30 min at room temperature. For stimulation of endogenous Ca2+ signaling cascades, cells were superfused with ES solution containing different histamine concentrations or high K+ (55 mM). Two-photon excitation of samples was at 760 nm (Fura-2), 800 nm (Fluo-4), or 920 nm (GCaMP3.0). Fluorescence emission was registered at wavelengths > 500 nm. For each sample 70 to 800 images were registered with scanning speeds between 0.5–1 images/sec. Images contained 256 × 256 to 512 × 512 pixels. Data were analyzed with Image J (https://imagej.nih.gov/ij/ (accessed on 21 March 2011), 1997–2018; National Institute of Health, Bethesda, Maryland, USA). A region of interest (ROI) was defined. The mean fluorescence intensity was calculated for all individual images of a sample and plotted versus time (=duration of the experiment). Using the maximal change in fluorescence evoked by the different ligand concentrations, concentration–response curves (∆F vs. ligand concentration) were calculated using Graph Pad Prism 5.04.To monitor changes of [Caribed in . Cells w2, and 95% humidity in a 500 µL medium. Primary cortical cells were transduced with rAAVs on day in vitro (DIV) DIV8 with different multiplicities of infection (MOI). The medium (500 µL) was completely exchanged with medium containing 107, 108, 109, 1010, or 1011 viral genomes to achieve MOIs of 2·102, 2·103, 2·104, 2·105, and 2·106 viral particles per cell (vp/cell), respectively. As a control, the same volume of 40% Iodixanol was added to individual wells. Three days post transduction (DPT), 100 µL medium was added. Cells were assessed for functional measurements or fixed for immunostaining on DIV14.Primary cortical cultures were prepared as previously described ,80. Briew/v) paraformaldehyde in PBS (PFA) at RT for 20 min and washed with 1x PBS (pH 7.4). Unspecific binding sites in HEK293 cells were blocked for 30 min in a phosphate buffer containing 0.5% (w/v) Triton-X100 and 5% (w/v) chemiblocker . Unspecific binding sites in neurons were blocked overnight in PBS containing 1% (w/v) BSA and 2% (v/v) heat inactivated goat serum. The following primary antibodies were used: rabbit (rb)-anti-calnexin  and (rb)-anti-MAP2 . Primary antibodies were visualized using donkey (dk)-anti-rbCy3  and dk-anti-rbAlexa(A)594  fluorescently-labeled secondary antibodies in blocking solution. Nuclei were stained with TOPRO-3 . Mitochondria were stained with 100 nM MitoTracker  for 20–30 min at 37 °C, 5% CO2, and 95% humidity immediately before fixation of the sample with 4% PFA. Samples were mounted in Aqua-Poly/Mount  and examined using a Confocal laser scanning microscope .Cells used for immunostaining were grown on cover slips, fixed with 4% (


Moreover, accumulating evidence indicates that the endoplasmic reticulum (ER) is extensively present in axonal terminals of neurons and plays a modulatory role in synaptic transmission by regulating Ca2+ levels. Familial Alzheimer’s disease (FAD) is marked by enhanced Ca2+ release from the ER and downregulation of Ca2+ buffering proteins. However, the precise consequence of impaired Ca2+ signaling within the vicinity of VGCCs (active zone (AZ)) on exocytosis is poorly understood. Here, we perform in silico experiments of intracellular Ca2+ signaling and exocytosis in a detailed biophysical model of hippocampal synapses to investigate the effect of aberrant Ca2+ signaling on neurotransmitter release in FAD. Our model predicts that enhanced Ca2+ release from the ER increases the probability of neurotransmitter release in FAD. Moreover, over very short timescales (30–60 ms), the model exhibits activity-dependent and enhanced short-term plasticity in FAD, indicating neuronal hyperactivity—a hallmark of the disease. Similar to previous observations in AD animal models, our model reveals that during prolonged stimulation (~450 ms), pathological Ca2+ signaling increases depression and desynchronization with stimulus, causing affected synapses to operate unreliably. Overall, our work provides direct evidence in support of a crucial role played by altered Ca2+ homeostasis mediated by intracellular stores in FAD.Neurotransmitter release from presynaptic terminals is primarily regulated by rapid Ca This specialized cascade underscores the importance of IP3Rs and RyRs in the regulation of the Ca2+-induced Ca2+ release (CICR) mechanism of the ER. Previous works have shown that CICR is necessary for ER stores to adequately influence spontaneous vesicle release and homosynaptic plasticity , on the other hand, accelerates the recruitment of release-ready vesicles, controlling the degree of synaptic plasticity  that extends over more than 100 nm  H,I. Prev WT mice . In part neurons H,I. Thus2+ sensors , which permit direct presynaptic recordings and are reported to have high structural similarities with their hippocampal counterparts  and Normal Coupling (NC) between the ER and AZ. The HC configuration corresponds to the downregulation of Ca2+ buffering proteins  by counting the number of vesicles released from the slow and fast release-ready pools (RRP), divided by the number of vesicles initially in both pools. In the FAD-affected synapse, we observe enhanced Pr over a wide range of VGCC expression in a dose–response manner  enhanced Ca2+ release through IP3Rs but NC (AD-NC), (2) enhanced Ca2+ release through IP3Rs with HC (AD-HC), (3) normal Ca2+ release through IP3Rs and NC (WT-NC), and (4) normal Ca2+ release through IP3Rs and HC (WT-HC). We notice that while HC causes a minor increase in the release probability and vesicles released in the WT synapse, it strongly affects both these features in the FAD-affected synapse  on Pr  at AZ in the FAD-affected synapse consistently across a wide range of VGCC expression, in agreement with several studies showing that ER Ca2+ channels can sculpt the spatiotemporal dynamics of exocytosis and consequently neuronal function . STP is assessed by determining the paired-pulse ratio (PPR): a classical measure of presynaptic modulation in response to paired stimuli separated by very short time intervals  on the baseline probability  selectively diminishes the reliability of intermediate-Pr synapses, suggesting that low and intermediate Pr synapses are more susceptible to FAD-associated Ca2+ disruptions. Importantly, the model predicts that the aberrant Ca2+ rise in AD-affected neurons may trigger hyperactivity over very short timescales (~30–60 ms) and lowers facilitation during prolonged (~450 ms) stimulation. We also report enhanced excitability in pathological synapses when simulated with higher coupling, which corresponds to a tighter feedback loop between the ER and AZ. Thus, our findings here provide a plausible explanation for why alterations in ER Ca2+ handling, which result in excessive efflux, induce a severe perturbation of neuronal processes that can in turn decrease the reliability of information encoded in the firing rate of neurons affected by FAD pathology  and, in turn, influence spontaneous release: a miniature form of exocytosis  from the ER and in turn increases the Pr.Many explorations into the Caocytosis ,29. Althpocampus ,116. In asticity . Shorterfunction ,119,120.r and PPR but suggests that the enhanced STP over rapid timescales observed in FAD merely indicates hyperactivity rather than increased synaptic reliability [2+ dynamics, which consequently causes them to exhibit enhanced excitation in a paired-pulse protocol. The traditional view claiming the massive reduction in the efficacy of excitatory synaptic transmission in early stages of AD has been challenged recently by several studies reporting aberrant Ca2+ homeostasis and hyperactivity in AD-affected neuronal networks [2+ release from intracellular stores plays an important role in the history-dependent neuronal hyperactivity observed during early stages of FAD.Consistent with experimental evidence, our model reproduces the well-established inverse relationship between the Piability ,121. Thenetworks ,100,101.2+ sensors controlling the timescales of synchronous and asynchronous release in CA3 pyramidal neurons, respectively [2+ affinity of Syt1 enable the remarkable temporal precision of synchronous release, where most vesicles immediately fuse with the membrane following stimulation. On the other hand, the slow kinetics of Syt7 promotes the progressive desynchronization of release later in the spike train [Our model shows that long high-frequency pulses trigger depression in FAD-affected synapses, which is governed by the depletion of RRP of vesicles. Experimental evidence reports three distinct molecular pathways for exocytosis . Indeed,ectively ,122. Theke train . Studieske train ,125,126.r range for hippocampal synapses, although lower-Pr synapses exhibit relatively elevated temporal precision in both WT and FAD-affected synapses. Thus, despite the high fidelity of hippocampal synapses, their conspicuously low response success may make them more susceptible to AD pathology. We hope that future experiments will uncover the molecular mechanisms underlying the pathological enhancement of susceptibility in low-Pr hippocampal synapses.CA3-CA1 presynaptic terminals are equipped with a conspicuously low release probability that allows them to maintain a delicate balance between facilitation and reliability, giving rise to finely tuned rate codes with remarkable temporal precision. Thus, perturbations of release mechanisms are expected to alter the fidelity of neural rhythms in AD. Indeed, cortical neurons with Aβ peptide expressed reduced N-methyl D-aspartate (NMDA) receptor density, resulting in the rapid and persistent depression of NMDA-evoked currents . Moreove2+ influx at the axon terminal [r, our model falls short of the measured values, suggesting that we cannot eliminate additional mechanisms when investigating the interplay between residual Ca2+, facilitation, and depression. Although IP3R-dependent modulation of cytosolic Ca2+ is usually adequate for explaining the critical aspects of ER Ca2+ release and regulation of neurotransmission, the upregulation of RyR expression and modulation of IP3Rs’ gating due to Ca2+ release through RyRs (and vice versa) are also thought to play a key role in the aberrant Ca2+ release from the ER, as well as the propagation of presynaptic signals [2+ signaling alterations that promote synaptic depression [2+ transients that can modulate LTP/LTD [2+ permeable leak channels formed by Presenilins that are disrupted in FAD-affected neurons [2+ gradients in synaptic terminals. Synaptic mitochondria are among the earliest targets in AD. Among other aspects, the ATP production and Ca2+ buffering capacities of mitochondria are severely disrupted [We remark that while our model is developed to be consistent with most observations in the hippocampal CA3-CA1 synapses, it does not incorporate the uneven distribution of P/Q-type, N-type, and R-type VGCCs specifically found in the AZ of central synapses ,127. At terminal . Thus, sterminal . Furtherterminal ,129. Ourterminal ,94. Thus signals ,22,130.  signals ,131. Furpression . However LTP/LTD ,133. Pre neurons . Anotherisrupted ,136. All2+ homeostasis in the axonal terminal and explore how exocytosis is affected in FAD. Motivated by the difficulty in probing signaling cascades at the AZ of small hippocampal synapses, our main goal was to build a comprehensive but simple framework for unravelling the role of enhanced Ca2+ release from the ER in SVR during FAD pathology. In addition to agreeing closely with several observations about the kinetics of IP3Rs, SVR, and synaptic plasticity in both WT and diseased synapses, our modelling work provides key insights into impaired presynaptic function in FAD. Specifically, we make five key predictions: r in response to a single AP is upregulated in FAD-affected synapses;The overall PShort-lived low-frequency stimuli promote potentiation in FAD-affected synapses;During sustained high-frequency stimulation, FAD-affected terminals exhibit enhanced depression;FAD-affected synapses operate less reliably, andr. The effect of FAD pathology is exacerbated in synapses with low to intermediate PIn summary, we have leveraged diverse experimental data to model Ca2+ dysregulation and loss of synaptic integrity in FAD, SVR therapeutics remain underdeveloped. For example, the current lack of validated in vivo biomarkers that can be used as proxies of synaptic dysfunction has directed most translational efforts towards indirect measures of synaptic integrity in AD [2+ dysregulation to provide better insights into the disease pathogenesis and therapeutic directions. Taken together with the aforementioned limitations, our analysis highlights the need for further studies investigating the role of perturbed Ca2+ signaling due to intracellular organelles such as the ER and mitochondria in cognitive deficits associated with AD and other neurodegenerative diseases.Although extensive experimental evidence corroborates our findings and demonstrates the significance of ER Caty in AD . Our fin


R, a cisplatin-resistant sub-line of SiHa and 3-methylcholanthrene–induced cervical cancer mice models. SiHaR exhibited higher MRP2, p-AktThr308, NF-κB, XIAP, and survivin expressions which cumulatively compromised cisplatin retention capacity and accumulated PEITC better than SiHa. SiHaR appeared to favor PEITC uptake as its accumulation rates were found to be positively correlated with MRP2 expressions. PEITC treatment in SiHaR for 3 h prior to cisplatin exposure revived intracellular platinum levels, reduced free GSH levels, generated greater ROS, and altered mitochondrial membrane potential compared to SiHa. Western blot and immunofluorescence results indicated that PEITC successfully downregulated MRP2 in addition to suppressing p-AktThr308, XIAP, survivin, and NF-κB expressions. In mice models, administration of 5 mg/kg body-weight PEITC priming dosage prior to treatment with 3 mg/kg body-weight of cisplatin remediated cervical histology and induced tumor regression in contrast to the group receiving the same dosage of cisplatin only. This suggested PEITC as a potential chemosensitizing agent in light of acquired cisplatin resistance in cervical cancer and established its candidature for Phase I clinical trial.Acquired cisplatin resistance in cervical cancer therapy is principally caused by reduction in intracellular drug accumulation, which is exerted by hyperactivation of the oncogenic PI3K/Akt signaling axis and overexpression of cisplatin-exporter MRP2 along with prosurvival effectors NF-κB and IAPs in cervical cancer cells. These activated prosurvival signaling cascades drive drug efflux and evasion of apoptosis for rendering drug-resistant phenotypes. Our study challenges the PI3K/Akt axis in a cisplatin-resistant cervical cancer scenario with phenethylisothiocyanate (PEITC) for chemosensitization of SiHa Cervical cancer, the fourth-leading cause of morbidity among women worldwide , is reported to cause maximum deaths among Indian women . Relapse and recurrence catered by failure in treatment owing to acquirement of resistance to chemo/radiotherapy is a common occurrence . ConventCisplatin  renders its action by attacking DNA to generate complex irreparable DNA adducts following ‘intracellular hydrolytic activation’ . PleomorThe Phosphatidylinositol 3 Kinase/Protein Kinase B or Akt (PI3K/Akt) signaling axis plays a pivotal role in conferring cells with CDDP-resistant properties by metabolically transforming them for reducing drug accumulation . AKT/pro. PEITC chemosensitizes many cancer cells by scavenging free radicals  potentiate apoptosis by negatively regulating prosurvival molecules . Reportsradicals . In biliof Mcl-1 . Additioof Mcl-1 . Thus, m2( ∼ 5%) incubator.SiHa cells were maintained in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics  at 37°C in a humidified COR) was developed from the parental SiHa cell line by ‘pulse treatment’ , was used in this study  treatment of virgin female Swiss Albino mice , each consisting of ten animals separated into two batches [No. of mice (n) = 5/cage x 2]. Group I was kept as an “untreated” control group where mice did not receive any treatment. Invasive cancer-bearing mice were randomized into Group II (no intervention), Group III (3 mg/kg body weight CDDP), Group IV (2.5 mg/kg body weight PEITC), and Group V (PEITC followed by CDDP). These doses were selected after proper dosimetry. During the concurrent chemocycles of two weeks, body-weight fluctuations and tumor-growth alterations were recorded periodically. Food and water were provided R cells after exposure to a wide range of PEITC (SIGMA Aldrich) and CDDP (CIPLA) concentrations as per the detailed description of the process provided in The MTT assay was performed in SiHa and SiHaR following 1, 2, 3, 4, 5, and 6 h of its administration was undertaken by the cyclocondensation assay (R cells were treated with 2 µM of PEITC (maximum tolerated dose or MTD). PEITC concentrations were determined by spectrophotometry against a standard curve , and acid-digested in concentrated nitric acid at 60°C (The frozen cell pellets (-20°C) of SiHa and SiHa at 60°C  for 2 h.R was performed following the trypan blue dye exclusion method. Accordingly, the cells were pelleted down by centrifugation at 1,500 rpm for 5 min after trypsinization. Equal volumes of cell suspension and 0.4% trypan blue stain were thereafter incubated for 1 min, followed by differential counting of the live and dead cells under a Phase Contrast Microscope (Olympus) using a hemocytometer. The results were graphically (% viable cells vs. treatment points) represented. The experiments were repeated thrice.Calculation of cell viability in SiHa and SiHaThe dissected mice cervix tissues were washed in cold normal saline (0.87%), fixed in 10% neutral buffered formalin , and processed for histology sectioning .R were seeded in 55-mm plates (2.5 × 106 cells) and over coverslips in 6-well plates (2.5 × 105 cells) for flow cytometry and microscopic analysis, respectively. The plates were trypsinized and incubated with 10 μM 2′,7′-dichlorofluorescein dihydroacetate  for 45 min, followed by flow cytometric analysis in FL1-H. Scatter plots and histograms were generated in replicates using Cell Quest software. Respective coverslips from the corresponding 6-well plates were scanned for qualitative analysis of generated ROS using an FITC filter under a fluorescent microscope (Leica).Intracellular ROS was quantitatively estimated by flow cytometry  followed by qualitative analysis under a fluorescent microscope. Equal densities of SiHa and SiHaR cells, seeded in densities of 2.5 × 105 cells/well in 6-well plates, were stained with 5 μg/ml of rhodamine (Rh-123) and incubated at 37°C for 30 min following PEITC and CDDP treatment. The results were generated spectrofluorimetrically  and represented graphically (fold-change) for each replicate point.SiHa and SiHaFree glutathione reductase [E.C1.8.1.7] levels were spectrophotometrically assessed as per the protocol of the glutathione assay kit  for triplicate experimental sets.The expression status of prosurvival effectors  and cisplatin exporter pump (MRP2) was comparatively studied by Western blotting after standardized laboratory protocol . The parIsolation of total cellular RNA was performed using TRIzol reagent (Invitrogen). cDNA was synthesized from 2 μg of total RNA using a RetroScript kit (Ambion/Applied Biosystem) which was amplified by PCR using respective forward and reverse primer sequences . The PCR5 cells/well) onto coverslips placed within 6-well plates for performing immunocytochemistry. PEITC and CDDP-treated cells were immunostained with respective antibodies and fluorophore-tagged secondary antibodies as per laboratory protocol  generated due to chronic treatment with 3 MC in animal models were spectrofluorimetrically quantitated according to the protocol given by AutoDock Vina  was utilp-value < 0.0001 was considered statistically significant.The mean values of the PEITC, CDDP, and PEITC + CDDP points were compared by factorial analysis of variance (ANOVA). The relationship between the studied parameters was analyzed by calculating Pearson’s correlation coefficient using the CORREL function of Microsoft Excel. Data were expressed as mean ± standard deviation (S.D.) A R, developed by “pulse treatment” of SiHa, displayed terminal resistance to the parental IC30 (3.5 µM) dose of CDDP , IC30 (3.5 µM), and IC50 (4 µM) CDDP concentrations for 24, 48, or even 72 h, respectively. Death of SiHaR cells in comparison to SiHa at IC70 (8 µM) and IC100 (11.5 µM) CDDP doses was notably less  compared with that of SiHa (367.97 µm2). As per Western blot results (R expressed remarkably higher levels of MRP2 (2.1 fold) and p-AktThr308 (1.91 fold), unlike the parental SiHa. A significantly higher ratio of p-Akt/Akt (1.7 fold) in SiHaR cells  indicated the contributory role of the upregulated PI3K/Akt pathway in the acquirement of the CDDP-resistant phenotype.Furthermore, a comparative morphological characterization of SiHa and SiHamination  vividly  results , SiHaR eR cells  indicatR cells. The MTT assay was therefore undertaken following treatment of cells with PEITC doses ranging between 0.5 and 3 µM for 3–12 h (data not shown). Accordingly, the time-point of 3 h was selected  than SiHa  for 3 and 4 h.  for exploring the association between their PEITC accumulation and CDDP-retention capacities  from 98.5% upon PEITC treatment ahead of CDDP treatment , total-Akt, XIAP, survivin, NF-κB (p65), NF-κB (p50), and MRP2 in combinatorial treatment modalities of PEITC and CDDP in comparison to single-agent treatments and untreated SiHaR. Respective band intensities  for chemoenhancing CDDP, the SiHa results  depictedensities  also revensities . This inensities  suggesterographs , PEITC rreatment .PEITC was screened for the top-ranked poses based upon the docking score and non-bonded contact potential with the target protein conformations. Docking results  delineatin vivo invasive cervical cancer model was explored by intraperitoneal administration of CDDP and PEITC either in combination or singularly  and subsequently decreased its weight  among the treatment groups. Nevertheless, sole treatment with 3 mg/kg body weight of CDDP failed to reduce the tumor size as the tumor weight remained unaltered, reflecting the acquirement of potential drug-insensitive nature. The results corroborated with the harmonizing patterns in their relative body weight fluctuations  supported the abovementioned viewpoints and aligned with pre-existing reports  promote the intracellular import of isothiocyanates . PEITC, eventive , and cheeventive . Correla reports . The premulation  as the dmulation . Among SR particularly. It was observed that the phosphorylation status of Akt had remarkably reduced in SiHaR with PEITC followed by CDDP treatment. Expression profiles (protein/mRNA) of downstream effectors of the signaling cascades aligned with those of p-Akt. The expression profiles of NF-κB (p50/p65) decreased, resulting in cumulative inhibition of XIAP, survivin, and MRP2. PI3K/Akt is reported to ubiquitously modulate the multidrug-resistant phenotype in cancer (R cells considerably reduced MRP2 accumulation in the membrane even upon CDDP treatment. This unveiled the root cause of increasing CDDP levels within the resistant cells upon PEITC pretreatment. A milieu of reference studies also reported that apparently, PEITC reduces the expression of drug exporters to promote the reversal of acquired chemoresistance (In silico observations pinpointing at specific interactions of PEITC with Akt, XIAP, and MRP2 proteins further established the role of PEITC as a chemosensitizer.Considering the pioneering role of the PI3K/Akt signaling cascade in orchestrating the scenario of acquired CDDP resistance in cervical cancer, it was intended to concentrate upon the ways in which PEITC modulated this cascade for altering MRP2 distribution in SiHan cancer . Therefosistance . In siliin vivo cervical cancer model, reiterations of similar results were attained. The mice group when treated with CDDP alone did not restrict the tumor growth, as evident from histopathological study and tumor images. However, prior PEITC administration alongside CDDP injection controlled tumor growth and improved the relative histology and cytology by permissibly surging systemic-ROS levels. These aggregated evidences were enough to confirm PEITC as a CDDP sensitizer in cisplatin-resistant cervical cancer. Although, detailed insight into PEITC-mediated cisplatin sensitization in in vivo set-up is mandatory. The present study has laid down a foundation for the candidature of PEITC as a cisplatin sensitizer and enhancer in Phase I clinical trial.Upon validation in a 3 MC induced


There have been a number of federal policies and guidance’s impacting diversity, equity, inclusion, and accessibility (DEI) in clinical research. While these are needed, they have not diminished the gaps related to clinical trial recruitment, research professional’s capacity for cultural competence, and clinical research professional role development. Mentoring and co-mentoring circles have traditionally been used in Medicine, but until now had not been used for workforce development of clinical research professionals (CRPs).We designed a six-session, monthly co-mentoring circle to take place at two academic medical centers to pilot an interinstitutional co-mentoring circle centered on storytelling videos of Black Voices in Clinical Research. This provided a DEI framework for discussions on role experiences, cultural competence, and role progression.Discussion: Co-mentoring circles can be useful tools to connect CRPs across complex research medical centers and provide support that may have a positive impact on role satisfaction and retention.Seven CRPs completed the DRC pilot. The participants positively evaluated the experience and made recommendations for future iterations. This framework for developing co-mentoring circles can serve as a toolkit for future CRP co-mentoring circles within and across institutions for workforce development. The Black Voices in Clinical Research storytelling videos provide a rich foundation for future discussion on DEI issues for CRPs and collaborating with participants. Since 2017, there has been an increasing series of policies and initiatives that address diversity, equity, inclusion, and accessibility (DEI) issues for clinical research workforce, workforce education and training, and participant recruitment   Revitalization Act required NIH-funded clinical trials to include women and minorities as participants , but thoClinical research professionals (CRPs) regularly face significant barriers to successful enrollment of women, children, underrepresented minorities, LGBTQIA+, and those with diverse abilities –18. EducTo ensure robust cultural competency in the workforce, it is important to have a working environment that encourages CRPs to become competent communicators and actors in the intersectional areas of diversity and culture. Cultivating a culturally competent workforce begins with valuing, supporting, and addressing the unique challenges experienced by each individual. Though all new professionals may encounter obstacles to gain employment in the field, staff from diverse backgrounds more often encounter unique barriers related to DEI challenges. Additionally, while there may be varying levels of generic support structures in place to help advance new careers, diverse staff encountering DEI specific challenges typically do not have focused supportive structures in place to further encourage their careers .3) that provides training to improve DEI. Using an innovative approach, the UF DC3 developed a series of storytelling videos entitled: Black Voices in Research [Several initiatives have come to fruition that address health inequities and aid in improving awareness and best practices. The Society for Clinical Research Sites has identified a Diversity Site Assessment Tool that can aid in awareness and benchmarking ,22. BeyoResearch . StoryteCo-mentoring is the merging of collaboration and mentoring . MentoriCo-mentoring circles are a type of group mentoring usually involving a small group of mentees (5 to 7 members), organized around a facilitator, and focused on topics supporting learning and development within an organization. Fostering these circles promises to create a more egalitarian mentoring culture, especially for CRPs who often lack any form of mentoring in their careers outside of their direct supervisors. CRP staff collaborate and engage in co-creating the circle. Co-mentoring circles draw on a model of relational mentoring which allows a “full range of processes, mechanisms, and outcomes of developing relationships” (p. 374) based on earlier work on Relational-Cultural Theory (RCT) . The fou3 Black Voices in Clinical Research [To explore an alternative workforce development strategy to address DEI, we created and piloted a collaborative DEI co-mentoring circle for CRPs working at two CTSA Hubs, aiming to create a model which stimulates reflection as well as sharing and solution finding for DEI issues of the workforce in clinical research management work. We used the DCResearch  as a bas3 Black Voices in Clinical Research, 2) stimulate reflection and discussion on issues resonated from the videos, and 3) discuss the participant’s own stories about entering and progressing in clinical research professional roles.We conceptualized the collaborative co-mentoring circle approach for DEI training for CRPs by assembling a team from both University of Florida CTSI and the Ohio State University Center for Clinical Translational Science (CCTS). Our steps to developing the co-mentoring circle is illustrated in Fig. We developed a project charter, activity plan, survey instruments, and recruitment plan. We also developed a facilitator guide with scripts and pre-work assignments. Finally, we drafted a co-mentoring circle agreement for participants. Our circle’s agreement included common commitments for co-mentoring circles Table . ParticiBecause of the reflective nature of the co-mentoring circles, individuals were encouraged to reflect and journal about topics raised during the meetings.We designed the meeting plan to allow rotation of facilitation by different facilitators from University of Florida (UF) and The Ohio State University (OSU) to avoid the appearance of a single “lead.” At project launch, we provided facilitators with instruction on how to lead a co-mentoring circle session to insure participant engagement, safety, and prompt discussion. Using focus group approaches, facilitators used a script to start the conversations and to prompt discussion without “leading,” and encouraged to share as a member of the circle without inserting a personal agenda or authority. This enabled a safe space for participants to view themselves as co-mentors who were co-creating the process through sharing and reflection. We held the sessions every four weeks, via Zoom, for six sessions.Our intake survey included quantitative and qualitative responses including demographics, experience level in clinical research, mentoring experiences, how they entered and progressed from their first role in clinical research, satisfaction in role progression, institutional factors, and any contemplations about leaving the institution. Descriptive statistics were planned to evaluate the initial intake survey and served to set stage to understand the group.The mid-point and exit surveys served to evaluate participant experiences in the process and satisfaction with the co-mentoring circle. Descriptive statistics were planned to evaluate these two surveys. The final session also served as an evaluation dialogue with participants about what worked well? What did not? What do they suggest for future co-mentoring experiences? What expectations were or were not met?In total, we had ten co-mentoring circle participants including one facilitator from UF and two from OSU. Seven of the participants, including facilitators, completed the intake survey. None of the participants had ever participated in a co-mentoring circle or a traditional mentoring experience. The demographic constitution of our group was primarily biracial, multiracial, or Black/African American. Half of the participants were Hispanic or Latino, and the majority of participants were female. In this cohort, there were varying levels of education and experience from bachelor’s degrees to Ph.D. candidates and levels of experience ranging from five years’ experience to more than two decades. As is common in most institutions, there were varying job titles.“A friend told me about a job. I got the job through word of mouth.”“The University posted an ad and I applied and got the job.”“I wanted to move to Gainesville FL, so I searched for jobs online and applied.”We asked about ease of getting a first job in clinical research. Three individuals rated it difficult (1) or very difficult (2) to get their foot in the door for their first job. Qualitatively, those who did not have difficulty shared how they got their first job:The respondents did recognize the institutions dedication to improve CRP diversity; however, for the other questions on role progression, performance review, workforce DEI, results were skewed to dissatisfied and very dissatisfied. Regarding the availability of CRP mentors, responses leaned equally from neutral to very dissatisfied. Table We also asked participants if they had thoughts about leaving the institution. Only one stated “Never,” four indicated “Sometimes,” and two indicated “Rarely.”At the end of week four, we solicited feedback from participants at midpoint; however, only five individuals completed the midpoint survey and there were no selections for “moderate amount of the time,” “not very much” or “never” Table .“The videos were insightful, emotional and motivating”“The videos motivated me to assist in the efforts of overcoming systemic racial prejudice in the academic institution environment as well in my direct community”3 Black Voices in Research speakers, and it made me feel less isolated and I learned of a community I never knew existed.”“I identified with the DC“I feel more empowered to further concentrate my efforts, however I can, in aiding mentees of underrepresented, multicultural, and first-generation populations in having a successful and enlightening college experience, free of racism, bias, discrimination and prejudice”In the final session, we had an open discussion evaluating the co-mentoring circle. The participants valued the videos as a “blueprint” and jumping off point for discussion.3 stories moved them to understand the importance of wellness and community, stating “I submitted and was funded to study the impact on the wellness and sense of community of Black faculty, staff and students at my institution through West African dance.”Collectively, participants stated that the emotional benefits of the co-mentoring circle included appreciation for “being heard,” “gaining inter-institutional perspectives”; “helped with sanity and mental health, emotional awareness, and “experiencing empathy, empowerment and hope.” Another participant stated that the DCThe participants wondered if six sessions were sufficient, although they agreed that the sessions should meet no more frequently than monthly, given their busy schedules. They thought future sessions should transition at session six and then use the circle to form actionable outcomes, such as setting and achieving attainable goals. They debated the size of the group, ten was almost too large and sharing may become limited, but that attrition reduced the ultimate number to seven. Many of the participants wanted to stay in touch after the sessions concluded and continue the support for one another in professional growth. Participants also expressed a willingness to facilitate future co-mentoring circles. Moreover, participants had several DEI-related suggestions to aid institutions to improve a culture of diversity and inclusion for clinical research professionals Table .Several publications exist to describe co-mentoring circles for clinical research scientists and women faculty ,29,34. HCo-mentoring circles can lessen disconnection by-products such as feeling unsafe, violated, stuck, or even disappointed . Pre-COV3 Black Voices in Clinical Research offered a meaningful platform for stimulating conversation, reflection, and motivating ideas and action. In offering DEI co-mentoring support, we recommend longer sessions (over the course of a year) with incorporation of role progression support and goal setting. This model should be considered for other types of CRP co-mentoring circles, both locally and across institutions, on a variety of topics such as publication development, onboarding groups, leadership, mentoring, and CRP team-science training.This project offers practical methods and lessons learned from forming co-mentoring circles, based on our pilot offering of a co-mentoring circle in addressing DEI disparities for CRPs working at two CTSA hubs. The DC