Ala gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a statistically robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNA Parkin, Bcl2l13, Ulk1, and Ulk2, to female mice with high levels of the pathogenic tRNAAla gene mutation. We identified a robust effect of Bcl2l13 on the selection process, weaker effects of Ulk1 and Ulk2, whereas Parkin had no statistically significant impact. Our study thus provides strong experimental evidence for distinctive roles of autophagy in germline purifying selection of mtDNA. Furthermore, the experimental strategy and statistical methods we have developed in the manuscript provide a novel framework for future studies of the enigmatic purifying selection process in mammals.We have addressed the role of autophagy on purifying selection of mtDNA by mating different autophagy-deficient mouse models, including knockouts of  Mutations in mtDNA are an important cause of mitochondrial disorders where about one in 200 healthy individuals carries a pathogenic mutation in mtDNA and one in 10000 individuals is affected by mtDNA disease  without Ala gene. In this mouse model, mean heteroplasmy levels are consistent across all tissues [A possible link between the genetic bottleneck and purifying selection is supported by recent evidence from the C5024T mouse model harbouring a pathogenic C to T substitution within the mitochondrial tRNA tissues ,7, which tissues . However tissues . This fi tissues . One appParkin  in mice harboring random mtDNA mutations has been suggested to affect the pathogenicity of mtDNA mutations in the striatum and it could possibly also play a role in controlling the inheritance of mtDNA [Bcl2l13 expression can rescue the mitophagy phenotype caused by Atg32 knockout in yeast [Drosophila melanogaster homologue of Ulk1 is necessary for germline selection of mtDNA in the fly [In mammals, several pathways are in place for the removal of mitochondria via autophagy, these might be partially redundant to ensure robustness of the process or allow the removal of mitochondria under specific conditions –12. Therof mtDNA . A repreof mtDNA –17. Intrin yeast . Eventuain yeast –20. It w the fly . Importa the fly ,22. In tBcl2l13, Ulk1, and Ulk2 transcripts was detected in mouse E12.5 primordial germ cells (PGCs), female germline stem cell (FGSCs), germinal vesicle (GV) oocytes and metaphase II (MII) oocytes, whereas expression of Parkin was detected in all stages besides PGCs [For PARKIN, BCL2L13, ULK1, or ULK2 to contribute to purifying selection, they need to be expressed when selection occurs. Segregation of mtDNA likely occurs during germ cell development, the exact timing is however not known. We therefore checked the expression of the genes encoding these autophagy factors at various stages during germ cell development in publicly available databases and datasets. As these proteins are conserved in mouse and humans, we queried mouse as well as human data. According to the GTEx Portal )  and the des PGCs . As the Parkin, Bcl2l13, Ulk1, or Ulk2 shifted the maximally tolerated mutation load in the offspring of heteroplasmic C5024T mice. To this end, we generated whole body Parkin-/- C5024T, Bcl2l13-/- C5024T, Ulk1-/- C5024T, and Ulk2-/- C5024T females along with Parkin+/+ C5024T and Bcl2l13+/+ C5024T litter mate control females, all on the same mtDNA and nuclear C57BL/6NCrl genetic background  and wanted to keep the animal use to a minimum, we did not generate Ulk1+/+ C5024T or Ulk2+/+ C5024T control groups. Instead, throughout our analysis we used both the Bcl2l13+/+ C5024T and Parkin+/+ C5024T cohorts as controls. It must be noted that out of all samples measured, one sample belonging to the Bcl2l13+/+ C5024T group exceeded the 80% threshold. This turned out to be a technical artefact as remeasurement of the sample revealed a heteroplasmy level below the threshold (1035:417 pup heteroplasmy (%): original measurement: 86; repeated measurement: 71). We did however not want to manually remove this outlier and it is hence included in the analysis. Analysis of the maximally tolerated mutation load in the different groups revealed that neither knockout of Parkin, Bcl2l13, Ulk1, nor Ulk2 in C5024T mothers had any effect on the maximal tolerated heteroplasmy  respectively (Parkin knockout group (-0.20 ± 0.34). However, the shift in the Bcl2l13 knockout group was closer to zero (-0.01 ± 0.32), and shifts in the Ulk1 and Ulk2 knockouts were higher than in both controls . Of note, none of the gene knockouts had any obvious effect on the normalized heteroplasmy variance of the pups , while the shifts in Bcl2l13-/-, Ulk1-/-, and Ulk2-/- were not . However, in the data, mothers have multiple pups (24.73 ± 9.76), and therefore each mother-offspring observation is not independent, violating standard statistical hypothesis test assumptions. Indeed, heteroplasmy shift distributions varied by mother, albeit not to the extent that a single mother would have an outsized effect on the shift distribution in the whole cohort , against all other groups indicated that only the Bcl2l13 and Ulk1 knockouts resulted in significantly different distribution of heteroplasmy shifts . To ascertain that our results were not because we restricted the control data to the six Bcl2l13+/+ C5024T mothers, we further generated all 210 possible subsampled control datasets, consisting of any six mothers and all their respective offspring from the Bcl2l13+/+ C5024T and Parkin+/+ C5024T groups. For example, one such subsampled control could consist of all four mothers in the Parkin+/+ C5024T group and two out of the six mothers in the Bcl2l13+/+ C5024T group. Another subsampled control could consist of one mother from the Parkin+/+ C5024T group and five mothers from the Bcl2l13+/+ C5024T group. We subsequently performed Kolmogorov–Smirnov tests for the Parkin+/+ C5024T and Bcl2l13+/+ C5024T control groups and each knockout group against each of the 210 subsampled controls. We measured how frequently the genotype groups were significantly different to the subsampled controls . Under the null hypothesis that heteroplasmy shifts are identically distributed between control and knockout groups, the median p-value would be 0.50. The proportion of subsampled control tests resulting in p-values below 1% and below 5% for each genotype group are depicted in Parkin+/+ C5024T and Bcl2l13+/+ C5024T control groups did not significantly differ from any of the 210 subsampled controls , indicating the knockout is indeed unlikely to affect the heteroplasmy shift , providing robust evidence of the Bcl2l13 effect on purifying selection , with median p-value = 0.002 below the significance threshold  showing a statistically significant difference, with a median p-value = 0.013 above the threshold . We therefore repeated the Kolmogorov–Smirnov test comparing Bcl2l13-/- C5024T to controls by resampling both knockout and control data with replacement. Knockout and control subsamples of different sizes were repeatedly generated, and the fraction of significant differences for each sample size was calculated , which is relatively well tolerated in the mouse. The impact of knocking out key autophagy proteins could be different for other mutations, especially for other mutations leading to more severe phenotypes. Secondly, as mentioned above, several pathways exist for the removal of mitochondria via autophagy. Unexplored pathways, mitigated by other players in the adapter-dependent pathway for example, may be fruitful avenues of further investigation . Thirdlyelopment . Functioelopment ,35. Theselopment .Overall, our findings establish a robust role for BCL2L13 in purifying selection of mtDNA in the female germline, and provide both an entry point to determine the effects of other autophagy modulators, and a methodological approach to further dissect molecular mechanisms for this essential and incompletely understood phenomenon.The study was approved by the Stockholm ethical committee (Stockholms djurförsöksetiska nämnd) under the ethical permit 2001–2018 and performed in accordance with the guidelines of the Federation of European Laboratory Animal Science Associations (FELASA).Bcl2l13 knockout mice were generated by the Karolinska Center for Transgene Technologies using the CrisprCAS9 system. Two guide RNAs  targeting exon 2 and 5 of Bcl2l13, respectively, were injected alongside Cas9 protein into C57BL/6NCrl zygotes twice. Out of the 38 animals born, three animals carried deletions around the guide RNA targeting exon 5. Out of these 3 founder animals, the animal carrying a 108 bp deletion  was chosen to establish the Bcl2l13 knockout mouse line. The C5024T mice were generated previously [Parkin knockout mice were obtained by crossing mice with Parkin exon 7 flanked by loxP-sites [Ulk1 and Ulk2 knockout mice were created by mating Ulk1FL and Ulk2FL mice  to β-actin-cre transgenic mice [ad libitum with a normal chow diet.Whole body xP-sites  to β-act2 followed by cervical dislocation. Liver was collected immediately, washed with phosphate-buffered saline (PBS), snap-frozen in liquid nitrogen, and stored at -80°C.Animals were euthanized by CO2; 0.5mM EDTA; 1x EDTA-free protease inhibitor cocktail (Roche); 1% NP-40 and 140 mM NaCl) and homogenized for 15 sec with the T 10 basic ULTRA-TURRAX system. Samples were centrifuged at 13000 rpm for 10 min at 4°C and supernatants were collected. Protein concentration was determined with the BCA protein assay kit (Pierce) and 75 μg of protein was resolved by electrophoresis using 12% Bis-Tris Plus NuPAGE gels (Invitrogen) and transferred to PVDF membranes (iBlot 2 system (Invitrogen)). BCL2L13 was detected (Proteintech 16612-I-ap) and GAPDH (ab8245) was used as a loading control.Livers were ground in liquid nitrogen using mortar and pestle. Powder was resuspended in 500 μL of lysis buffer  and var(·) denote the sample mean and sample variance respectively.Normalised heteroplasmy variance for each mother was calculated asn = 6 when testing all genotype groups simultaneously, and n = 5 when testing the Bcl2l13+/+ C5024T control against the other five genotype groups.Across this study, Wilcoxon’s rank tests and Kolmogorov–Smirnov tests on the heteroplasmy shift were carried out at the 5% significance level. A Bonferroni multiple-testing correction was applied with Bcl2l13 knockout data of equal size were generated by sampling 500-fold with replacement. Sample sizes considered ranged from n = 50 to n = 235 at intervals of five.When investigating sample size effects, control and S1 FigWhile a range of normalized variances was observed (0.003 to 0.041), gene knockouts did not appear to have an effect on the pup heteroplasmy variance.(TIF)Click here for additional data file.S2 FigSolid lines represent the mean shift across each genotype group, and dotted lines represent the mean ± 1.5 st.dev. In every genotype group, mother-to-pup heteroplasmy shift varied by mother. Nevertheless, no mothers had a significant proportion of outlier shifts, and no individual mother had an outsized effect on the overall group shift distribution.(TIF)Click here for additional data file.S3 FigNNT wildtype mothers (WT) and mothers carrying the NNT mutation (NNT) were comparable . (B) When the subsampling analysis was repeated excluding the mothers carrying the NNT mutation, over half of tests  remained significant, with median p-value 0.005 below the significance threshold.(A) Heteroplasmy shift distributions between (TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


The computational modelling of this mechanism has mainly been investigated using coupled differential equations (DEs). However, there is a lack of computational investigation into (1) how the different formulation of coupled DEs capture the Ca2+ movement in the cytosol and sarcoplasmic reticulum (SR), (2) the buffer and dye inclusion in both compartments, and (3) the effect of buffer and dye properties on the calcium behaviour. This work is set out to explore (1) the effect of different coupled formulation of DEs on spark/blink occurrence, (2) the inclusion of improved sarcoplasmic buffering properties, and (3) the effects of cytosolic and sarcoplasmic dye and buffer properties on Ca2+ movement. The simulation results show large discrepancies between different formulations of the governing equations. Additionally, extension of the model to include sarcoplasmic buffering properties show normalised fluorescent dye profiles to be in good agreement with experimental and amongst its one- and two-dimensional representations.Several computational studies have been undertaken to explore the Ca Ca2+ releases from the SR into the cytosol appear as sparks in the cytosol and as blinks in the SR. The successive activation of RyRs increases the local Ca2+ concentration in the cytosol. This process is called Ca2+ transient and the sequential activation of Ca2+ results in spontaneous propagating waves of Ca2+ which occur in ventricular cardiac myocytes mainly due to Ca2+ overload. This occurs under pathological conditions affecting the heart’s normal function which may lead to heart failure and ventricular arrythmias  and ordinary differential equations (ODEs). Ca2+ sparks are stochastic in nature  has supressed Ca2+ spark activity as well as caused earlier termination of these sparks. Nevertheless, the effect of different types and level of buffering and dye properties on the Ca2+ dynamics is not considered to be fully explored. This is especially important since these properties may influence calcium handling and contractility   2+ spark2+ concentrations are represented by x and y directions; 2+ leak from the cytosol to the exterior of the cell and sarcoplasmic Ca2+ leak from the SR to the cytosol; 2+-ATPase. The last three terms are given as 2+ efflux from the cytosol to the extracellular medium and from the SR into the cytosol.The cytosolic and sarcoplasmic Ca2+ release from a Ca2+ release unit (CRU) is determined by 2+ release channel based on the current of the CRU, 2+ fluorescent indicator dye and endogenous stationary buffers. These are given as 2+ bound dye, 2+ bound buffer, 2+ spark occurrence, 2+ sensitivity parameter and The flux of Ca2+ sparks and blinks are two different views of the same elementary release events. Ca2+ blinks are important events for the termination of sparks. An ideal model should be able to estimate the fluorescent values in the cytosol as well as that in the SR. In the previous section, the fluorescent dye and buffers in the SR are not taken into account. Modelling of such coupling has not been fully explored. Therefore, model presented in the previous section is modified and extended to accommodate for an improved estimation of the Ca2+ blinks with the normalised fluorescent profile in the SR. In addition to the equations used in the previous section, new equations are introduced for estimating the sarcoplasmic Ca2+ concentration, Ca2+ bound fluorescent indicator, 2+ bound buffer, 2+ concentration in Eq.   3) are dThe two-dimensional system is set to be isotropic  and two-dimensional (bottom panels) models, respectively. From initiation to activation of neighbouring channels there is some delay for Ca2+ near the neighbouring channels to reach activation threshold. Once the neighbouring channels are activated, there is an increase in Ca2+ around the region which explains the decrease then slight increase in concentration from spark initiation. The results for the sarcoplasmic Ca2+ shows a drop to between 441 and 482.2 µM for the one-dimensional model and between 397 and 441.1 µM for the two-dimensional case. This is a large reduction in the differences between the two models compared to our previous work  as opposed to PDEs for modelling Ca2+ sparks and waves reproduces more accurate full-width at half-maximum (FWHM) property of the Ca2+ spark (Chen et al. 2+ from the extracellular medium to the cytosol occurs as regular pulses through L-type channels. The effect of this in the current work is addressed by including two columns of points sources in the middle of the domain which accounts for only one pulse, but this can be extended to regular intervals of pulses. Due to the focus of the work, this has not been accounted for.Most mathematical models using stochastic partial differential equations, focus on studying the occurrence of individual Ca


Teaching Point: When a renal angiomyolipoma (AML) is incidentally detected on imaging, the venous system should be assessed for intravascular fat component. A 61-year-old female was admitted to the emergency department for epigastric pain following epigastric hernia surgery. An abdominal unenhanced computed tomography (CT) demonstrated a reoccurrence of epigastric eventration. As an incidental finding, two round well-defined lesions of fat density (–78 HU) containing tortuous foci of enhancing vessels features strongly suggestive of renal AMLs  and leading to surgical treatment or embolization is spontaneous retroperitoneal hemorrhage sometimes complicated by hypotensive shock.A rare case of AML showing liposarcomatous transformation has also been reported. Aggressive patterns with intravascular growth is a rare condition only described in a couple of case reports . Mostly Even a benign, and relatively frequently encountered, AML can rarely exhibit aggressive behavior, with intravascular potentially life-threatening fat extension. The radiologist should pay attention to this and attentively review the renal veins and IVC for a fat density component that suggests intravascular extension or embolization . Therape


Acute myeloid leukaemia (AML) is an aggressive disease of the bone marrow with a poor prognosis. Evidence suggests long established chemotherapeutic regimens used to treat AML are reaching the limits of their efficacy, necessitating the urgent development of novel targeted therapies. Canonical Wnt signalling is an evolutionary conserved cascade heavily implicated in normal developmental and disease processes in humans. For over 15 years its been known that the central mediator of this pathway, β-catenin, is dysregulated in AML promoting the emergence, maintenance, and drug resistance of leukaemia stem cells. Yet, despite this knowledge, and subsequent studies demonstrating the therapeutic potential of targeting Wnt activity in haematological cancers, β-catenin inhibitors have not yet reached the clinic. The aim of this review is to summarise the current understanding regarding the role and mechanistic dysregulation of β-catenin in AML, and assess the therapeutic merit of pharmacologically targeting this molecule, drawing on lessons from other disease contexts. Acute myeloid leukaemia (AML) is an aggressive and heterogeneous clonal disease of haematopoietic stem/progenitor cells (HSPC) in the bone marrow with approximately 3,200 cases annually in the UK (Cancer Research UK statistics). The disease predominates in older adults (60 years plus) and is characterised by the uncontrolled proliferation and arrested differentiation of myeloid committed blasts in the bone marrow. Patients experience the rapid onset of symptoms originating from impaired myelopoiesis including immunosuppression, defective haemostasis, and severe anaemia, and will ultimately succumb to bone marrow failure without timely medical intervention. Despite improvements in overall AML survival over the past 60 years, the prognosis for older patients, or those harbouring adverse gene mutations or cytogenetic events, remains poor . With li+ AML amino} benzoate (MSAB) as a potent inhibitor of TCF reporter activity in HCT116 cells . Furthermin/+APC mouse models. Provided PROTACs can permeate the membranes of myeloid cells with similar efficiency, the employment of PROTACs seems an entirely sensible strategy to reducing the excessive β-catenin protein levels observed in AML cells.PROteolysis-TArgeting Chimaeras (PROTACs) have emerged as a promising strategy for degrading a protein of interest (POI) and have already been trialled for β-catenin in alternative settings. PROTACs are heterobifunctional molecules consisting of a POI ligand chemically linked to an E3 ligase ligand. Upon capture of the target protein by the PROTAC, the closely associated E3 ligase polyubiquitinates the POI stimulating its degradation in the proteasome . Liao etSimilarly, along the ubiquitination theme, deubiquitinases (DUBs) work through antagonising the actions of E3 ligases by removing ubiquitin groups from target proteins, thus promoting their stability. DUBs such as USP2A , USP4 1, USP9X ,141 and Finally, our group recently performed the first β-catenin interactome in myeloid cells, representing the first exploration of β-catenin protein partners in haematopoietic cells of any kind . From thThe rationale for exploring Wnt signalling therapeutics in AML is both sound and justified, however β-catenin has proved a challenging component to pharmacologically target in this setting. This challenge is a reflection of the genetic heterogeneity of AML where a multitude of mechanisms have been proposed to dysregulate the molecule in blasts and LSCs. These mechanisms are more diverse, elusive, and intricate than the relatively simple genetic mutations that occur to single Wnt signalling components  in solid cancers. Only the full and thorough characterisation of these mechanisms in AML cells would inform effective drug design; however, this may be an impractical task given the considerable inter-tumour heterogeneity displayed between AML patients. In which case, for now the continued development of novel agents that bind and directly destabilise the excessive β-catenin protein in AML cells holds the most promise, since only minimal levels/activity of the protein are required to sustain the maintenance and development of normal HSC. The ongoing molecular characterisation of β-catenin in a haematopoietic context will be vital in revealing new vulnerabilities that can be exploited through combinatorial approaches to extinguish β-catenin activity in AML.


Vβ3 integrins (using large necrotic tumors). The αVβ3 integrin-targeting Cy5.5-RAFT-c(-RGDfK-)4 was used as a reference. In vivo and ex vivo fluorescence imaging, whole-tumor section imaging, fluorescence microscopy, and multiplexed fluorescence colocalization analysis were performed. The ITD of fluorescent dye-labeled pHLIPs was heterogeneous, having a high degree of colocalization with necrosis. A direct one-to-one comparison of highly magnified images revealed the cellular localization of pHLIP in pyknotic, karyorrhexis, and karyolytic necrotic cells. pHLIP and hypoxia were spatially contiguous but not overlapping cellularly. The hypoxic region was found between the ITDs of pHLIP and the cRGD peptide and the Ki-67 proliferative activity remained detectable in the pHLIP-accumulated regions. The results provide a better understanding of the characteristics of ITD of pHLIPs, leading to new insights into the theranostic applications of pHLIPs.pH (low) insertion peptides (pHLIPs) have been developed for cancer imaging and therapy targeting the acidic extracellular microenvironment. However, the characteristics of intratumoral distribution (ITD) of pHLIPs are not yet fully understood. This study aimed to reveal the details of the ITD of pHLIPs and their spatial relationship with other tumor features of concern. The fluorescent dye-labeled pHLIPs were intravenously administered to subcutaneous xenograft mouse models of U87MG and IGR-OV1 expressing α The fluorescent pHLIP constructs, AF546-pHLIP  was synthesized as reported previously [The 27-residue peptide pHLIP variant 3 (ACDDQNPWRAYLDLLFPTDTLLLDLLW) was synthesized and fluorescently labeled based on the methods previously reported ,38. Brieeviously .The human glioblastoma U87MG cell line was purchased from the American Type Culture Collection . The human epithelial ovarian adenocarcinoma IGR-OV1 cell line was obtained through a Material Transfer Agreement #1-5734-18) from the National Cancer Institute Developmental Therapeutics Program Tumor Repository . The two cell lines were cultured and maintained as previously described -18 from .nu/nu mice (4–5 weeks old) were purchased from CLEA  and bred in a specific pathogen-free facility. Animal procedures were approved (Protocol No. 13-1022-9) by the Animal Ethics Committee of the National Institutes for Quantum Science and Technology  and were conducted according to institutional guidelines. Tumor models were established by subcutaneous injection of 5 × 106 U87MG or IGR-OV1 cells in the right flank of the mice. Subsequent experiments were performed when the tumor diameter reached 10–15 mm (3–4 weeks after xenografting).Female athymic BALB/cAJcl-n = 3–5/group) were anesthetized and imaged prior to and at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h after intravenous injection (via the tail vein) of IR800-pHLIP (5 or 10 nmol) [max/emmax: 740–790/810–860 nm) and at the same settings .The VISQUE™ InVivo Smart-LF imaging system  was used. Tumor (U87MG or IGR-OV1)-bearing mice (10 nmol)  with an max/emmax: 630–680/690–740 nm).Ex vivo imaging was additionally performed at 4 h ,39 afterImage processing (pseudocoloring and superimposing) and measurements of fluorescence intensity of the region of interest (ROI) were performed using VISQUE. The ROIs were drawn to encompass the tumors, and mean surface intensities were calculated.A cocktail of either AF546-pHLIP + IR800-pHLIP (5 nmol each) or that of IR800-pHLIP + Cy5.5-RaftRGD  was administered to the mice and the mice were examined at 4 h post-injection. Pimonidazole, a commonly used exogenous hypoxia marker in histochemistry , was administered intravenously to the mice 1 h before euthanizing. Tumors were removed and cryo-sectioned in sequential sections (10 µm thickness) as previously described . SectionFrozen sections of tumors treated with AF546-pHLIP, IR800-pHLIP, and pimonidazole were air-dried without further processing and scanned at 800 nm using the Odyssey CLx NIR Imaging System  to visualize IR800 fluorescence throughout the section. Representative samples were further imaged using the Keyence BZ-X800 fluorescence microscopy system  with Texas-Red and Cy7 filter sets to obtain the whole-section images of AF546-pHLIP and IR800-pHLIP, respectively. Sections were then subjected to double staining (acetone fixation) of the microvascular biomarker CD31 and pimonidazole using rat anti-CD31  and rabbit anti-pimonidazole , followed by Alexa Fluor 594–conjugated goat anti-rat IgG  and IR800–conjugated goat anti-rabbit IgG  as previously described . Nuclei Based on the whole-section images, ROIs (representing differing patterns of pHLIP distribution) were selected for high-resolution imaging using the remaining adjacent sections. One section -fixed and H&E-stained) was used to study the morphology of the ITD of pHLIP, and another (CD31 and pimonidazole double-stained) to clarify the spatial relationship between pHLIP and hypoxia in the context of the microvasculature. The PFA fixation, mentioned below, was preferentially used (when necessary) to better retain the cellular structure and tissue integrity. All corresponding images of the same ROI-defined sites were sequentially captured using a 20× objective lens on the Keyence, as mentioned above.After regional imaging, the pimonidazole and CD31 double-stained sections were subjected to H&E staining (if possible). Regional fluorescence colocalization images of pHLIP, pimonidazole, CD31, and DAPI were produced in parallel with the corresponding images of H&E staining. From these images, three representative ROIs with: (i) negligible accumulation of both pHLIP and pimonidazole, (ii) high accumulation of pHLIP, and (iii) high accumulation of pimonidazole were further magnified to detect the morphological details of pHLIP localization at the cellular level.To investigate the possibility that the ITD of pHLIP was attributed to the targeting of lipid droplets (reportedly found in the necrotic area of cancer ), represTo investigate the possibility of the ITD of pHLIP being attributed to the targeting of tumor-associated macrophages , repRepresentative images of AF546-pHLIP-accumulated regions (20× objective lens) were captured. Subsequently, double staining (PFA fixation) was performed by using rabbit anti-Ki-67  and anti-CD31. Corresponding images were acquired using Cy7 and TRITC filters for Ki-67 and CD31 staining, respectively.The ITD of IR800-pHLIP and Cy5.5-RaftRGD across the whole sections of tumors was visualized using the Odyssey at 800 and 700 nm, respectively. Regional bright-field and NIRF images (20× objective lens) or tiled images (viewed in a wider field) were captured with the Keyence. The fluorescence of IR800-pHLIP was read as described above, while Cy5.5-RaftRGD was detected using a Cy5.5 filter set. After imaging, the sections were subjected to CD31 and pimonidazole double staining, and the corresponding images were acquired as described above.The previously obtained images of the objects  were registered and merged using Adobe Photoshop 23.3.2 software. Intensity plot profiles of the fluorescence signals were created and superimposed using ImageJ 1.53t software . The spatial relationship between the imaging components was then visually determined.t-tests (unpaired and two-tailed) were used for two-group comparisons . p < 0.05 was considered significant.Quantitative data are presented as the mean ± standard deviation (SD). Student’s p < 0.001 at 1 h post-injection; p < 0.01 at 1 h post-injection; The ITDs of AF546-pHLIP, IR800-pHLIP, hypoxia (pimonidazole staining), microvessels CD31 staining), and nuclear DAPI staining were examined at 4 h post-injection of the cocktail of AF546-pHLIP and IR800-pHLIP.  stainingRepresentative images of the ITD of AF546-pHLIP, lipid droplets, and hypoxia obtained from the same sites in the same sections of tumors were compared. In general, as shown in Representative images of the ITD of AF546-pHLIP and CD11b staining (macrophages and activated lymphocytes) obtained from the same sites in the same sections of tumors were compared. In general, as shown in The ITD of IR800-pHLIP, Cy5.5-RaftRGD, hypoxia, microvessels, and nuclear DAPI staining was examined at 4 h post-injection of the cocktail of IR800-pHLIP and Cy5.5-RaftRGD. Vβ3-targeting cRGD peptide, and others of concern by conducting a series of imaging from the mouse whole-body to tumor-cell level, using our unique high-resolution multiplexed fluorescence colocalization analysis. This study unambiguously demonstrated the ITD patterns of pHLIPs and their spatial relationship with necrosis, hypoxia, and the cRGD peptide. A pHLIP-based anticancer agent is a promising candidate for a combination of cancer cell-targeted therapy and hypoxia-targeted therapy.Extracellular acidity-targeting pHLIPs are under development for cancer imaging and therapy. An in-depth investigation of the ITD of pHLIPs is of paramount significance for providing basic information toward understanding therapeutic limitations and facilitating a rational combination therapy design. Owing to the lack of a histological biomarker for extracellular acidity, studies on the ITD of pHLIPs have so far focused on comparisons with other related features of the tumor microenvironment. If the relationships are to be fully comprehended and unambiguously described, it is imperative that a histological comparison of multiple parameters is performed based on the same whole section, rather than preferentially selected small regions of adjacent sections ,46. To tVβ3-positive necrotic xenografts  were selected for the current study because we focused attention on the detailed ITD of pHLIPs associated with necrosis, hypoxia, and integrins. On the other hand, some studies employed tumor models with more aggressive phenotypes (more acidic), such as metastatic xenografts, allografts, and spontaneously developed tumors in transgenic mice [For this study, it was necessary to analyze tumors that had developed hypoxia and necrosis for optimally comparing the ITD of pHLIP; hence, large-sized tumors (10–15 mm in diameter) were studied. We validated the tumor-targeting ability of IR800-pHLIP by in vivo and ex vivo NIRF imaging in two different human tumor models . An identical ITD was observed between AF546-pHLIP and IR800-pHLIP at both the whole-section and enlarged area level . FurtherThe ITDs of pHLIPs in necrotic U87MG and IGR-OV1 xenografts were heterogeneous throughout the tumor section , consistSome efforts have been made to clarify the spatial relationship of pHLIPs with necrosis and/or hypoxia ,31,32. HThe mechanism of action of pHLIPs—targeting cancer cells by exploiting the extracellular acidic microenvironment—is well understood ,16,47,48The spatial relationship between pHLIP and hypoxia is easier to understand in terms of the corresponding microvasculature . Our res225Ac  since pHLIPs have been reported to be labeled with a range of radiometals  via peptide modification [Given that hypoxia is associated with increased tumor aggressiveness and poor prognosis and that hypoxic tumor cells are resistant to treatment, this common feature of solid tumors represents a compelling therapeutic target ,52,53. Vfication ,32,33,34225Ac, it is also imperative to lower the risk of nephrotoxicity, as pHLIPs can accumulate highly in the kidney  have been introduced to prevent renal damage related to peptide-based radiotherapy. We previously showed that the co-injection of Gelofusine (succinylated gelatin) and L-lysine (positively charged basic amino acid) with 64Cu-RaftRGD caused a 30–50% reduction in mouse renal radioactivity levels [225Ac-DOTATOC for somatostatin-receptor-expressing cancers, the amino acid mixture of lysine and arginine formulated in gelatin solution was co-administered as a renal protective cocktail [It should be noted that when developing pHLIP-based radiotherapeutics armed with Cys-Var3  and 89ZrCys-Var3  have beey levels . In paticocktail .Vβ3- and hypoxia-targeted radiotherapies [64Cu-RaftRGD targeting αVβ3 on cancer cells [64Cu-ATSM targeting hypoxic metabolism [225Ac), in addition to producing a uniform ITD of radioactivity, hypoxic tumor cells and/or cancer stem cells  and a cRGD peptide (in normoxic regions) along with a spatially complementary relationship between the two peptides , we woulherapies ,37. As mtabolism ,6 has detabolism . Therefo regions ,59) woulIn addition to the previously discussed limitation of this work, there are at least three more notable limitations. First, this work focused on only one member of the pHLIP family (variant 3), although this is a representative member and is reported more frequently in the literature. Second, the ITD characteristics of pHLIPs were investigated in a small number of tumor types (two xenograft models), thus requiring further studies using more tumor types, including more aggressive phenotypes such as allografted tumors. Nevertheless, we hope that researchers in the field will use the study protocols established herein to identify the ITD characteristics of newly developed pHLIP-based drugs toward future clinical translation. The third limitation is that pHLIP compounds have also been reported to target the tumor–stroma interface ,45, but We performed a comprehensive investigation  with two tumor mouse models, unambiguously revealing the details of the ITD of a representative pHLIP (variant 3). The spatial relationship of the ITD of pHLIP with multiple parameters was clarified in a straightforward manner using a unique high-resolution multiplexed fluorescence colocalization analysis based on the same whole tumor sections. The ITD of pHLIP was heterogeneous, with increased accumulation in the regions of necrosis by localizing to various morphological patterns of necrotic cells. There was a spatially contiguous but nonoverlapping relationship (at least at the cellular level) between the ITD of pHLIP and hypoxia. Notably, an intermediate position of hypoxia was revealed between the ITD of pHLIP and a cRGD peptide. These results provide a better understanding of the ITD characteristics of pHLIPs, which may lead to new insights into the theranostic applications of pHLIPs. Based on the close spatial relationship of hypoxia with pHLIP and cRGD peptides, we propose an alternative approach, a new “pHLIP + cRGD” combination for tackling treatment-resistant hypoxia via a potential crossfire of the two peptide-based therapeutics.


However, the processes regulating Ca2+ puff amplitude (average ∼0.5 ΔF/F0) and duration  have yet to be elucidated. A recent study in JBC by Smith and Taylor determined that Ca2+ puff amplitude is independent of IP3R cluster density and that the termination of IP3R Ca2+ puff is regulated by IP3 dissociation, illuminating the steps of this regulatory dance.Concerted openings of clustered inositol 1,4,5-trisphosphate receptors (IP IP3R activity is finely tuned not only by mechanisms that control IP3 production but also by other molecules and proteins that bind to and regulate IP3Rs . Upon IP3 binding, Ca2+ that is first released by the IP3R binds to high affinity Ca2+-binding sites in neighboring IP3Rs, thus amplifying the rising phase of the Ca2+ puff. As Ca2+ concentration increases during the rising phase of Ca2+ puffs, it binds to low affinity site in the IP3R, inactivating further Ca2+ release. This process can be reinitiated from the same cluster following IP3R recovery from inactivation or activation of IP3Rs in the cluster that never underwent Ca2+-dependent inactivation. This regenerative process is further tuned by IP3 abundance, which correlates with Ca2+ puff frequency. Although many studies have characterized the kinetics of IP3R-mediated Ca2+ puffs, the mechanisms which control their amplitude and termination have not been determined.Clear identification of IPclusters . These l3R3, harboring an N-terminal SNAP-tag (SNAP-IP3R3) to assess Ca2+ puff amplitude and termination ; this not only allowed rapid release of IP3 but also a tight control of total IP3 concentration. Using this approach, Smith and Taylor were able to determine a) how IP3R3 expression affects receptor localization, b) how IP3R3 density in a microdomain influences the amplitude and frequency of Ca2+ puffs, and c) how IP3 dissociation impacts Ca2+ puff termination  is likely capped by microdomain-dependent regulation of IP3Rs. This regulation was suggested by Smith and Taylor to be dependent on both interactions between IP3Rs, limiting the number of open IP3Rs, and Ca2+-dependent IP3R inactivation during the rising phase of Ca2+ puffs. This is supported by previous data showing that rapid Ca2+ buffering increases the number of active IP3Rs in a microdomain . Although IP3R3R568Q and WT IP3R3 show similar Ca2+ puff amplitude, IP3R3s with lower IP3 affinity showed reduced Ca2+ puff frequency, shorter duration, faster decay time, and increased latency to puff initiation. Thus, reducing IP3R affinity for IP3 results in faster termination of Ca2+ puffs. This supports previous work showing that IP3R Ca2+ puff duration correlated with IP3 affinity of IP3R subtypes (IP3R2>IP3R1>IP3R3) .The authors also examined if dissociation of IPy IP3R3R58Q. Altho3R3s expression and ligand concentration, Smith and Taylor illuminated how IP3R3 density and IP3 dissociation influence Ca2+ puff frequency, amplitude, and termination. However, in physiological systems, IP3Rs function in a complex during conditions of graded molecular interactions and posttranslational modification. Thus, studies examining IP3R-mediated Ca2+ puff kinetics with more intact cellular milieu that are beginning to emerge will help to delineate physiological IP3R function. For example, Kras-induced actin-binding protein interaction with IP3R has recently been shown to regulate the amplitude of Ca2+ puffs and the number of active IP3Rs within a cluster  are activated by different intracellular signals, the independence of Ca2+ release rise time and amplitude on receptor cluster density is maintained in both channels (2+-induced closure (inactivation) of these receptors, which would likely cap Ca2+ puffs/sparks at an equivalent amplitude even in microdomains with a high receptor density (3Rs in a microdomain is also constrained by inter-receptor interactions and/or other allosteric pathways. Although Ca2+ release during puffs/sparks is tightly regulated in the microdomain, puff/spark initiation and frequency correlate with receptor density (3R and RyR regulation underlie the importance of Ca2+ puffs/sparks to cellular function. The findings by Smith and Taylor take a significant step towards fully elucidating the complex and coordinated regulation of endogenous IP3Rs during Ca2+ puffs.This study illuminates the conserved and carefully choreographed spatio-temporal dance of microdomain Cachannels , 9. This density . Additio density , 9. ThesThe authors declare that they have no conflicts of interest with the contents of this article.


The ontogeny of the heart describes its development from the fetal to the adult stage. In newborn mammals, blood pressure and thus cardiac performance are relatively low. The cardiomyocytes are thin, and with a central core of mitochondria surrounded by a ring of myofilaments, while the sarcoplasmic reticulum (SR) is sparse. During development, as blood pressure and performance increase, the cardiomyocytes become more packed with structures involved in excitation–contraction (e-c) coupling (SR and myofilaments) and the generation of ATP (mitochondria) to fuel the contraction. In parallel, the e-c coupling relies increasingly on calcium fluxes through the SR, while metabolism relies increasingly on fatty acid oxidation. The development of transverse tubules and SR brings channels and transporters interacting via calcium closer to each other and is crucial for e-c coupling. However, for energy transfer, it may seem counterintuitive that the increased structural density restricts the overall ATP/ADP diffusion. In this review, we discuss how this is because of the organization of all these structures forming modules. Although the overall diffusion across modules is more restricted, the energy transfer within modules is fast. A few studies suggest that in failing hearts this modular design is disrupted, and this may compromise intracellular energy transfer.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’. The pumping action is performed by cardiomyocytes, specialized cells with myofibrils that contract to perform mechanical work in response to an elevation of Ca2+ during the excitation–contraction (e-c) coupling. As the heart must work continuously, contracting to pump blood and relaxing to refill, for energy supply, it depends mainly on ATP from oxidative phosphorylation in the mitochondria.The heart must work continuously to pump blood around in the body to reach all the capillaries, where it provides the tissues with oxygen and nutrients as well as takes away COIn newborn mammals, cardiac performance is relatively low. As it increases during ontogeny, the cardiomyocytes change morphology, e-c coupling and energetics to enhance performance. As there are relatively few studies on neonatal mammals, we can learn a lot from studying cardiomyocytes from other species with similar characteristics. In particular, the cardiomyocytes from fishes bear resemblance to cardiomyocytes from neonatal mammals. In the following, we will describe the ontogeny of cardiomyocytes in mammals in terms of morphology, e-c coupling and energy transfer. When possible, we will draw parallels between cardiomyocytes from neonatal mammals and fishes.In most mammal species, cardiac performance in terms of stroke work is lower at birth than in adults. During development, blood pressure and cardiac output increase, and thus the heart must perform more mechanical work. In rats, resting diastolic blood pressure increases from approximately 20 to approximately 60 mmHg . In rabbThe pressure developed by the left ventricle in neonatal mammals is similar to the mean ventral aortic blood pressure of 30–50 mmHg in trout, cod and several other fish species . This is. 2During development, the mammalian cardiomyocytes grow in size and change shape to become cylindrical. The main cylinder of the cells has a diameter of 20–30 µm. They sometimes branch and can thus connect to several adjacent cells through the intercalated discs at the end of each branch. As the cells grow in size, the sarcolemma forms invaginations termed transverse tubules (t-tubules) ,39,41. T. 32+ influx into the cytosol across the sarcolemma through L-type Ca2+ channels (LTCCs). This Ca2+ influx triggers the Ca2+ induced Ca2+ release (CICR) from the SR through ryanodine receptors (RYRs). For the cell to relax again, Ca2+ is removed from the cytosol by the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA), the Na+/Ca2+-exchanger (NCX) operating in forward mode and the plasma membrane Ca2+ ATPase (PMCA). During development, there is a change in the relative contribution of these Ca2+ flux pathways.The e-c coupling changes in conjunction with the morphology of the cardiomyocytes. As the heart rate increases, the action potential (AP) shortens . In adul2+ enters the cell through LTCCs as well as reverse action of the NCX (NCXrev). However, NCXrev is the dominant transsarcolemmal Ca2+ influx pathway, and the main influx pathway coupled to CICR [2+ transient [2+ transients rise faster at the edges of the cell than in the centre [In neonatal cardiomyocytes, Ca to CICR . This sc to CICR ,40. CICRransient . Owing te centre .2+-fluxes are also relatively more important than SR Ca2+-cycling [2+-fluxes in cardiomyocytes from neonatal rabbits and fish are similar. In trout cardiomyocytes, NCXrev is also the main transsarcolemmal Ca2+ influx pathway contributing approximately 30% of the total Ca2+ influx, whereas LTCC contributes approximately 20%, and CICR 50% to the overall Ca2+ transient [2+-transient also rises faster at the edges than in the centre of the cell [2+-cycling through the SR than sedentary species [A similar pattern of e-c coupling is found in fishes, where sarcolemmal Ca-cycling . The Ca2ransient . In trouthe cell . However species .2+-influx through LTCCs. Concomitant with the development of t-tubules, the co-localization of NCX and RYR gradually declines [2+ transient [2+ transients are spatially homogeneous, with a synchronous rise at the edges and in the centre of the cell [In adult mammalian cardiomyocytes, the e-c coupling changes to become more reliant on CICR from the SR triggered by Cadeclines , while cdeclines ,40. CICRransient ,35, and the cell .2+ dynamics. In adult cardiomyocytes, the majority of LTCCs in the sarcolemma and RYRs in the junctional SR co-localize internally, at the t-tubules [2+ microdomains in which the Ca2+ concentrations are much higher than in the surroundings, and this is critical for the LTCC regulation of CICR. Indeed, Ca2+ release events are delayed in loose dyads, in which the distance between RYR and LTCC is larger than in compact dyads [2+ transient [2+ release and, thus, contraction [The changes in e-c coupling, that are observed during ontogeny, rely not only on changes in protein expression, but to a large extent also on the cardiomyocyte morphology. As noted above, the SR forms a continuous network throughout adult mammalian cardiomyocytes , and the-tubules , where t-tubules ,47. Withct dyads . An incrransient –51. Thustraction –54.2+-fluxes in e-c coupling change with adrenergic stimulation enhancing cardiac performance during the fight-or-flight response. Stimulation of β-adrenergic receptors interacting with Gs-protein activates adenylyl cyclase to produce cyclic AMP (cAMP), which in turn binds to and activates protein kinase A (PKA). PKA phosphorylates several proteins involved in e-c coupling such as the LTCC, RYR, phospholamban and troponin I. This results in an increased LTCC open probability and Ca2+-influx [2+ uptake, as the phosphorylation of phospholamban removes its inhibition of SERCA [2+-current [2+ transients, reflecting the functional state of the SR [2+-current in fish cardiomyocytes, but the magnitude of the response is species-dependent [2+-influx [The Ca+-influx  and a faof SERCA . The res-current , but altf the SR . Adrenerependent ,59. In f+-influx , a direc. 42+ and PMCA consumes 1 ATP per Ca2+. The NCX does not consume ATP directly but is coupled through the electrochemical gradient of Na+ to the Na+/K+ ATPase, and thus indirectly consumes 1 ATP per Ca2+ transported out of the cell. Usually, the main consumption of ATP is by myosin ATPase as the sarcomeres contract. However, this varies with the mechanical load, heart rate and inotropic state. The ATP consumption of actomyosin is directly related to the pressure developed by the heart and the volume of blood pumped, and there is a linear relationship between cardiac oxygen consumption and the pressure–volume area of the working heart [Whereas energy for the ion fluxes during excitation comes from their electrochemical gradient, the re-establishment of ion gradients during relaxation costs energy in the form of ATP. SERCA consumes 1 ATP per 2 Cang heart . It has ng heart ,62, 30% ng heart .in utero, and hearts of newborn mammals are more hypoxia-tolerant than in the adult stage [The sources of ATP in the heart also change during ontogeny. In the fetal stage, cardiac metabolism is adapted to the slightly hypoxic conditions lt stage . Of the lt stage . Fish allt stage ,67. Aftelt stage . In the lt stage .. 5A key question in cardiac energetics concerns energy transfer, i.e. how ATP and ADP circulate between ATPases and mitochondria, and whether the energy transfer changes in response to the changes in intracellular structure during the development. Energy transfer can occur by direct diffusion of ATP and ADP, and/or by creatine kinase (CK) facilitated diffusion.+ ↔ ATP + creatine. In the adult mammalian heart, there is one mitochondrial isoform (Mi-CK) and three cytosolic isoforms . Mi-CK is bound to the outer surface of the inner mitochondrial membrane and forms complexes with the voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM) and adenine nucleotide translocase (ANT) in the inner mitochondrial membrane [+/K+ ATPase [ATP [CK catalyses the following, reversible reaction: ADP + phosphocreatine + Hmembrane . The cytmembrane , SERCA [membrane , the Na++ ATPase  and the ase [ATP . The binase [ATP . Indeed,The role of CK in the heart is still a subject of research. It was suggested to be more important as a spatial energy buffer facilitating energy transfer between mitochondria and ATPases, because it generates an additional diffusional circuit between ATPases and mitochondria . Further. 6M ADP is estimated from recordings in the respirometer, where the respiration of permeabilized fibres or cardiomyocytes is recorded while stepwise increasing the concentration of ADP until the respiration rate is maximal. Permeabilized fibres are prepared from small pieces of the heart wall, which are gently dissected with fine tweezers to separate small bundles of cells [M ADP is the ADP concentration that stimulates respiration to half of the maximum respiration rate. In isolated mitochondria, which are taken out of their structural setting, the apparent KM ADP is 5–20 µM, but permeabilized fibres or cardiomyocytes from adult mammals have a higher apparent KM ADP of approximately 200–400 µM [M ADP indicates the difficulty with which ADP diffuses from the medium outside the cells to the mitochondrial inner membrane [As probably the simplest way to assess diffusion in cardiomyocytes, the apparent Kof cells . Fibres of cells . If the 0–400 µM ,75,77–79membrane , and theM ADP in permeabilized cells compared to isolated mitochondria indicates that this overall diffusion is restricted [M ADP would correspond to the concentration gradient between the solution and the mitochondrial inner membrane required to sustain the mitochondrial ADP-consumption through ADP diffusion. From outside the cells, ADP can encounter several barriers that may prolong or hinder the diffusion. It was suggested that unstirred layers surrounding permeabilized fibres result in unphysiologically long diffusion distances before ADP in the solution has even encountered the cells [M ADP is caused by barriers inside the cells [M ADP in permeabilized trout fibres and cardiomyocytes with fibres having a much higher KM ADP [M ADP can be impacted by the relative activities of mitochondrial ATP synthesis and surrounding ATPases. As shown by Kongas et al. [M ADP.The higher apparent Kstricted , as the he cells . In addihe cells . Differer KM ADP . This war KM ADP . In addis et al. , increasM ADP is very useful to get an impression of the overall diffusion restrictions within the cell, experiments on fibres or where the relative contributions of ATP synthesis and ATP consumption could change, such as during a treatment, must be interpreted with caution. However, as noted above, quantitative analyses of data from isolated, permeabilized rat cardiomyocytes have demonstrated the intracellular nature of the diffusion barriers. Knowing the identity and location of these barriers is important for understanding the physiological role of the CK system.Thus, while estimating the apparent K. 7M ADP in adult cardiomyocytes was owing to diffusion restriction caused by the OMM. The physiological advantage would be that energy transfer of ADP/ATP between mitochondria and ATPases would take place through the CK system [In the 1990s, it was suggested that the high apparent KK system . DiffusiK system  in additK system . Thus, iM ADP of respiration as well as Mi-CK expression [M ADP. Again, this backed up the theory that diffusion restriction by the OMM shifted energy transfer to take place through the CK system.The importance of CK as a facilitator of energy transfer in muscles with intracellular diffusion barriers was supported by the finding that its acute inhibition lowered the phosphorylation potential near ATPases to such an extent that cardiac function was impaired . Furtherpression ,88. Thus. 8There are several transgenic mouse models, in which CK is inhibited by knockout of its different isoforms (CK KO ), or of M ADP was the same as in wild-type (WT) [M ADP was lower than in WT, and mitochondria seemed to intercalate between the myofibrils [M ADP as well as mitochondrial organization was the same as in WT littermates [M ADP. This is because the OMM is not the only diffusion barrier within cardiomyocytes.If diffusion restriction by OMM caused channelling of energy transfer through the CK system, and this was crucial for cardiomyocyte function, it is conceivable that inhibition of the CK system would lead to compensatory changes in the permeability of the OMM. In permeabilized cardiac fibres from transgenic mice lacking Mi-CK, the apparent Kype (WT) . In fibrofibrils . Howevertermates , and thetermates . Thus, i. 9More recently, it has become clear that intracellular diffusion restriction is not just owing to the OMM but has multiple causes. The search for multiple factors imposing diffusion restrictions intensified in 2001, when experimental works demonstrating that there is an unexpected coupling between ATPases and mitochondria in the cardiomyocytes were published ,107,108 2 s−1 in water and approximately 200 µm2 s−1 in physiological solution containing bovine serum albumin. The overall diffusion coefficient is 24 and 35 µm2 s−1 in the radial and longitudinal direction [2 s−1) [2 s−1) [2 s−1 [The modules are not completely isolated from each other, but the barriers between them cause a considerable slowing of the overall diffusion in adult cardiomyocytes. Note, that the term ‘overall diffusion’, means diffusion across several modules. This is shown in irection . This esirection , but ver [2 s−1)  and impo [2 s−1) . While t) [2 s−1 . Thus, dM ADP [The modular design has the effect that when recording the respiration of permeabilized cardiomyocytes while adding ADP to the solution outside the cells, peripheral modules restrict the diffusion to more central modules. Indeed, when changing the ADP concentration outside the cardiomyocytes, the peripheral mitochondria respond before the central mitochondria, as is observed in pictures of how mitochondrial autofluorescence changes with the concentration of ADP outside the cell . A threeM ADP . This alM ADP . Thus, tThere are several advantages of the modular design of cardiomyocytes. In terms of energy transfer, it reduces diffusion distances and leads to what is termed ‘direct adenine nucleotide channelling’. In adult cardiomyocytes, direct adenine nucleotide channelling from mitochondria to myosin ATPase and SERCA is almost or as efficient a source of ATP as the CK system ,104. In While most of the modelling approaches focus on intermyofibrillar mitochondria for the sake of simplicity, it should be noted that during the functional recordings of respiration or autofluorescence, it is not possible to distinguish them from perinuclear or subsarcolemmal mitochondria. The three different subpopulations of mitochondria have their own morphology and biochemistry . As subs. 102+-uptake is receiving increased attention [The barriers between modules explain approximately 50% of the overall diffusion restriction with the remaining 50% being at the level of the OMM, but it is not possible to distinguish whether it is owing to the SR and/or the OMM . The OMMttention ,130.2+-uptake is a close association between the SR and the mitochondria. This allows the formation of microdomains with high Ca2+ concentrations, as is necessary for Ca2+ uptake through the mitochondrial Ca2+ uniporter, which has a low Ca2+ affinity of 10–30 µM [2+ concentration has not been measured in cardiomyocytes, but mathematical modelling suggests that mitochondrial Ca2+-uptake does not play a role in e-c coupling on a beat-to-beat basis [2+ transport and energy transfer. Some studies have suggested that the SR is continuous with the endoplasmic reticulum (ER), and may carry out the same functions such as protein synthesis and folding [The structural prerequisite for mitochondrial Ca10–30 µM . To the at basis . Howeverat basis . Three-dat basis ,134. In at basis . On the at basis . Further folding . This is folding . Thus, t folding . The ext folding . It woul3-receptors, and VDAC2 interacting with RYR2 are aimed at Ca2+ shuttling, whereas VDAC1 interaction with ANT and hexokinase is aimed at metabolite shuttling [2+, while outside these contact regions, VDAC1 and ANT form couplons for exchange of ATP/ADP [Recent studies suggest that submitochondrial heterogeneity in VDAC isoform distribution should also be considered. Although the roles of the different VDAC isoforms may to some extent be overlapping, they are also distinct. It seems that VDAC1 interacting with IPhuttling . It is l ATP/ADP . If most ATP/ADP ,104 suggin vivo, where the cardiomyocytes receive multiple substrates for the citric acid cycle and oxidative phosphorylation. Glutamate reduces the open probability of VDAC [M ADP [in vivo continuously contract and relax against a load. Tubulin, being an important part of the cytoskeleton, is a known regulator of VDAC permeability [in vivo. In permeabilized cardiomyocytes, the fraction of VDAC molecules that were accessible to ADP was surprisingly small, approximately 2% [If the diffusion restriction by the OMM does not limit the performance of myosin ATPase and SERCA, it raises questions about the physiological role of the CK system. As noted above, transgenic mouse models suggest that Mi-CK is not needed to facilitate energy transfer across the OMM, at least at baseline levels of activity. This speaks in favour of that the main role of the CK system in the heart is as a temporal energy buffer, regenerating ATP when the ATP-demand exceeds the ATP generation by mitochondria. This could happen during abrupt changes in workload such as during a fight-or-flight response. The role of the CK system as a spatial energy buffer is still not clear. It is conceivable that spatial energy buffering might be necessary at high workloads. As noted above, the performance of CK and creatine-deficient hearts is limited at high workloads ,102. Mea of VDAC , and it C [M ADP  is owingeability  and coulately 2% , but thi. 11M ADP is approximately 80 µM [M ADP of 100–200 µM [As noted in the beginning, cardiomyocytes from newborn mammals are slender and have a single, peripheral ring of myofilaments surrounding a central core of mitochondria, and a sparsely developed SR. Their morphology is very similar to that of trout cardiomyocytes . In permly 80 µM . As meas0–200 µM ,38. This0–200 µM . Again, In terms of facilitated energy transfer by CK, it should be noted that in neonatal mice and rabbits, Mi-CK activity is low, and it is not yet coupled to respiration ,142. TroM ADP increases [During development, the cardiomyocytes loose most of their cytosolic space to become densely packed with t-tubules, myofilaments, SR and mitochondria . As the ncreases ,30 indic. 12M ADP is lower [There are few functional studies of what happens to the diffusion barriers in diseased hearts. After acute ischaemia and coronary artery ligation, the apparent Kis lower ,144 indiis lower ,144, butis lower ,39,49,52is lower ,146.. 132+ concentrations than in the cytosol, and these are crucial for rapid and adequate Ca2+ signalling in e-c coupling. The picture is more complicated when looking at energy transfer, where it is counterintuitive that the OMM and perhaps the SR restrict diffusion of ADP and ATP between ATPases and the mitochondria. However, the overall organization of the SR and mitochondria together with protein dense parts of the sarcomeres form modules within the cells. This modular design keeps diffusion distances relatively short. Thus, whereas e-c coupling relies on microcompartments for better communication through Ca2+, energy transfer relies on macrocompartments for a tight communication between ATPases and mitochondria. Studies on mice, in which the CK system is inhibited, suggest that the modular design of cardiomyocytes ensures a sufficient energy transfer at baseline workloads. However, in failing hearts with disorganized structures, energy transfer may be compromised as the modules are disrupted.Whereas most of this review has focused on diffusion barriers and how they govern energy transfer within the cell, it started with a description of how the overall morphology, e-c coupling and energetics change during ontogeny in mammals. In addition, parallels were made between cardiomyocytes from fishes and neonatal mammals. The latter information was included to illustrate the bigger picture: cardiomyocytes with similar workloads—such as in fishes and neonatal mammals—are remarkably similar in both morphology, e-c coupling, energetics and energy transfer. Thus, studies of different species can provide information about general principles in cardiac physiology. As mammals develop, cardiac performance increases and the cardiomyocytes adapt to the higher workload. They grow in diameter and become more structurally packed. Multiple interchanging rows of myofilaments and mitochondria take up approximately 90% of the cell volume and are intersected by t-tubules and the SR. The juxtapositioning of the SR with t-tubules and mitochondria forms microcompartments with higher Ca


We further showed that the combination of 8GL and an mTOR inhibitor synergistically elicited AML cell differentiation in vitro. Importantly, 8GL alone or in combination with an mTOR inhibitor remarkably impaired tumor growth and extended mouse survival in an AML xenograft model accompanied by enhanced cell differentiation. Notably, genetic or pharmacological activation of GPR132 triggered the differentiation of human primary AML cells. In summary, this study demonstrated that activation of orphan GPR132 represents a potential strategy for inducing myeloid differentiation in AML patients.Blocked cellular differentiation is a critical pathologic hallmark of acute myeloid leukemia (AML). Here, we showed that genetic activation of the orphan GPCR GPR132 significantly induced cell differentiation of AML both in vitro and in vivo, indicating that GPR132 is a potential trigger of myeloid differentiation. To explore the therapeutic potential of GPR132 signaling, we screened and validated a natural product 8-gingerol (8GL) as a GPR132 agonist. Notably, GPR132 activation by 8GL promoted differentiation and reduced colony formation in human AML cell lines with diverse genetic profiles. Mechanistic studies revealed that 8GL treatment inhibits the activation of the mammalian target of rapamycin (mTOR), a regulator of AML cell differentiation blockade, via activating GPR132-G AML is the most common type of acute leukemia affecting adults, with an estimated number of new cases and deaths in the United States of 20,240 and 11,400, respectively, in 2021 . In the G protein-coupled receptors (GPCRs) are the largest and most diverse family of cell–surface proteins in humans . These tBlocked cell differentiation is a critical pathologic feature of AML . In this2 at 37 °C. Cell lines were identified by STR profiling and were confirmed to be mycoplasma free.HL60, MV4-11, Kasumi1, THP1, HEK293, and CHO cell lines were obtained from the Stem Cell Bank . NB4 and OCI-AML3 cell lines were obtained from the American Type Culture Collection (ATCC). AML cell lines were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin solution . HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), and CHO cells were cultured in DMEM/F12 medium. All cells were maintained in a humidified incubator with 5% COGPR132 in GPR132-Tango  was subcloned into the pLVX-TetOne-Puro vector. HEK293 cells were transfected with Tet-On-GPR132, psPAX2 , and pMD2.G , and then the lentivirus supernatant was collected. Human AML cell lines were resuspended in a medium containing 70% lentivirus supernatant, 10% FBS, 20% fresh RPMI 1640 medium, and 1 μg/mL polybrene . Subsequently, infected cells were subjected to puromycin selection (1 μg/mL). GPR132 expression was induced by treatment with 1 μM doxycycline.A tetracycline (tet)-induced gene expression system in AML cells was established using a Tet-On lentiviral vector . Briefly, the full-length cDNA of https://www.benchling.com/academic/): sg1 (5′-AGGGCCTCTAGGGAACCGTG-3′) and sg2 (5′-GGGGTGCCAGGAGTCCCCAG-3′). In brief, sgRNA sequence was subcloned into the lentiCRISPR v2  and lentivirus supernatant was produced as described above. Totally, 5 × 106 AML cells were suspended with 2 mL medium containing 70% lentivirus supernatant, 10% FBS, 20% fresh RPMI 1640 medium and 1 μg/mL polybrene, and then selected with 0.5 μg/mL puromycin.GPR132 knockout cell lines were established by CRISPR–Cas9 system . Two dis3 per well) and treated with indicated concentrations of compounds for 72 h. Cell viability was then determined by MTS assay , and optical density was measured at 490 nm using a CytationTM 5 Imaging Multi-Mode reader . Cells were counted for proliferation determination. Cell viability and cell proliferation were uniforms as a percentage versus control (vehicle set at 100%).Cell viability and proliferation assay were performed as described in our previous study . BrieflyThe in vitro synergistic study was performed as described previously . BrieflyThe cellular morphology of AML cell lines or human primary AML cells was assessed by Wright–Giemsa staining as described in our previous study .Treated AML cell lines or human primary AML cells were incubated with 1 mg/mL nitro-blue tetrazolium (NBT) (Sigma) for 30 min at 37 °C and then smeared to coverslip followed by horizontal centrifugation of 10 s at 1500 rpm. The NBT formazan was imaged by microscope (Olympus) or quantified by measuring absorbance at 560 nm.TM  [3 cells per well) in methylcellulose media and pre-treated with indicated concentrations of compounds at 37 °C. Cells were allowed to grow for 5–7 days, and formed colonies were photographed under an inverted microscope . Formed CFU was counted and uninformed as a percentage versus control (vehicle set at 100%).We used the colony formation assay to assess the ability of cells to form colonies and to measure the proliferative potential of AML cells. A colony-forming unit (CFU) is defined as a viable clonogenic cell cluster with more than 50 cells. Colony formation assay was performed as described previously by using a methylcellulose-based medium Methocultologies) . In brie6 cells per well and treated with different compounds for 72 h. Subsequently, treated cells were resuspended in phosphate-buffered saline containing 1 mM EDTA and 2% FBS, and incubated with FITC-conjugated anti-CD11b or PE-conjugated anti-CD14 (BioLegend) for 30 min in the dark at room temperature. Flow cytometer data were collected by BD FACS Caliber  and analyzed through FlowJo .The cell surface markers of differentiation were evaluated by flow cytometry as described in our previous study . AML celTreated cells were washed three times with PBS and lysed at 4 °C with radioimmunoprecipitation lysis buffer (RIPA)  containing protease and phosphatase inhibitors . Protein concentrations were measured using bicinchoninic acid assay . Proteins were subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with specific primary antibodies overnight at 4 °C, followed by exposure to corresponding secondary antibodies. Full-length western blots were provided in the Supplemental Material.TM RT reagent Kit  according to the manufacturer’s protocol. Quantitative real-time PCR was performed using SYBR green reaction mixture in the ABI Q3 fast real-time PCR system . The gene expression was analyzed via the ΔΔCt method after normalization to GAPDH expression. The primers used in this paper were listed in Supplemental Table RNA isolation and reverse transcription from tumor tissues were conducted by using TRIzol reagent kit  and PrimeScript4 cells per well. After 24 h, the medium was refreshed, and cells were incubated with compounds at indicated concentrations for 18 h. Subsequently, cells were lysed with Passive Lysis buffer  for 15 min at room temperature in a shaker at 70 rpm. The cell lysate was transferred to white 96-well plates, and the bioluminescence was determined immediately after the addition of luciferase substrate  with a CytationTM 5 Imaging Multi-Mode reader .Tango assay was performed as described in our previous study . CHO cel4 cells per well). After overnight incubation, cells were treated with varied concentrations of compounds for 24 h. Luciferase activities were measured by Luciferase Assay System  using CytationTM 5 Imagine Multi-Mode reader.cAMP-responsive element (CRE) reporter assay was performed as described in our previous study with a minor modification . CHO cel® β-arrestin recruitment assay was performed [4 cells per well. Twenty-four hours later, cells were loaded with Nano-Glo Live Cell Reagent  in a CO2-independent medium at 37 °C. Then, compound stock solutions were dispensed to 96-well plates, and luminescence signals were monitored continuously for 1 h or more at 37 °C with a CytationTM 5 Imagine Multi-Mode Reader .To detect ligand-induced interaction between GPR132 and β-arrestin, a NanoBiterformed . HEK293TSix-to-eight-week-old female BALB/c Nude mice and NSG mice were obtained from the National Rodent Laboratory Animal Resources . All animal treatments were conducted according to Institutional Animal Care and Use Committee guidelines and under an institutional protocol approved by East China Normal University. All mice were blinded to the group allocation during the experiments.7 Tet-on-GPR132 HL60 cells or control HL60 cells were subcutaneously injected into the mid-right flank of BALB/c Nude mice. When tumor volume reached 300 mm3, mice xenografted with Tet-on-GPR132 HL60 or control HL60 were randomized into 2 groups (6 mice/group), respectively, and administered with doxycycline (1 mg/mL Dox in drinking water) or vehicle for 2 weeks.To examine the in vivo role of GPR132 in AML, 1 × 107 HL60 cells were subcutaneously injected into the mid-right flank of BALB/c Nude mice. For 8GL alone, mice were randomized into 3 groups (7 mice per group) and intraperitoneally injected with 8GL (30 mg/kg and 60 mg/kg) or with the vehicle daily for continuous 2 weeks; for 8GL in combination with everolimus, mice were randomized into 4 groups (6 mice per group) and intraperitoneally injected with vehicle, 8GL (30 mg/kg) and/or everolimus (3 mg/kg) daily for two continuous weeks. Tumor volume and mouse body weight were recorded every 2 days.To evaluate the in vivo anti-AML activity of 8GL alone or in combination with mTOR inhibitor everolimus, 1 × 107 HL60-Luc cells in 0.1 mL serum-free RPMI 1640 medium on day 0. Two days after inoculation, the mice were randomly divided into four groups of 6 mice and intraperitoneally injected with vehicle, 8GL (30 mg/kg), and/or everolimus (3 mg/kg) daily for 5 continuous weeks. Leukemia burden was assessed using noninvasive bioluminescence imaging using a Xenogen IVIS-200 Optical in vivo imaging system . Bioluminescence intensity was recorded on Day 0, Day 7, and Day 14.NSG mice were intravenously inoculated with 1 × 10st People’s Hospital, or Xinhua Hospital, Shanghai Jiao Tong University School of Medicine. Written informed consent was obtained from the patients and approved by the Ethics Committee for Medical Research (IRB) at Shanghai Jiao Tong University School of Medicine. The patient’s information was provided in Supplemental Table + cells were enriched via immunomagnetic bead selection. Cells were inoculated in IMDM with 20% calf serum supplemented with SCF, IL-3, IL-6, Flt-3L, and TPO.Human samples were supplied by the Department of Hematology at the 1GPR132 overexpression or knockdown in human primary AML cells was performed as described previously . The GPRGPR132, ADGRL4, GPR37, and GPR6) were performed using the GSE12417 (GEO database) or BEAT-AML cohort (http://vizome.org/aml/). Elder AML patients aged over 50 were selected for the Kaplan-Meier analysis. The significance of survival curves was calculated using a log-rank test in the GraphPad Prism 7.The dataset of AML was downloaded from TCGA (TCGA-LAML), GEO DataSets , and Vizome database (BEAT-AML cohort). Gene Set Enrichment Analysis was performed using GSEA software (GSEA v4.1.0). GEPIA2 was utilized for gene expression correlation analysis . PrognoSt-test and represented as mean ± SD unless noted otherwise. The analysis of TCGA-LAML or BEAT-AML RNA-seq data for the correlation between GPR132 and AML markers was performed using a two-tailed Pearson’s correlation test. The analysis of the survival data in the PrognoScan database was performed using the log-rank (Mantel–Cox) test. The P values were designated as significant , and non-significant (P > 0.05). No statistical methods were utilized to determine the sample size. Sample sizes were chosen based on previous experimental observations. The sample sizes of each experiment were shown in the figure legend. No data were excluded from the analysis. Detailed materials and methods are provided in the supplementary materials.All statistical analysis was performed with unpaired Student’s ADGRL4, GPR6, GPR37, and GPR132, were correlated with the survival of AML patients in three distinct gene sets of the AML cohort , GPR37 (P = 0.51) and ADGRL4 (P = 0.51) are not associated with the prognosis of AML patients  predicted a superior prognosis, suggesting a potential protective role of GPR132 in AML  [A list of orphan GPCRs   (Supplem cohort) , 31.Fig.We further determined the expression level of GPR132 across normal hematopoiesis development using a publicly available dataset [To explore the role of GPR132 in AML, we performed gene set enrichment analysis (GSEA) using The Cancer Genome Atlas (TCGA) AML dataset . Intrigu+ in both HL60 (Tet-On-GPR132) and MV4–11 (Tet-On-GPR132) cells  classification  cells. Notably, we found that GPR132 overexpression following doxycycline treatment significantly impaired the growth of HL60 (Tet-On-GPR132) xenograft Fig. . However50) value of 9.00 μM  Fig. . MoreoveMolecular docking revealed that 8GL interacted with residues Y49, W98, F122, and L294 of GPR132 Fig. . MutatioSPI1 and CEBPA in HL60 and MV4–11 cells  and MV4–11 , 8GL could induce differentiation in other AML cell lines, such as OCI-AML3  and THP1  , compared to the control group , whereas the drug regimen involving 8GL and everolimus significantly impaired tumor growth (62.50 ± 4.03% for drug pair) Fig. . SimilarTo further examine the activity of the combined therapy in vivo, we generated a systemic leukemia model by intravenous injection of HL60-Luc cells. Tumors in the vehicle-treated group grew rapidly and diffused throughout the mouse body by day 14. A single treatment with 8GL or everolimus retarded tumor growth, whereas the combination therapy showed superior anti-tumor activity than either of the single drug used alone Fig. . The surNext, we investigated whether GPR132 could enhance the differentiation of human primary AML cells. We initially overexpressed GPR132 in human primary AML cells and demonstrated that GPR132-overexpressing cells had ~25‒57% fewer colony numbers than control cells, as determined by an in vitro colony forming assay Fig. . Overexp+ differentiated AML cells  [Several endogenous oxygenated free fatty acids, including 9(S)-HODE, have been reported to be potential ligands of GPR132 with micromolar potency (9(S)-HODE: EC = 9 μM) , 65. How = 9 μM) . Thus, tIn this study, natural 8GL was identified as a GPR132 agonist. We further observed that 8GL impaired leukemic cell growth and extended mouse survival in preclinical AML models, likely through direct activation of orphan receptor GPR132. Notably, 8GL effectively triggered the differentiation of human primary blast cells derived from AML patients. These comprehensive preclinical results demonstrated 8GL’s anti-leukemic activity as a GPR132 agonist. Considering the chemical skeleton differences distinguishing 8GL and other reported in vitro GPR132 agonizts, as well as it is in vivo safety and efficiency in AML models, our results support a rationale for exploiting more potent and specific GPR132-targeted 8GL derivatives for the treatment of different types of AML. A recent study has demonstrated that EGFR/STAT/ERK pathway is involved in the anti-cancer activities of 8GL in colorectal cancer in vitro . Thus, fThe role of cAMP in AML signaling and its clinical significance has been previously described. For example, cAMP induces cell growth arrest and enhances chemotherapy-induced AML cell differentiation . In a clDysfunctional mTOR signaling is closely involved in AML progression . Here, wIn conclusion, our results demonstrated that genetic or pharmacological activation of the orphan receptor GPR132 induces leukemic cell differentiation, providing a potential new therapeutic approach to treat patients with AML. Our results also demonstrated that GPR132 activation by 8GL acts synergistically with an mTOR inhibitor by co-targeting mTOR signaling in vitro and in vivo. The in vivo safety and efficacy of 8GL warrant consideration of novel, optimized GPR132 agonizts for AML differentiation therapy.Supplemental materialsFull length western blotsReproducibility checklist


More than just a buzzword in business and education, mentoring is a complex interactional process that, at its best, fosters personal and professional development for all involved. In other words, a good mentoring relationship can be both transformative and reciprocal. This raises the question of what is (and is not) a good mentoring relationship? Moreover, how does one enter into and capitalize on the benefits of mentorship? While some mentoring relationships are intentionally created, others seem to evolve so organically that participants are not immediately aware of mentoring’s presence. The current presentation looks at the nature of mentoring relationships within the context of gerontology and geriatrics education from both the mentor and mentee perspectives.


Urticaria is a symptom of acute skin allergies that is not clearly understood, but mast cell histamine is hypothesized to cause swelling and itching. Omalizumab, an anti-human IgE antibody that traps IgE and prevents its binding to high-affinity IgE receptors, is effective in treating urticaria. We recently experienced a case of urticaria refractory to antihistamine therapy in which the peripheral-blood basophil count responded to omalizumab therapy and its withdrawal. Furthermore, the peripheral-blood basophils showed an unexpected increase in the expression of a cell surface activation marker. This phenomenon has been reported by other analyses of basophil and mast cell dynamics during omalizumab treatment. Here, we analyze these observations and formulate a hypothesis for the role of basophils in urticaria. Specifically, that activated basophils migrate to the local skin area, lowering peripheral-blood counts, omalizumab therapy alters basophilic activity and causes their stay in the peripheral blood. We hope that our analysis will focus urticaria research on basophils and reveal new aspects of its pathogenesis. Urticaria is a common skin disease characterized by itchy, red, raised bumps or hives. It is generally classified based on its duration, acute (≦ 6 weeks) or chronic (>6 weeks), and the relevance of eliciting factors, spontaneous (no specific eliciting factor involved) or inducible (specific eliciting factor involved) . Based oThe etiology and pathogenesis of CSU remain largely unexplored. However, there is a consensus that cutaneous mast cells play a major role by releasing histamine, which acts on blood vessels and nerves to cause swelling and provoke itching, respectively. Antihistamine is the standard treatment for urticaria and antihistamine-resistant patients are well-controlled with omalizumab, an anti-human IgE antibody, which reconfirms that IgE is important in the pathology of urticaria, even in CSU with unknown triggers.We recently reported a case with a 6-year history of severe CSU, whose peripheral basophil count was persistently low  for more than 1 year . Upon urThe contribution of basophils to allergic reactions, acquired immunity, and autoimmune diseases is better understood , 5, thouFCER1A that codes α-chain of FcϵRI, CPA3 that codes carboxypeptidase A3, and HDC that involves histamine synthesis.The fact that basophils express FcϵRI and release histamine, together with observation that the basophils in peripheral blood inversely correlate with urticarial rash severity, suggests that basophils play a role in CSU. Indeed, as omalizumab has become more frequently used in the treatment of CSU refractory to antihistamine therapy, a number of reports have examined potential predictors of its efficacy –10. ManyThe phenomenon that basophils are inversely correlated with CSU severity is not specific to omalizumab treatment, as it was also observed in CSU patients treated with antihistamines . This reIn clinical practice, basophils account for a very small percentage of peripheral blood cells, which makes it difficult to define an expected value. However, basophil fluctuations can indicate urticaria activity, at least on a patient-by-patient basis.in vitro  onset of omalizumab’s therapeutic effects mirrors basophil dynamics and corresponds with changes in the basophil population leads us to hypothesize that basophils play a large role in CSU pathology Figure 2RT-I, IK, NM, KK and NK contributed to conception and design of the study. RT-I and NK wrote the first draft of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This study was supported by a Grant-in-Aid for Young Scientists (B) to IK (Grant No. 20K17364).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SLC and ABC could produce interindividual differences in clinical outcomes. A systematic review was performed to evaluate the influence of SLC and ABC polymorphisms and their combinations on efficacy and safety in AML cohorts. Anthracycline intake was especially influenced by SLCO1B1 polymorphisms, associated with lower hepatic uptake, showing higher survival rates and toxicity in AML studies. The variant alleles of ABCB1 were related to anthracycline intracellular accumulation, increasing complete remission, survival and toxicity. Similar findings have been suggested with ABCC1 and ABCG2 polymorphisms. Polymorphisms of SLC29A1, responsible for cytarabine uptake, demonstrated significant associations with survival and response in Asian populations. Promising results were observed with SLC and ABC combinations regarding anthracycline toxicities. Knowledge of the role of transporter pharmacogenetics could explain the differences observed in drug disposition in the blast. Further studies including novel targeted therapies should be performed to determine the influence of genetic variability to individualize chemotherapy schemes.Antineoplastic uptake by blast cells in acute myeloid leukemia (AML) could be influenced by influx and efflux transporters, especially solute carriers (SLCs) and ATP-binding cassette family (ABC) pumps. Genetic variability in  Acute myeloid leukemia (AML) is a clinically and biologically heterogeneous hematologic malignant disease characterized by an excess of blast cells in bone marrow and blood. Approximately 60–80% of young AML patients achieve complete remission (CR) using conventional 3 + 7 schedules of anthracyclines and cytarabine, which might be followed by an allogeneic hematopoietic stem cell transplant  to prevent relapse ,2. UnforSLC and ABC transporters may play a promising role in drug exposure and have been associated with clinical response and toxicity  or “organic anion transporters” [MeSH] or “organic cation transport proteins” [MeSH]) and “acute myeloid leukemia” [MeSH].ABC and/or SLC polymorphisms and clinical response to chemotherapy; and (3) AML studies analyzing the impact on safety of ABC and/or SLC polymorphisms.Studies that fulfilled the following criteria were included: (1) studies based on clinical data in AML patients ; (2) AML studies analyzing the associations between Our systematic search obtained 569 citations from databases and journals and 21 records were identified through other sources . Of the SLCO1B1) and the organic cation transporter SLC22A16 .The intake by blast cells and other tissues of the antineoplastics employed in AML therapy and other xenobiotics is mediated by SLC transporters, a family that includes more than 400 transporters. Different SLC transporters have been related to anthracycline uptake, especially the organic anion transporter polypeptide-1B1  is predominantly expressed in the liver and is involved in the hepatic uptake and plasma clearance of several organic anionic compounds, including anthracyclines and other drugs such as statins ..SLC28A3 SLC29A1 gene) is responsible for up to 80% of cytarabine influx in blast cells. Schemes with high doses of cytarabine (2–3 g/m2 daily), used in consolidation or intensification therapy, can saturate the pump-mediated transport of hENT1 with concentrations >10 µmol/L and produce free diffusion into the cell . Sud safety  [20,56,6ABCC2 expresses MPR2, an export pump localized to the apical membrane of polarized cells, especially those hepatocytes with functions in biliary transport. This protein appears to contribute to the drug resistance of different anticancer drugs including anthracyclines [ABCC2 have been correlated with anthracycline toxicities in other malignancies: cardiotoxicity in non-Hodgkin lymphoma (rs45511401) [ABCC2 rs8187710, without any significant influence in response or toxicity [cyclines . Polymor5511401) , in surv5511401)  and in p5511401) , febrile5511401)  and leuc5511401) . In AML toxicity .ABCC3 encodes a protein that may play a role in biliary transport and the intestinal excretion of organic anions, which is also related to drug efflux. The expression of ABCC3 was found to be significantly higher in AML patients resistant to daunorubicin [ABCC3 polymorphisms , whereas other ABCC3  SNPs showed a higher DFS [ABCC3, rs4793665 [ABCC3, rs4148405 [orubicin . Clinicaorphisms  [16,18,8s4148405 .ABCC10 and ABCC11 genes encode the MRP7 and MRP8 pumps which can efflux cytarabine in blast cells [ABCC10 and ABCC11 in AML populations. Sorafenib, an FLT3 inhibitor employed in AML, produces the inhibition of ABC pumps, avoiding the efflux of cytarabine by MRP7 and MRP8 pumps and thereby increasing the cytarabine-sensitivity of blast cells [The st cells ,50. Unfost cells ,90.ABCG2 gene expresses the “breast cancer resistant protein” (BCRP), a well-known ABC pump responsible for anthracycline efflux [ABCG2 was observed in 33% of blast cells and this BCRP expression correlated with a worse prognosis and lower OS [ABCG2 SNPs are rs2231137 and rs2231142, and the minor alleles of these SNPs are related to a reduced level of BCRP expression [ABCG2 in an AML cohort with daunorubicin  [ABCG2 genotypes in AML  . Severals in AML  [18,56,6n cohort  and no in cohort . On the carriers ,98 and ccarriers . Similarorphisms . No effege study .SLC wild-type genotypes , ensuring the anthracycline uptake in cells, with the variant genotypes of ABC pumps , avoiding anthracycline expulsion [ABCB1 and SLC polymorphisms, including higher hepatic and renal toxicities, mucositis and neutropenia, as well as a higher incidence of induction death  [ABC pumps was not explored.Most of the included pharmacogenetic studies employed the candidate genes approach based on the pharmacologic pathway of the drugs. The drug intake depends on the combination of input and output transporters, but only a few studies analyzed the genetic variability of both types of carriers together. A recent study explored the combination of xpulsion . Severalon death . All of CB1 SNPs . In additoxicity , reproduinotecan ,100 and inotecan ,102. RegA, etc.) , but theSLC and ABC families play crucial roles in the absorption, disposition and elimination of antineoplastic drugs. In AML, the expression of these transporters has been proposed as one of the main drug resistance mechanisms and has been widely studied for standard chemotherapy 3 + 7 schedules based on anthracyclines and cytarabine. However, the impact of genetic variability in the SLC and ABC genes remains controversial. This review aims is to demonstrate that polymorphisms in transporter genes may have a potential impact on the clinical outcomes of AML therapy.Transporters of the SLC carriers in AML therapy; promising findings were obtained with polymorphisms in the SLCO1B1 and SLC29A1 genes. Variant alleles of SLCO1B1 were correlated with a lower function, decreasing anthracycline hepatic uptake and metabolism [SLC29A1, responsible for cytarabine uptake, showed a relevant impact on CR and survival rates, especially in Asian populations [Despite this, only a few studies have analyzed the role of tabolism ,11 and stabolism ,13,14. Pulations ,43,44,45ABCB1 have been widely studied in AML, demonstrating a clear association with lower pump function, as well as higher CR and survival rates in meta-analyses [ABCB1 polymorphisms in anthracycline-related toxicities remains more controversial in AML, with scarce relevant findings [ABCC1 [ABCG2 polymorphisms [Meanwhile, the variant alleles of analyses ,74. The findings ,62 and wfindings ,23,75,76s [ABCC1 ,62,82,83orphisms ,62,97,98SLC or ABC genes only demonstrate a minor effect that could be affected by their combination [SNP–SNP combinations of transporters could play a crucial role in characterizing the anthracycline pathway, which involves complex pharmacokinetic and pharmacodynamic mechanisms, although this was only evaluated in a Caucasian AML cohort . In addibination . Combinabination ,44.ABC pumps in anthracycline pharmacokinetics has been suggested in vitro [ABCB1 and ABCG2 polymorphisms [CDA genotype, the main enzyme responsible for liver metabolism of cytarabine [ABCB1 and SLCO1B3 [SLCO1B1 521T>C SNP reduced methotrexate clearance [ABC and SLC SNPs in drug bioavailability have found the same limited evidence of PK studies in the AML context [The influence of in vitro ,80 and sin vitro , but a porphisms . Furthertarabine . In chro SLCO1B3 . Similarlearance . Previou context ,107,108.SLC and ABC and their combinations) in AML studies, which was briefly explained in this recent review [The influence of genetic variability in AML therapy has been previously analyzed by other authors, especially focused of the main SNPs of the cytarabine and anthracycline metabolic pathways ,109,110 t review .SLC and ABC genes should be analyzed in further studies involving these novel therapies. In this line, a higher response to gemtuzumab ozogamicin was reported with the variant alleles of ABCB1 in a pediatric cohort [Most of the reported pharmacogenetic studies were performed in patients treated with a standard 3 + 7 scheme with a candidate genes approach. The importance of pharmacogenetics for the multiple new drugs recently approved for AML treatment remains unknown. Although these therapies are more tolerable than classical antineoplastics, potential drug–drug interactions involving P-gp, BCRP and OATP transporters have been described . The genc cohort , but no c cohort .SLC and ABC) with AML outcomes and safety profiles. Unfortunately, most of these studies were observational and involved retrospective cohorts, and only anecdotally were these transporter genes analyzed together with metabolic enzymes, molecular targets and DNA repair genes. In the future, randomized clinical trials on larger populations including those of different age, ethnic and therapy groups should be developed in order to validate the clinical benefit of pharmacogenetics in AML patients.In conclusion, pharmacogenetic studies based on candidate genes have reported relevant associations between SNPs in transporters (