In microorganisms, fungi, and plants, H+/Ca2+ antiporters (CAX) are known to play key roles in the homeostasis of intracellular Ca2+ by catalyzing its efflux across the cell membrane. Here, we reveal that the bacterial CAX homolog YfkE transports Ca2+ in two distinct modes: a low-flux H+/Ca2+ exchange mode and a high-flux mode in which Ca2+ and phosphate ions are co-transported (1:1) in exchange for H+. Coupling with phosphate greatly accelerates the Ca2+ efflux activity of YfkE. Our studies reveal that Ca2+ and phosphate bind to adjacent sites in a central translocation pathway and lead to mechanistic insights that explain how this CAX alters its conserved alpha-repeat motifs to adopt phosphate as a specific “transport chaperon” for Ca2+ translocation. This finding uncovers a co-transport mechanism within the CAX family that indicates this class of proteins contributes to the cellular homeostasis of both Ca2+ and phosphate.Ca 2+ and inorganic phosphate (Pi) together in a 1:1 ratio, exchanging them for H+ and becoming a high-flux antiporter when coupled with Pi.Bacterial CAX antiporter homolog YfkE can transport Ca Ca2+ signals are typically initiated by Ca2+ influx into the cytoplasm via selective membrane channels and is further augmented by Ca2+ efflux from intracellular organelles such as the endoplasmic reticulum. Sustained high levels of cytosolic Ca2+ are however lethal. Calcium-cation antiporters (CaCA) are key to restore Ca2+ homeostasis. This family of membrane transporters, which includes H+/Ca2+ antiporters (CAX) and Na+/Ca2+ exchangers (NCX), sequester and actively export Ca2+ out of the cytosol, powered by the influx of H+ or Na+ down transmembrane electrochemical gradients4.Calcium ions are among the most abundant metal ions in nature and are crucial for many important functions in cells, where Ca3. For example, three CAX homologs, known as CAX1-3, have been identified in Arabidopsis, and more than twenty others in apple trees5. In plant cells CAX proteins are present in both the plasma and tonoplast membranes and facilitate the movement of cytosolic Ca2+ either out of the cell or back into the acidic vacuoles, in response to various stimuli including cold, salinity, and soil pH changes8. CAX homologs are also ubiquitous in microorganisms, although their physiological roles require further investigation. Ca2+ is involved in a broad range of bacterial processes including chemotaxis, cell division, and sporulation;9 Ca2+ signaling has also been implicated in bacterial infection and host–pathogen interactions10. CAX proteins are likely to play a central role in these processes, as they appear to be the major Ca2+ efflux systems in bacteria. Therefore, elucidating the transport mechanism of CAX will help understand how these organisms maintain Ca2+ balance in response to Ca2+ perturbations and signaling.CAXs are ubiquitous in bacteria, fungi, and plants+ and Ca2+ recognition. X-ray structures have recently begun to reveal the molecular basis for the mechanism of this class of antiporters, namely those of YfkE from Bacillus subtilis, VCX1 from Saccharomyces cerevisiae, and CAX_Af from Archaeoglobus fulgidus13. These structures suggest a mechanism wherein helices TM2-3 and TM7-8 adopt distinct arrangements that alternately expose recognition sites for H+ and Ca2+ to either side of the membrane. These three structures however capture a similar state in the alternating-access mechanism, namely one that is inwardly open , in which two conserved glutamate residues important for the H+/Ca2+ exchange activity are adjacently located in a central ion-binding site. These two carboxylate residues coordinate one Ca2+ ion in the VCX1 structure11. Whether the structures of YfkE and CAX_Af capture an unliganded state or one bound to H+ was not immediately apparent, although the two carboxylate residues (E72 and E255 in YfkE) adopt different conformations compared to the Ca2+-bound state of VCX1. Recently, our studies for YfkE have also uncovered that its alternating-access mechanism is allosterically regulated by Ca2+ binding to an intracellular Ca2+ mini-sensor12.Structurally, all CAX proteins appear to share a common basic architecture, consisting of eleven transmembrane (TM) helices. Two conserved sequence motifs, referred to as α-repeats, are found in helices TM2-3 and TM7-8 and seem to provide sites for H2+ transport mechanism for YfkE, and possibly other CAX family members. We demonstrate that YfkE features a transport mode wherein Ca2+ and inorganic phosphate (Pi) are co-translocated in a 1:1 ratio, in exchange for H+. Coupling to Pi transforms YfkE from a low-flux to a high-flux antiporter. To gain insights into this Ca2+-Pi co-transport mechanism, we utilize multiple biochemical and biophysical approaches together with computer simulations and experiment-based molecular modeling. Our results lead us to conclude Ca2+ and Pi bind to a shared recognition site within the transporter interior, which includes E72 and E255 as well as other polar residues in the vicinity. E72 and E255 can however bind H+ instead, explain the ability of YfkE to harness the H+ gradient to drive Ca2+ and Pi transport. Our results also enable us to rationalize why translocation of Pi anions accelerates the Ca2+/H+ exchange reaction.Here, we reveal a type of Cai is an important nutrient and is abundant in the cells for the synthesis of nucleotides, energy, and phospholipids. We provide an example that a transporter protein concurrently facilitates the membrane translocation of Ca2+ and Pi. Our findings not only reveal a unique transport mode within the CAX family, but also suggest its physiological role in the homeostasis of both Ca2+ and phosphate in cells.P2+ transport activity using inside-out vesicles, after an outward H+ gradient was established by adding NADH, which activates H+ pumping into the vesicles by the electron transport chain. As expected, the resulting time course showed that Ca2+ was transported into the YfkE vesicles  to the external solution  strongly accelerated the Ca2+ transport activity of YfkE by eightfold 2, the major form of calcium phosphate compounds at neutral pH, is highly soluble in aqueous solutions. Consistently, we observed no Ca2+ accumulation in control vesicles devoid of YfkE even when Pi was present  and no influx was detected when Ca2+ was absent   Fig. . These ri transport activity of YfkE WT was significantly decreased when the Ca2+ concentration was reduced from 0.5 mM , based on analysis of the transport kinetics , yielding a binding affinity of ~1 mM  to our transport assays completely abrogated Ca2+ uptake into the inside-out vesicles, in the absence or the presence of Pi  and polar residues such as threonine, serine and histidine15. The Ca2+ recognition site in the interior of YfkE, formed by E72, E255 and surrounding polar residues, is the only region suitable for Pi recognition in the transmembrane domain of the protein . Specifically, we labeled positions G56 (intracellular TM1-2 loop) and C3 (amino-terminus) with terbium chelate and Atto-465, respectively, using thiol-reactive chemistry , while keeping the Atto-465 acceptor in C3 . LRET measurements report on changes in the distance between a fluorescent donor and its acceptor, manifested as luminescence signals of different lifetimes. Through this approach, we sought to ascertain whether recognition of Catry Fig. . Based otry Fig. . Additio C3 Fig. . As obse2 Å Fig. . Importa2+ and Pi in a manner that results in competition with H+, as is the norm for a coupled ion antiporter. The first step in this process was to determine whether the existing structure of inward-facing YfkE12 captures a H+-bound state. If so, we reasoned that this experimental structure would provide a good foundation to model the state bound to Ca2+ and Pi; indeed, available structural data for membrane antiporters show that alternate substrate-bound forms of the same functional state differ primarily in the configuration of the sidechains forming the substrate binding sites, with no major changes in the protein backbone.To provide a structural context to these biochemical and biophysical measurements, we resorted to molecular modeling. Specifically, we sought to construct a hypothetical model that might explain how YfkE recognizes both Ca+ binding since it is conserved in H+-coupled CAXs, but not in Na+-coupled NCXs12. As negative controls, we also probed E233 and H226, which are exposed on the protein surface and are thus not expected to deviate significantly from their inherent protonation propensities in solution, for a given pH. As summarized in Table To that end, we carried out a series of all-atom molecular dynamics simulations designed to quantify the likelihood of protonation of selected side chains within the transporter interior  directly coordinates Ca2+, while E109 (E72 in YfkE) indirectly interacts with the ion via three water molecules  compared to WT . The model also incorporates information derived from a survey of the Protein Data Bank, specifically from a protein of known structure that also recognizes Ca2+ and Pi concurrently, namely a Ca2+ -dependent hydrolase known as PON116. This protein exhibits a donut-shaped structure with a central site where Ca2+ and Pi are coordinated, which appears strikingly similar to the binding site in YfkE and VCX1 . The model shown in Fig. 2+ concentration are a universal cellular signaling strategy. Ca2+-mediated signaling is however not only dependent on the magnitude of the changes in concentration but also on the specific rate, frequency, and spatiotemporal patterns of those changes. Generation of these complex signals in response to stimuli requires the concerted action of Ca2+ channels, transporters, and pumps on the plasma membrane and the membranes of intracellular organelles, so as to regulate Ca2+ influx and efflux17. In unicellular organisms and higher plants, CAX antiporters provide one of the primary mechanisms for maintaining this complex Ca2+ balance18.Fluctuations in cytosolic Ca+/Ca2+ exchange activity of the bacterial CAX homolog YfkE can be greatly accelerated in the presence of Pi. Furthermore, our results using both E. coli vesicles  is modestly reduced. Bound Pi does not interact with E72 directly, according to our hypothetic structural model, but it is proximal to E255  of the equilibrium between mono-anionic H2PO41− and di-anionic HPO42− species  has also been proposed for a H+/phosphate transporter29. It is thus conceivable that this kind of H+ hopping mechanisms are common among Pi-coupled transporters. Despite the availability of multiple structures of CAX13, the stoichiometry of H+ vs Ca2+ has been elusive since its determination remains technically challenging. In this study, we found that the transport activity of a CAX homolog is regulated by the presence of Pi, which is itself a protonatable species. Careful characterization of H+ coupling in each transport mode will therefore be required to dissect the interplay between Pi, Ca2+ and H+ in this exchange mechanism.Elevation of cytosolic [Pway Fig.  as suggeway Fig. . The mec255 Fig. c, d. Thuies Fig. . Interes18. All CaCA homologs feature the two sequence motifs known as α-repeats (TM2-3 and TM7-8) featuring the two carboxylate residues used for H+/Na+/Ca2+ coordination . These motifs are however not strictly conserved, and sequence variations therein appear to modulate the substrate selectivity of different transporters and ultimately dictate whether the coupling ion in the exchange cycle is H+ or Na+30 . E54 and E213, the two counterpart carboxylate residues in the Na+/Ca2+ exchanger NCX_Mj, can also bind H+ in the outward-facing state, but the transporter is then inhibited32 as it cannot access the inward-facing state33, in line with the fact that NCX_Mj does not transport H+33. In contrast, Na+ binding facilitates that conformational transition enabling NCX_Mj to harness the inward Na+ gradient. The correspondence between transport and binding specifies is therefore non-trivial and requires mapping of the energetics of the complete alternating-access cycle. Furthermore, sequence variations also result in more complex ion dependencies. For example, in brain cells NCKX catalyzes the co-transport of K+ and Ca2+ in exchange for Na+. This K+ dependence has been attributed to a single aspartate residue within the second α-repeats34. In mitochondria, the Na+/Ca2+ exchanger NCLX can also transport Li+ in exchange of Ca2+, and this transport mode is dependent on a single amino acid substitution in the second repeat35. Our results suggest that H256, conserved among many CAX proteins  but replaced by leucine/threonine in Na+/Ca2+ exchangers  cells harboring a pET28a-YfkE vector were grown in Luria broth medium at 37 °C to A600 of 0.4. Mutations were generated using a standard site-directed mutagenesis approach and confirmed by sequencing. Protein expression was induced by adding 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) for 2 h at 25 °C. Cells were washed with buffer containing 10 mM Tris-HCl pH 7.3, 140 mM KCl, 0.5 mM DTT, 250 mM sucrose. Cell rupture was processed by a single passage through a C3 homogenizer (Avestin) with low pressure (4000 p.s.i.). After removing cell debris by centrifugation, supernatants were centrifuged using a Ti45 rotor at 40,000 rpm for 1 h to pellet membrane vesicles. Vesicles were homogenized in the same buffer and then quickly frozen in liquid nitrogen before use.Inside-out vesicles (ISOs) were prepared by a low-pressure homogenization method. Briefly, 2+ and Pi transport activities of YfkE were measured using inside-out vesicles. Briefly, membrane vesicles were diluted in a buffer (pH 8.0) containing the same components. Before assays, an outward H+ gradient was established by adding 5 mM NADH to vesicles for 10 min. Although it is possible that a small number of right-side-out vesicles are present in our samples, the electron transport chain in these vesicles cannot be energized as NADH is membrane impermeable. Therefore, the only activity measured is that of the inside-out vesicles. For measuring Ca2+ transport activity, 5 mM cold potassium phosphate was also added to the vesicles. To measure phosphate transport activity, 0.5 mM cold CaCl2 was added instead. Transport assays were triggered by adding 45Ca2+ or 32P-Pi (Perkin Elmer) as indicated in the reactions at room temperature. Reactions were terminated by filtration through a nitrocellulose membrane (0.22 µm) on a Millipore filtration manifold and then washed immediately with buffer. The filters were air-dried and counted in a liquid scintillation counter to determine transport activity. Kinetic analysis was performed by data fitting into the Michaelis–Menten single exponential model using the software Graphpad Prism 9. For vesicle assays, membrane vesicles prepared with BL21(DE3) cells harboring empty vector were used as control. For kinetic assays of mutants, the concentration of vesicles was adjusted individually to obtain useful data for model fitting.Both CaE. coli C41(DE3) strain in auto-induction medium38 at 25 °C overnight. Cells were suspended in a buffer containing 20 mM Na phosphate, pH 7.4, 500 mM NaCl, and 20 mM imidazole and then ruptured by three passages through a C3 homogenizer (Avestin) at 15,000 p.s.i. Membrane fractions were collected as described above and suspended in lysis buffer. Membrane fractions were solubilized by adding 1% (w/v) n-dodecyl-β-maltoside (DDM) at 4 °C for 1 h and incubated with Ni-NTA resin . Resins were washed with buffer supplemented with 60 mM imidazole and 0.05% DDM and then eluted with buffer supplemented with 400 mM imidazole and 0.05% DDM. The eluted protein was further purified using a Superdex-200 10/300 GL column  equilibrated in a buffer containing 20 mM HEPES (pH 7.4), 500 mM NaCl and 0.05% DDM.The proteins of YfkE WT and variants were expressed and purified using the same approach. Briefly, protein expression was carried out in E. coli total lipid (Avanti polar lipids Inc.) as follows: Lipids solubilized in chloroform were first dried under inert gas to form a thin lipid film in a glass tube and further kept under vacuum overnight to remove residual chloroform. The lipid film was then resuspended in 20 mM HEPES (pH 7.4), 100 mM NaCl and then sonicated on ice until the suspension became clear. After two freeze–thawing cycles, generated large multilamellar vesicles were passed through a 400 nm polycarbonate filter for 11 times to form unilamellar liposomes using a mini-extruder (Avanti Polar Lipids Inc). Prior to reconstitution, liposomes were first destabilized by adding 5 µl 10% Triton X-100 (wt/vol) per 1 mg lipids at room temperature for 30 min. The YfkE protein was mixed with detergent-destabilized liposomes in a 1:10 (w:w) ratio on ice for 20 min. Detergent was removed by adding bio-beads to the mixture (80 mg beads for 1 ml sample) to form proteoliposomes overnight at 4 °C with gentle agitation. Next day, fresh bio-beads were added for another 3 h to finalize the reconstitution.Proteoliposomes were prepared using i transport activity, 200 µl 4 mg/ml YfkE-reconstituted proteoliposomes (pH = 7.4) were diluted into 400 µl buffer containing 20 mM HEPES (pH = 8.0), 100 mM NaCl, 5 mM 32P-phosphate (Perkin Elmer), and 0.5 mM CaCl2 (or 2 mM EDTA as control) at room temperature. At indicated times, reaction was terminated by filtration through a nitrocellulose membrane (0.22 µm) on a Millipore filtration manifold and then washed immediately with buffer. The filters were then air-dried and counted in a liquid scintillation counter to determine transport activity.To measure P32Pi was added to the beads in the presence of 0.5 mM Ca2+ for 10 min. The beads were then washed extensively using a protein buffer without Pi. The proteins were eluted using 250 mM imidazole and the radioactivity was measured using a scintillation counter.Prior to assays, 10 µg purified His-tag YfkE proteins were mixed with 100 µl Ni-NTA beads. 40 mM Protein conformational changes in the native membrane environment were detected using LRET in ISOs. To ensure specific labeling, two endogenous cysteine residues (C34 and C293) were substituted with valine using site-directed mutagenesis to generate a cysteine-free YfkE construct. A pair of cysteine residues were then introduced for fluorophore labeling: one cysteine residue C3 was inserted at the N-terminus and another cysteine residue was placed at the position of 56 or 202 based on the YfkE structure. An amino-terminal flanking sequence immediately after C3 includes a His6 tag to increase the distance between the donor and receptor for optimal LRET signals and a thrombin cleavage site for background normalization.2 and/or 5 mM potassium phosphate (pH 7.3) were mixed with vesicles.ISO was prepared using the approach described above. Vesicle labeling was carried out using thiol-reactive chemistry for 1 h at room temperature in the dark using 200 nM maleimide derivative of donor and acceptor fluorophores. The fluorophores used are terbium chelate (Invitrogen) and Atto-465 (Sigma). After labeling, vesicles were dialyzed twice against a buffer containing 10 mM HEPES pH 7.3, 140 mM KCl, and 250 mM Sucrose for 2 h at 4 °C to remove excess probes. Before fluorescence scanning, 0.5 mM CaClOrigin software (OriginLab Corp). The sensitized emission of the acceptor was detected before and after thrombin cleavage, a well-established technique used to subtract background fluorescence41. Specifically, after obtaining acceptor lifetime measurements, five units of bovine thrombin  were added to the cuvette and allowed to cleave the N-terminal labeled fluorophore. Cleavage was completed 1–3 h following the addition of thrombin. The donor-acceptor labeled sample was excited at 337 nm, and the emission was detected at 508 nm. Distances were calculated using the Förster equation . Each experiment is reported as an average of three measurements for a given state, with each measurement having an average of 99 pulses from the flash lamp. Data were collected using Fluorescan software  and analyzed using tion Eq. ):1\docum2+ and Pi binding affinity was determined using isothermal titration calorimetry (ITC). Proteins were decalcified by adding 10 mM EGTA and then separated using size-exclusion chromatography. The Ca2+ or Pi solution was prepared in the same buffer. ITC assays were performed by titrating Ca2+ into a solution of 5 or 10 μM YfkE protein on a VP-ITC device . All assays used the same ITC program: The system was thermally equilibrated at 25 °C; after an initial delay of 120 s, serial injections (10 μl each) were done with a spacing time of 240 s at 307 rpm stirring speed. Each measurement was corrected with a background titration in which the ligand was titrated into a buffer solution. Data fitting was carried out using Origin software .Ca12. All simulations were conducted with NAMD 2.942 using the CHARMM36 force field for protein and lipids44. All simulations were carried out at constant temperature (298 K) and semi-isotropic pressure (1 atm), using periodic boundary conditions and an integration time step of 2 fs. Long-range electrostatic interactions were calculated using PME, with a real-space cut-off of 12 Å. Van der Waals interactions were computed with a Lennard-Jones potential, cut-off at 12 Å with a smooth switching function taking effect at 10 Å. The specific protein construct studied is a single protomer including residues 1–177 (TM-TM5) and residues 200-351 (TM6-TM10); the seemingly unstructured cytoplasmic loop connecting TM5 and TM6 was truncated. The protein construct was embedded in a pre-equilibrated hydrated palmitoyl-oleoyl-phosphatidyl-choline (POPC) lipid bilayer using GRIFFIN45 and enclosed in an orthorhombic box of ~88 × 88 × 95 Å in size. The resulting simulation system contains approximately 76,700 atoms, including 207 lipid molecules and a 100 mM KCl buffer  through a set of repulsive potentials acting at a certain threshold distance. Dual topologies (protonated and deprotonated) were introduced for both the protein sidechain and its equivalent in solution. The Free-Energy Perturbation (FEP) algorithm was then used to alter the protonation states of both sidechains, concurrently but in opposite directions; thus, the resulting free-energy value reflects the increase or decrease in the protonation propensity of the protein sidechain with respect to what is intrinsic for that sidechain type in solution 46.We analyzed protein X-ray structures that fulfill the following criteria: (i) the resolution is 3.0 Å or better; (ii) the sequence identity with a previously selected structure is lower than 70%; (iii) the structures have one calcium and one phosphate ion bound, in a common binding site ; and (iv) the binding site is flanked by two acidic and one histidine side-chain, like in YfkE. This survey resulted in one structure, namely that of the mammalian serum paraoxonase 1 (PON1), a calcium-dependent hydrolase (PDB entry 3SRE)2+ and phosphate was generated with MODELLER v9.1347. The procedure followed was akin to that used in homology modeling, except that here the target was the unknown structure of the Ca2+/phosphate-bound state, and the template was the known structure (PDB 4KJR)12. Only the structure of the four TM helices flanking the binding site  was remodeled. To specifically model the geometry of the binding site, with Ca2+ and phosphate bound, we added a set of geometric restraints inferred from the abovementioned structures of PON1 and NCX_Mj30, and Ca2+/phosphate transport studies of wild-type and mutated forms of YfkE. Specifically, the latter have shown that the following mutations cause significant changes in measured KM and/or Vmax values, without abrogating transport: G68A, N69A, N99A, N252A, H256A, and Q281A. Thus, restraints were applied to the following distances between Ca2+ and the protein, using a harmonic potential acting at distances equal to or greater than 2.6 Å (and a ‘deviation’ of 0.1 Å): (1) to each of the four oxygen atoms from the carboxylate groups of E72 and E255; (2) to the nearest of the oxygen atoms of phosphate (modeled as H2PO4−); (3) to the carbonyl of G68; and (4) to the carbonyl oxygen of the N69 sidechain. Taken together, these four restraints imply a Ca2+ coordination number of 7. Analogous restraints were applied to the distances between the phosphorus atom and (5) Nδ of H256 at 3.9 Å, (6) Cγ of N252 (4.5 Å), and (7) Cγ of S278 (4.5 Å); and to the distance between (8) Oε of Q281 and Nδ of N69 (3.2 Å).; and (9) that between Cγ of N252 and Cγ of N99 (4.2 Å). Finally, to impose double-bi-dentate interactions between Ca2+ and E72 and E255, a restraint was also applied to (10) the dihedral angles formed by Ca2+ and atoms Cδ, Oε1, and Oε2 of E72 and E255 . We generated an ensemble of 2000 models in total and analyzed this ensemble through clustering based on pairwise RMSD; this RMSD calculation included all non-hydrogen atoms of residues N69, E72, N99, N252, E255, H256, S278, and Q281, plus the Ca2+ and phosphate ions, and clusters were defined by an RMSD threshold value of 0.6 Å. 15 clusters comprising more than 10 models resulted from this analysis, which add up to 92% of the models.The molecular model of YfkE bound to Can = 3) to ensure data reproducibility. All data points and error bars (standard deviations) are provided in each figure and Supplementary Data if applicable.All experiments were performed at least three times (Further information on research design is available in the Supplementary MaterialDescription of Additional Supplementary DataSupplementary Data 1Reporting Summary


AML is a type of leukemia with a very unfavorable prognosis. Some of the new therapeutic targets that are being investigated by researchers worldwide are chemokines of the CXC subfamily, which includes CXCL12. Although this chemokine has been very well studied, other CXC chemokines has been less frequently examined in AML. There is also a lack of a review summarizing the role of CXC chemokines other than CXCL12 in AML. For this reason, this review describes the significance of the ligands for receptors CXCR1, CXCR2, CXCR3, CXCR5, and CXCR6 in AML. The focus is on clinical aspects as well as molecular cancer processes in AML.Acute myeloid leukemia (AML) is a type of leukemia known for its unfavorable prognoses, prompting research efforts to discover new therapeutic targets. One area of investigation involves examining extracellular factors, particularly CXC chemokines. While CXCL12 (SDF-1) and its receptor CXCR4 have been extensively studied, research on other CXC chemokine axes in AML is less developed. This study aims to bridge that gap by providing an overview of the significance of CXC chemokines other than CXCL12  in AML’s oncogenic processes. We explore the roles of all CXC chemokines other than CXCL12, in particular CXCL1 (Gro-α), CXCL8 (IL-8), CXCL10 (IP-10), and CXCL11 (I-TAC) in AML tumor processes, including their impact on AML cell proliferation, bone marrow angiogenesis, interaction with non-leukemic cells like MSCs and osteoblasts, and their clinical relevance. We delve into how they influence prognosis, association with extramedullary AML, induction of chemoresistance, effects on bone marrow microvessel density, and their connection to French–American–British (FAB) classification and FLT3 gene mutations. Acute myeloid leukemia (AML) is a type of leukemia with very unfavorable prognoses . It is eDue to the unfavorable outlook, research is being conducted to develop new therapeutic approaches for AML. One such avenue of research involves examining extracellular factors, including chemokines.CXC chemokines (α-chemokines) belong to a subfamily of chemokines and are chemotactic cytokines responsible for guiding immune system cells  7]. The. The7]. CXCL1, also known as growth-regulated oncogene (Gro)-α, melanoma growth stimulatory activity (MGSA),CXCL2, also known as Gro-β,CXCL3, also known as Gro-γ,CXCL5, also known as epithelial cell-derived neutrophil-activating factor 78 (ENA-78),CXCL6, also known as granulocyte chemoattractant protein 2 (GCP-2),PPBP) gene in the form of pro-peptide, which is then proteolytically shortened to connective tissue-activating peptide III (CTAP-III), β-thromboglobulin (β-TG), and CXCL7 . ,10. 8,10CXCL16 is synthesized as a transmembrane protein . In thisIn adult AML patients, the bone marrow exhibits higher levels of CXCL16 compared to healthy individuals . Bone maAdditionally, AML cells secrete CXCL16, with the lowest expression observed in AML cells with the FAB M3 phenotype ,10. HighFLT3 gene mutation also display lower CXCR6 expression than those without this mutation [NPM1 gene mutation [AML cells exhibit higher expression of CXCR6 than control cells . The levmutation ,10,51. SThe level of CXCR6 expression in the blood of AML patients may be associated with better outcomes , but conThe significance of the CXCL16–CXCR6 axis in AML oncogenesis is not fully understood. CXCL16 may directly affect AML cells, and in some patients, it may increase AML cell proliferation . HoweverCXCL17 is a recently discovered chemokine that remains poorly studied. This chemokine can activate GPR35  and promhttps://ualcan.path.uab.edu, accessed on 25 June 2023) showed that the expression levels of CXCL17 and GPR35 in AML cells do not influence patient outcomes [p = 0.058) towards worse outcomes was observed with higher GPR35 expression on AML cells. On the GEPIA portal , higher GPR35 expression on AML cells was associated with poorer patient outcomes [Currently, the role of CXCL17 and GPR35 in AML has not been thoroughly investigated. Data from the UALCAN portal  in AML therapy. This should be a direction of clinical research in the near future.Regarding other α-chemokines, CXCL16 appears promising from a clinical perspective. Elevated expression of this chemokine in AML cells is strongly correlated with poorer patient prognoses. Therefore, it is essential to investigate how CXCL16 participates in AML tumorigenesis. Additionally, evaluating the potential therapeutic benefits of targeting CXCL16 in AML patients should be a priority. Developing new drugs that target the axes of ligands–CXCR2, ligands–CXCR3, and CXCL16–CXCR6 may significantly improve the outcomes for AML patients.


The importance of Zn2+ as a signaling molecule in cardiovascular functioning is gaining traction. In the heart, Zn2+ plays important roles in excitation–contraction (EC) coupling, excitation–transcription coupling, and cardiac ventricular morphogenesis. Zn2+ homeostasis in cardiac tissue is tightly regulated through the action of a combination of transporters, buffers, and sensors. Zn2+ mishandling is a common feature of various cardiovascular diseases. However, the precise mechanisms controlling the intracellular distribution of Zn2+ and its variations during normal cardiac function and during pathological conditions are not fully understood. In this review, we consider the major pathways by which the concentration of intracellular Zn2+ is regulated in the heart, the role of Zn2+ in EC coupling, and discuss how Zn2+ dyshomeostasis resulting from altered expression levels and efficacy of Zn2+ regulatory proteins are key drivers in the progression of cardiac dysfunction.Increasing evidence suggests that Zn This review will focus on research specific to the cardiovascular system with a focus on the role of intracellular Zn2+.Zinc is an essential trace element that is proposed to interact with >10% of the human proteome . It is eg events . As more2+ plays an emerging but important role in heart function, including excitation–contraction (EC) coupling , the cells were unable to control the amount of Zn2+ taken up. This was indicated by an increase in total zinc within the cells over time, which was not observed when albumin was present in the media . MTs are low-molecular-weight, cysteine-rich proteins that play important roles in metal homeostasis and in the protection against intracellular heavy metal toxicity and oxidative stress at levels sufficient to induce cell damage. In humans, there are four main MT isoforms  that are encoded by genes located on chromosome 16q13  designated to the solute carrier family 30A (SLC30A) and zrt-, irt-related proteins , grouped as solute carrier family 39A  in cardiomyocytes  binding, resulting in MTF-1 translocation to the nucleus and subsequent activation to bind DNA and initiate MT expression  and calreticulin, which also bind Ca2+  may be involved in Ca2+ signaling across the mitochondria and ER , whereas Zn2+ favors protein binding sites that additionally possess N- and S-ligands , it is important to point out that Zn2+ is typically present (both intracellularly and extracellularly) at a lower concentration than Ca2+ and Mg2+. This, together with the respective affinity of a particular site/region for each metal determines which will bind . We have previously shown that the type-2 ryanodine receptor (RyR2) has both high-affinity Zn2+ activation sites and low-affinity Zn2+ inhibition sites. Although the inhibitory action of Zn2+ is likely a consequence of Zn2+ binding to the divalent inhibitory site of the channel, at least some of the activatory sites are distinct from the Ca2+ binding sites  with moderate affinity  exhibiting an average Kd ≈ 300 µM  through LTCC in a manner that is attenuated by ZnT1  involving H9C2 cells and isolated murine cardiomyocytes in combination with Ca2+ and Zn2+ chelators, the group reported that Ca2+ mobilization triggers a reduction in ZIP13 protein expression. This reduction of ZIP13 was reported to activate Ca2+/calmodulin-dependent protein kinase II and contribute to I/R injury.In 1995, Atar and colleagues demonstrated through use of live cell imaging and electrophysiology that Znthe LTCC . While the heart . However by ZnT1 . A subse by ∼40% . The Mor by ∼40% . This wohycardia . Recent 2+ movement, and is implicated in atherosclerosis and heart failure  is located on the S/ER in cardiac cells, has also been linked to intracellular Ca >300 µM . As well2+ from the S/ER. An action potential travels down the transverse tubule of a cardiomyocyte where depolarization activates LTCCs, leading to Ca2+ influx . We also showed that Zn2+ modulated both the frequency and amplitude of Ca2+ waves in cardiomyocytes in a concentration-dependent manner and that reduction of the [Ca2+]i to subactivating concentrations failed to abolish Ca2+ waves in the presence of 1 nM Zn2+. These data suggest that RyR2-mediated Ca2+ homeostasis is intimately related to intracellular Zn2+ levels. In the heart, RyR2 channels operate in closely packed clusters  is a 23-kD transmembrane protein found in the S/ER and nuclear membranes of cardiac muscle cells and other tissues including skeletal muscle, epithelial cells, and the brain  in cultured cortical neurons resulted in the release of Zn2+ from thapsigargin-sensitive stores, suggesting that sequestration of Zn2+ into the S/ER is important in regulation of intracellular levels and that Zn2+ is released following agonist stimulation . It has also been shown that MT inhibits doxorubicin-induced mitochondrial cytochrome c release and caspase-3 activation in cardiomyocytes  is an active substance in the drug Entersto, which is used to treat chronic heart failure . Sacubitl domain , linking2+ chelation. The TACT trial (NCT00044213) investigated the effect of chelation therapy using EDTA on the occurrence of subsequent cardiovascular events in participants with previous myocardial infarction  over other divalent metals in plasma including Ca2+ (Kd ∼10−11 M) due to the high affinity EDTA has for Zn2+ (2+ to improve patient outcome in myocardial infarction and heart failure has not yet resulted in development of new cardiovascular disease treatments. In addition, Zn2+ levels cannot be used as a biomarker for cardiovascular disease as several factors including dietary intake and blood glucose levels can alter plasma Zn2+ concentration and zinc handling (2+ in the short term, for example, during myocardial infarction, would help to attenuate the damage observed postmyocardial infarction.There have also been trials examining the usefulness of Znfarction . EDTA isand Cu2+ . Reactivfor Zn2+ . The trifor Zn2+ . The resfor Zn2+ . This trfor Zn2+ . The tarhandling . However2+-binding proteins in the heart provides novel insights into the regulation of cellular Zn2+ and its role as a signaling molecule in cardiac tissue. The ability of Zn2+ to act as a regulator and/or activator of cellular Ca2+ channels suggests a new and important role for Zn2+ in cardiac function under both physiological and pathological conditions, raising the suggestion that correction of Zn2+ dyshomeostasis may be a novel therapeutic strategy to combat cardiovascular diseases.The role of ZIPs, ZnTs, and Zn2+, there has been relatively little work investigating the biological function of Zn2+ in the heart. Consideration of accurate [Zn2+]i measurements should be emphasized as failure to acknowledge dynamic Zn2+ changes could lead to significant overestimation of [Ca2+]i. Indeed, many of the tools routinely used to measure Ca2+ also bind Zn2+, challenging us to consider how many processes driven by Ca2+ may also be in part, attributable to Zn2+ (2+ fluxes and the ability of these methods to distinguish Zn2+ from Ca2+ in biological systems, the field of zinc biology is currently advancing rapidly (for a comprehensive overview of different Zn2+ sensors, see 2+ and Ca2+ homeostatic mechanisms and their roles in heart disease. However, more work is needed to fully understand the role of Zn2+ in the heart. This includes better understanding of cellular Zn2+ dynamics, how Zn2+ is regulated, and the biological targets of labile Zn2+. This will require a greater appreciation of the spatio-temporal patterning of intracellular Zn2+ fluxes in the heart and how these relate to cardiac functioning in health and disease.In comparison to Ca to Zn2+ . Thanks


Ca2+ entering into excitatory cells binds to Ca2+-binding proteins, which are classified into two groups of Ca2+ sensors: C2 domain and EF hand proteins. Within transient Ca2+ elevation, each Ca2+ sensor, having different affinity and binding speeds to Ca2+, determines the timing of protein–protein interactions to produce the specific action of each organ. This Special Issue aims to focus on investigations ranging from the control of Ca2+ entry at the plasma membrane of excitatory cells to detailed studies on the intracellular mediation of Ca2+-binding proteins that catalyze organ-specific action. The submitted papers are studies of Ca2+ channels and Ca2+ dynamics controlled by Ca2+ elevation in neurons, cardiomyocytes and osteosarcoma. Thus, recent findings of Ca2+ channels’ regulation and Ca2+ dynamics controlled by Ca2+ elevation in neurons are discussed before summarizing the papers in this Special Issue.Signals of nerve impulses are transmitted to excitatory cells to induce the action of organs via the activation of Ca2+ transients is a change in the local density of VGCCs i ≥ 10 µm . In. In2+ trtability B 3]. Fa. Fa2+ trrneurons C 4]. BK. BK2+ trl family . Within  ≥ 10 µm . In preseins/RIM , major amembrane . Neurotr2+ signaling are a complex built between L-type VGCCs at the plasma membrane and RyRs in the ER, well known from muscle cells i via Ca2+-mediated communication between STIM proteins in the ER membrane and VGCCs in the plasma membrane i. The Ca2+ clearance of the glutamate-induced Ca2+ transient was slower than that of the high K+-induced Ca2+ transient, and followed by the Ca2+ rebound. The time course of the Ca2+ clearance and the Ca2+ rebound depended on the duration of glutamate exposure. The application of glutamate, but not high K+, increased [Na+]i. In addition, the Ca2+ rebound was abolished by ouabain, which inhibits Na+/K+-ATPase (NKA), monensin, which increases Na+ by activating NKA, Na+-free solution, or nimodipine, which blocks L-type channels. These results suggest that glutamate-induced Ca2+ rebound originates from Na+ loads through activated NKA. Ouabain or Na+-free solution also slow Ca2+ clearance, apparently by retarding the Na+/Ca2+-exchanger (NCX)-mediated Ca2+ extrusion. Thus, the time cause of the glutamate-evoked Ca2+ response is controlled by glutamate-induced Na+ loads and NKA and NCX activation. The glutamate-activated NKA promotes Na+ extrusion and mediates rebound Ca2+ suppression, which accelerates Ca2+ clearance. In the absence of external Na+, Ca2+ clearance is still slower for the Ca2+ response to glutamate than for high K+, suggesting the participation of additional Ca2+ handlers in the slower Ca2+ clearance under this condition.The suprachiasmatic nucleus (SCN) is the central clock that coordinates peripheral oscillators to control circadian rhythms in mammals . Photic ynthesis ,29. In tynthesis , using a2+-dependent release of transmitters and peptides. Such a release is named extrasynaptic because it occurs in the absence of synaptic structures. In this Special Issue, De-Miguel reviewed the cooperative actions of three Ca2+ sources on somatic exocytosis [2+ entry through L-type VGCCs. For action potential frequencies above 5 Hz, the summation of Ca2+ transients on individual action potentials activated the second calcium source: RyRs produced CICR. The resulting Ca2+ tsunami activated mitochondrial ATP synthesis to fuel the transport of vesicles to the plasma membrane. Serotonin released from the vesicles maintained a large-scale exocytosis by activating the third Ca2+ source; serotonin autoreceptors coupled to phospholipase C promoted inositol 1,4,5-trisphosphate (IP3) production. Activated IP3 receptors in peripheral ER released Ca2+ that promotes vesicle fusion. The machinery for somatic exocytosis has a striking disadvantage. The essential Ca2+ releasing ER near the plasma membrane prevents vesicle transport, drastically reducing the thermodynamic efficiency of the ATP expenses and elevating the energy cost of release.The soma, dendrites, and axon of neurons may display the Caocytosis . The som2+ is a critical second messenger in almost all cell types [2+ plays a central role in the nervous system, where it is closely linked to the regulation of numerous neuronal functions such as neurotransmission, neuronal excitability, or gene expression [2+ levels can also activate a series of harmful mechanisms for the cell, such as alterations in mitochondrial functioning and the generation of free radicals, which can eventually cause cell death through apoptosis [2+ homeostasis. Pesticides have been shown to alter Ca2+ homeostasis, mainly by increasing its intracellular concentration above physiological levels. Pesticide-induced Ca2+ overload occurs through two main mechanisms: the entry of Ca2+ from the extracellular medium through the different types of Ca2+ channels present in the plasma membrane, or its release into the cytoplasm from intracellular stocks, mainly from the ER. It has also been observed that intracellular increases in Ca2+ concentrations are maintained over time, because pesticides inhibit the enzymes involved in reducing its levels. Thus, the alteration of Ca2+ levels can lead to the activation of various signaling pathways that generate oxidative stress, neuroinflammation and, finally, neuronal death. In this Special Issue, Costas-Ferreira and Faro summarize the main mechanisms of pesticides’ actions on neuronal Ca2+ homeostasis , as well as the positive feedback mechanism, known as Ca2+-dependent facilitation (CDF) [2+-binding protein, causes Ca2+-dependent facilitation (CDF) and the inactivation (CDI) of the Cav1.2 channels. Ca2+-free CaM (apoCaM) also contributes to the regulation of Cav1.2 channels. A role of apoCaM in the channel ‘rundown’ phenomena and the related repriming of channels, and CDF, as well as the role of Ca2+/CaM in CDI, has been identified. Furthermore, CaM indirectly affects channel activity by activating CaM-dependent enzymes, such as CaM-dependent protein kinase II and calcineurin (a CaM-dependent protein phosphatase). In addition, Cav1.2 channels’ trafficking are regulated through the intrinsic properties of isoforms of the α1 subunit of the channel, the associated auxiliary subunits, and regulatory proteins such as CaM. However, Kameyama concluded that the role of CaM in the trafficking of Cav1.2 channels remains controversial and further studies are required.VGCCs are divided into high-voltage-activated L-type , P/Q-type (Cav2.1), N-type (Cav2.2), and R-type (Cav2.3) channels because they are activated by relatively large depolarizations, as well as low-voltage-activated T-type  channels, because they are activated by relatively small depolarizations. Among VGCCs, Cav1.2 is the dominant type in the cardiac working muscle, while Cav1.3 and Cav3.2 are dominant in pacemaker  cells. Caon (CDF) . Calmoduon (CDF) . The dir2+—one of the key factors of mitochondrial energetics—is strictly maintained within an appropriate range [2+ in cardiomyocytes is balanced by an influx via the Ca2+ uniport activity and by an efflux via the Na+-Ca2+ exchange or the H+-Ca2+ exchange activities in which the former plays the major part [+-Ca2+ exchanger, NCLX, was reported to supply Ca2+ to the sarcoplasmic reticulum (SR)/ER, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes and cellular Ca2+ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca2+ handling proteins, and their physiological impact. In this Special Issue, Takeuchi and Matsuoka examined the tissue, focusing on the interaction of NCLX with an SR Ca2+ pump (SERCA) in cardiomyocytes [+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes well. These results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes. The functional coupling plays pivotal roles in the SR Ca2+ dynamics and in the generation of automaticity in cardiomyocytes.The heart possesses huge amounts of mitochondria which serve to supply ATP via oxidative phosphorylation, meeting the cardiac energy demand. The mitochondrial Cate range . The mitjor part ,40. The myocytes . A bimol2+ entry (SOCE) promotes cancer progression by changing Ca2+ levels in the cytosol or the ER. Stromal interaction molecule 1 (STIM1), which initiates SOCE by sensing severe ER Ca2+ store depletion, is upregulated in several types of cancer and responsible for cancer cell migration, invasion, and metastasis. In this Special Issue, Lin et al. [2+ elevation may increase cell migration. However, constitutively active STIM1 dramatically increased the Ca2+ influx, calpain activity, and turnover of FA proteins, such as the focal adhesion kinase (FAK), paxillin, and vinculin, which impede the cell migration ability. In contrast, dominant negative STIM1 decreased the turnover of FA proteins as its wild-type variant compared to the cells without STIM1 overexpression while promoting cell migration. These results suggest that cancer cells need an appropriate amount of Ca2+ to control the assembly and disassembly of focal adhesions by regulating calpain activity. On the other hand, overloaded Ca2+ results in excessive calpain activity, which is not beneficial for cancer metastasis.The dysregulation of store-operated Can et al.  investig2+ dynamics controlled by various mechanisms for cell and organ-specific action.Papers in this Special Issue entitled “Calcium Channels and Calcium-Binding Proteins” demonstrated broad effects of Ca


The t   with the resulting RUNX1- RUNX1T1 rearrangement is one of the most common cytogenetic abnormalities in acute myeloid leukemia (AML). It is associated with favorable prognosis. The t   is an uncommon translocation, fuses the gene for the nucleophosmin (NPM) to the retinoic acid receptor α(RARA) and was described essentially in acute promyelocytic leukemia (APL) variant. We present the case of a 19-year-old male patient who developed an AML with t   associated to t  . Morphology and immunophenotype of the leukemic cells were compatible with AML. The patient received chemotherapy based on cytarabine and anthracycline without all-trans retinoic acid (ATRA) followed by allogenic stem cell transplantation in first remission. To the best of our knowledge, this is the first report of an association between a rare translocation t  and t  in AML. In this report, we will discuss the prognosis of this association as well as the treatment. Acute myeloid leukemia (AML) is the most common acute leukemia in adults and counts for about 80% of all cases . CytogenHere we report an unusual association of t  with t  in AML and we try to discuss the prognosis of this association and then the treatment.3 consisting of 10% neutrophils, 10% lymphocytes, and 80% blasts. The hemoglobin was 6.9 g/dl and the platelet count was 10 000/mm3. Coagulation test showed disseminated intravascular coagulation (DIC) with a prolonged activated partial thromboplastin time of 45 sec, prolonged prothrombin time and an increased d-dimers concentration of 1460 mg/mmol. Bone marrow analysis revealed increased blasts (98%) with typical morphology of AML type 1 according to the FAB classification: The cytoplasm is moderately basophilic, sometimes containing fine granules and/or Auer bodies  indicating the allogeneic transplantation of stem cells. RT-PCR for RUNX1- RUNX1T1 before the allograft was negative. The patient is so far alive in persistent complete remission.A 19-year-old male patient presented in December 2017 with a one month history of fever and asthenia. Physical examination was normal; particularly absence of lymphadenopathy and splenomegaly. The white blood cell count was 42 000/mmr bodies  and 2. Fr bodies . RT-PCR The t , is reported to be found in 5–10% of all AML cases . On the On the immunological level of AML with t 8; 21), the cells, usually tend to express the following markers: high levels of CD34, HLA-DR, myeloperoxidase (MPO) and CD13 (7). A relative weak expression of CD33 was found, and aberrant lymphoid associated markers  expression was reported with variable prognosis implications ; 21, the. MoreoveIn few cases with a morphological diagnosis of APL, patients have variant chromosome translocations, which fuse RARA gene with partner genes other than PML, such as in the variant translocation t   that fuses the N-terminus of nucleophosmin (NPM1) gene at 5q35 to the retinoic acid receptor alpha at 17q21 , 12. In In APL with the classic reciprocal translocation t   which is characterized by fusion gene transcript PML-RAR-alpha, patients are responsive to differentiation treatment with all-trans retinoic acid (ATRA) . In our Unbalanced translocation der  resulting in a TP53 loss , whole-aUp to now only 7 cases with balanced t   translocation and the underlying NPM1/RARA fusion have been identified, remission obtained with chemotherapy associated or not with ATRA. The different cases were detailed in −4) after consolidation which led to the decision to recommend allogenic stem cell transplantation. Our patient didn’t receive ATRA in combination with chemotherapy (Marketing Authorization in Tunisia for only APL) and perhaps if he received it, he would have negative residual disease after the consolidation and therefore no indication for allogenic stem cell transplantation. The prognosis is difficult to assess, and further accumulation of data on such cases is desirable in order to be able to conclude.In two studies , 11 Tab, the ATRWe have described a patient with a novel balanced t   translocation emerging with t  in AML. To our knowledge, this additional chromosomal aberration has not yet been described. This aberration is thought to play a crucial role in prognosis. The efficacy of ATRA is controversial. Therefore, further accumulation of data on such cases is warranted to enable more comprehensive and conclusive evaluations.


Acute leukemias remain a challenge, in spite of improvements in diagnosis and treatment. Fully establishing the depth and extent of the relative impact of the molecular mechanisms of disease progression and the pathways to recurrent disease will require decades of research, until our comprehension enables the routine development of simple and effective cures. This special issue was aimed to address progress in the characterization of the molecular basis of acute leukemias and to explore potential links with disease course. The articles presented in this issue deal with the subject in general from two main perspectives.Wang et al., investigated the effect of Nrf level on chemotherapy success in adult B-ALL patients and demonstrated a direct negative relationship between Nrf level and vincristine therapy response, for the first time. Mechanistically, they have uncovered that vincristine treatment causes an increase in the Nrf2 mRNA and protein levels in adult B- ALL patients, elevated Nrf2 decreases pro-apoptotic BAD protein level in a PI3K/AKT dependent manner and consequently decreases vincristine chemotherapy sensitivity. We report a retrospective study of pediatric ALL patients performed by Feng et al., on the adverse effect of CDKN2A/B deletions on clinical outcomes in pediatric B-ALL patients. Since the reports about the clinical effect of CDKN2A/B deletions in pediatric ALL are controversial, Feng et al. have conducted a comprehensive analysis of CDKN2A/B deletions in 599 pediatric B-ALL patients and consequently demonstrated that CDKN2A/B deletions were associated with older age at diagnosis, higher white blood cell counts, and prominent hepatosplenomegaly. Moreover, they also demonstrated that the patients with CDKN2A/B deletions had the worst clinical outcomes, especially in the TP53 deletion carriers. Zhang et al., establish a transcriptome-wide m7G methylome profile for AML and identified N7-methylguanosine modification of messenger RNA as a potential source of acute myeloid leukemia (AML) drug resistance. They showed that there were significant differences in the genes modified by m7G methylation in resistant AML cells, and the number of methylated genes and peaks in drug-resistant cells was greater than those in non-resistant cells. Zhang et al., explained why in moderate-to-severe lower respiratory tract infections, lung microbiome changes in children with hematological malignancies are a source of negative disease course. Hong et al., investigated for the probable correlations of TIM-3 expression between leukemic blasts and T lymphocytes since TIM-3 is thought to be involved both in the self-renewal of leukemic stem cells and the immune escape of AML cells. They demonstrated that TIM-3 expression levels of leukemic blasts and T lymphocytes in the bone marrow of de novo AML patients correlate with the presence of core binding factor (CBF) translocations, without having a detectable impact on the clinical outcome. Zhao et al. investigated the function of ECHDC3, which is upregulated in CD34+ progenitor cells of AML after chemotherapy, but whose prognostic significance and function are as yet unknown. They demonstrated that ECHDC3 alters the bone marrow microenvironment by inducing changes in the composition of infiltrating immune cells. Furthermore, they also showed that knocking down ECHDC3 in AML cells by RNAi promoted the death of leukemia cells with cytarabine and doxorubicin. Nilius-Eliliwi et al., demonstrate the power of optical genome mapping (OGM) to yield novel clinically significant results, including information helpful in disease monitoring and AML disease biology. They applied a “next-generation diagnostic workup” strategy with OGM and whole-exome sequencing (WES), and detected a DDX3X: MLLT10 gene fusion, which would otherwise be missed by routine diagnostics. They also discovered several aspects of lineage ambiguity, not shown by standard diagnostics: these included deletions of SUZ12 and ARPP21, as well as T-cell receptor recombination. Their findings could explain an aggressive disease course. Schwarz et al., characterized a method to inhibit the constitutive activity of oncogenic receptor FMS-like tyrosine kinase 3 internal tandem duplication (FLT3ITD) in AML cells, by disrupting oligomerization of receptor protein tyrosine phosphatase PTPRJ. They showed that interfering with PTPRJ self-association may be used as a tool to restrict oncogenic FLT3 activity, affecting FLT3-driven cell proliferation and clonal growth. Yuan et al., presented a hypothesis that germline mutation in RecQ-like helicase can be involved in hereditary predisposition to acute leukemias. They performed whole exome sequencing in peripheral blood mononuclear cells in a familial leukemia case, and for the first time to our knowledge, found a germline RECQL mutation potentially involved in hereditary predisposition to acute leukemia. The hypothetical biological process in which RECQL gene mutation (rs146924988) affects DSBs repair and mediates the generation of fusion genes provides a new understanding for the pathogenesis of leukemia, and highlights the necessity for next-generation sequencing-based screening of genes involved in this process in potential HPS patients. Xu et al., used weighted gene co-expression network analysis, extracting gene co-expression modules to relate them to clinical features, and identified TRIM32 as a potential driver of unfavorable AML disease course. Consequently, they demonstrated that TRIM32 is highly expressed in AML cells compared with cells from healthy donors and Knockdown of TRIM32 significantly inhibited the proliferation of AML cell lines, in vitro. Jiang et al., investigated the role of mitochondria-related metabolic reprogramming in the occurrence, development, drug resistance, and recurrence of acute myeloid leukemia. They analyzed immune infiltration and immunosuppressive genes and demonstrated that the mitochondria-related gene risk signature of AML patients was strikingly positively correlated with an immune cell infiltration and expression of critical immune checkpoints, indicating that poor prognosis might be attributed to an immunosuppressive tumor microenvironment. Takeda et al., presented a case of repeated lineage switches in acute leukemia and they reported that dynamic lineage conversion from ALL to AML occurred after clofarabine monotherapy was provided as a fourth induction regimen. They proposed that a monoallelic deletion and a frameshift mutation in TP53 gene accompanied by MLL gene amplification may have contributed to lineage plasticity and therapeutic resistance in this case, the first reported case of acute leukemia presenting with lineage ambiguity and MLL gene amplification.The first perspective in this Research Topic is the identification of factors that associate with a negative disease course, and potentially affect drug efficacy. Zhong et al., investigated molecular patterns related to ferroptosis in AML cells. They integrated the genome information of 992 AML specimens, including expression profile chip data and high-throughput sequencing data, and analyzed the characteristics of immune cell infiltration, lipid metabolism, and inflammation development in the tumor microenvironment of AML patients. They used RSL3, a targeted inhibitor of GPX4, in AML cell lines HL-60 and THP1, to explore the biological effects of inducing ferroptosis. They demonstrated that RSL3 induced cell death in a dose-dependent fashion, indicating that GPX4 is a potential target for AML treatment. Li et al., constructed a cuproptosis-related lncRNA signature, to classify AML patients into high and low risk and revealed multiple signaling pathways, especially immune-related processes, were found to be significantly enriched in the high-risk group. In addition, their results indicated for AML patients in the high-risk group had a lower sensitivity to a range of anti-leukemia agents, including cytarabine, methotrexate, etoposide, and ABT-263 , while indicating a higher sensitivity to drugs like rapamycin, bortezomib, Erlotinib, even though some of them are currently not in clinical use for the treatment of AML. Tomaz et al., noted a significant induction of apoptotic AML cell death after treatment with the non-steroidal anti-inflammatory drug nimesulide, either alone or in combination with prednisolone. They also demonstrated that nimesulide potentiates the cytotoxic in vitro effect of several chemotherapy drugs used in AML, including cytarabine. They report the case of a patient with AML who presented a partial response after utilization of nimesulide, characterized by complete clearance of peripheral blood blasts and an 82% decrease of bone marrow blasts associated with myeloblast differentiation. Weighted correlation network analysis of serial whole-transcriptome data of cell lines treated with nimesulide revealed that the sets of genes upregulated after treatment with nimesulide were enriched for genes associated with autophagy and apoptosis, and on the other hand, the sets of downregulated genes were associated with cell cycle and RNA splicing.The second perspective in this Research Topic concerns AML cell death, and generally, pathways to block the uncontrolled proliferation of acute leukemia disease clones. Yin et al., presented a survey of novel biomarker candidates, and their critical comparison to representative existing biomarkers. They concluded that integrated analysis of AML biomarkers from cytogenetics, molecular biology, and pathophysiology is conducive to the correct evaluation of prognostic factors to achieve the precise implementation of individualized hierarchical treatment. Gu et al., discussed the roles of Cancer-associated fibroblasts (CAFs) in acute leukemia. Although, CAFs can originate from diverse cell types, leukemia transformation is rare in patients with non-fibrotic myeloproliferative neoplasms. However, it is common in patients with myelofibrosis, which is also associated with an unfavorable disease course. Consequently, they concluded that CAFs in bone marrow may correlate with myelofibrosis, promote leukemia progression, and induce chemoresistance.We also received 2 comprehensive review articles that discuss the established knowledge with current literature. Acute Leukemias: Molecular Characterization, Leukemia-Initiating Cells, and Influence of the Microenvironment “ we thank all contributing authors and hope you enjoy these interesting papers.As the guest editors for the Research Topic “All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.


Measurement of body temperature is essential for the study of metabolic changes and inflammation using experimental animals. Although measuring the rectal temperature is often used, this method is not only time-consuming but also stressful for animals. In addition, it is not possible to use this method to make a constant measurement of body temperature. In the current study, we developed an all-in-one packaged telemetry system, which consists of a temperature sensor, transmitter, balun, and a 3-axis accelerometer. The telemetry sensor was easily implanted subcutaneously in rabbits housed in the animal facility while temperature changes were continuously recorded through a personal computer. Our telemetry sensor system may be useful for monitoring body temperature in experimental animals such as rabbits.Continuous body temperature measurement is an important means of studying inflammation and metabolic changes using experimental animals. Although expensive telemetry equipment for collecting multiple parameters is available for small animals, readily used devices for mediate- or large-sized animals are rather limited. In this study, we developed a new telemetry sensor system that can continuously monitor rabbit body temperature. The telemetry sensor was easily implanted subcutaneously in rabbits housed in the animal facility while temperature changes were continuously recorded by a personal computer. Temperature data obtained by the telemetry was consistent with the rectal temperature measured by a digital device. Analysis of body temperature changes of unstrained rabbits, either under the normal condition or fever induced by endotoxin confirms the reliability and usefulness of this system. Measurement of body temperature along with heart rate, blood pressure, and physical activity is essential for the study of metabolic changes and inflammation using humans and animals because body temperature is one of the fundamental parameters for assessing the health status ,2. The bThe telemetry sensor system was made as described previously with a modification . The curBody temperature changes were monitored by the sensor subcutaneously implanted in the posterior neck of rabbits . The temad libitum. All animal experiments were approved by the Animal Care Committee of the University of Yamanashi (No. A3-50). For implantation of the sensors, three rabbits were anesthetized by intravenously injecting ketamine  at 2.5 mg/kg and medetomidine  at 0.05 mg/kg. The fur of the posterior cervical region was shaved  (2 μg/kg body weight) dissolved in saline solution via an ear vein and measured body temperature changes as described above. To compare the rectal temperature with the temperature measured by the sensor on the neck, the rectal temperature of the same conscious rabbits without immune challenge was measured every 6 h a day using a digital thermometer . The mean values of triplicate measurement were used for analysis.Japanese White rabbits (Kbs:JW) were purchased from Kitayama Labes . We used female rabbits aged 22–34 months for the current experiments. The rabbits were individually housed under a light cycle with 12 h light/12 h dark at 23 °C room temperature. They were fed a standard regular diet  and water s shaved A. After s shaved B and thes shaved C. The bop < 0.05 was considered statistically significant.Data are expressed as the mean ± SD. The Pearson correlation coefficient was performed using SPSS software version 21  for evaluating the correlation between rectal temperature using a digital thermometer and neck temperature measured by the sensor. p < 0.001)  . RegardlWe further investigated the usefulness of the sensor system for testing pyrogen-induced high fever. As shown in We noticed that when the telemetry sensor system is applied, the distance between the receiver-PC and antenna needs to be considered. The principle is “the nearer, the better” because the signals captured by the receiver is significantly reduced due to the presence of other radio wave interference in the same facility . Our stuIt has been reported that the body temperature of rabbits is characterized by an inconspicuous circadian rhythm and always reaches the bottom in the morning and rises the peak during midnight ,25, whicIn conclusion, the simple telemetry sensor system was developed for monitoring the body temperature of rabbits. This sensor system will become a useful tool for measuring animal body temperature for testing new anti-inflammatory drugs.


The relationship between the anatomy of the interradicular space and success in regenerative therapy of furcation defects is discussed in this paper. The goal of this retrospective, multicenter clinical study is to clinically evaluate the relationship between the interradicular conformation and regenerative therapy success with the use of a novel measurement method.One hundred thirty-eight radiographs of mandibular molars with furcation defects that had been treated with regenerative therapy were collected from six clinical centers. Data on the type of therapy and clinical parameters before and after treatment (follow-up of at least 12 months) were collected. The radiographs (before surgery and at least 12 months postoperatively) were measured with a visual evaluation method by a blind operator using graphics software.Success, defined as a reduction in horizontal and vertical furcation involvement, decrease in probing depths, and increase in clinical attachment level, was statistically assessed on 138 regenerated molars sites and were related to clinical variables such as age, sex, center, and treatment. No correlation was found between success in regenerative therapy and the conformation of the interradicular space, measured with a visual ratio method and a standard linear measurement.At the univariate analysis, the parameters that had a correlation with success were center, extent of furcation involvement, treatment, and sex. The use of enamel matrix derivative (EMD) seemed to be the most favorable therapy, with increase in CAL gain and reduction of vertical or horizontal furcation involvement.The regenerative outcome was not significantly influenced by the anatomy of furcation. The center, the degree of furcation involvement, sex, and treatment (EMD) were significantly associated with higher success of periodontal regeneration. Regenerative therapy of furcation defects is one of the biggest challenges of periodontal surgery. Different regenerative strategies have been proposed to improve the prognoses of teeth with furcation involvement (FI), including guided tissue regeneration , bone grWe recently proposed a new method to measure the interradicular anatomy  taking iThis is a multicenter, retrospective clinical study with the main goal of investigating a possible relationship between interradicular conformation and the clinical outcomes of regenerative therapy. The data were collected from six clinical centers. A novel evaluation method  was usedi.Radiographs taken with a paralleling techniqueii.Good image definition (root margins clearly distinguishable)iii.Complete view of the interradicular space and root apicesiv.Infrabony defect of at least 3 mmv.Absence of endodontic lesionsvi.Interproximal bone level coronal or at the same level as the roof of the furcationRadiographs were included in the present study only if they met the following inclusion criteria:A total of 138 periapical radiographs of mandibular molars that met the inclusion criteria and that had been treated with regenerative therapy were collected from six clinical centers. The following de-identified data were retrieved from each center: (i) regenerative technique performed; (ii) patient demographic characteristics, including age and sex; (iii) initial and final vertical probing depth (PD) at the midfacial aspect; (iv) midfacial gingival recession depth (REC); (v) midfacial clinical attachment level (CAL), obtained adding REC to PD; and (vi) horizontal and vertical furcation class at the last follow-up. The success of the therapy was defined as the presence of clinical attachment level gain and concomitant improvement of the FI  compared to baseline.A calibrated operator who was unaware of the center, the technique performed or the final clinical outcome, carried out measurements on X-rays of interradicular conformation using the method described by Limiroli et al.  and the Roof furcation width (RFW): the ratio between the diameter of the osculating circle  and the line from the roof of the furcation to an imaginary line passing through the tips of the roots .Interradicular furcation width (IFW): the ratio between a horizontal line drawn at the maximum width of the interradicular septum and the vertical line.Two ratios were calculated:The two ratios represent the radicular roof width and the interradicular width Fig. .Fig. 1ExIn addition to the newly proposed method, the interradicular conformation was also assessed using the method described by Bowers : in thisp = 0.05.The collected data were described using measures of central tendency and dispersion. Quintile values were calculated for interradicular conformation measurements . The relationship between these variables was also investigated using the linear correlation coefficient. The relationship between the interradicular conformation measurements and treatment outcomes was evaluated with these as control variables: center, sex, age, and type of treatment. A multivariable logistic model was used to study this relationship in the descriptive tables. The association between the variables was measured with the chi-squared test. The statistical significance threshold was set at Six centers provided 138 radiographs of 138 mandibular molars with FI treated with periodontal regeneration (Table The distribution of cases exhibiting CAL gain (Rec plus PD) and those also obtaining an improvement of the furcation involvement (“successful cases”) after treatment are depicted in Table The interradicular space conformation (RFW and IFW) was calculated on the 138 radiographs. The values of both ratios in study population overlapped the conformation range and distribution of a standard population. Table Parameters were then considered in a combined manner, and the sample of subjects resulted was divided into four groups. The first group of 11 subjects was the unsuccessful one, presenting a lack of CAL gain and no degree of reduction of furcation involvement. The second group of 11 subjects presented only degree of reduction of furcation involvement. The third group of 18 subjects showed only CAL gain. The fourth group of 98 subjects demonstrated all 3 positive variables success, degree of reduction, and gain in CAL. Distribution of RFW and IFW related to the groups was then assessed. The overlapping confidence intervals showed no significant association. The same groups described above were then compared with the variables horizontal FI and vertical. The result confirmed the previous association between success and reduction of furcation involvement variables. Moreover, crossing both the variables horizontal and vertical FI with success, it was concluded that 97% of subjects presenting success in the regenerative procedure also obtained an improvement in both vertical and horizontal furcation.Degree III horizontal furcation defects, considered to be subject to more difficulty in obtaining regeneration, were then removed, to evaluate a possible negative influence. The relationship between variables horizontal/vertical FI and RFW/IFW was considered: unsuccess resulted in a lower number confirming the III degree furcations as the more uncertain to be treated with regeneration, but even in this scenario, the confidence interval values were overlapping for both groups, not recording a statistically significant difference in the influence of RFW and IFW even without degree III horizontal furcation defects. The association between RFW and IFW relating to the variables Hf and Vf was related to success or unsuccess. The overlapping values of the confidence intervals indicated no statistically significant association.At this point, as a control to the RFW/IFW measurement technique, a standard linear measure (MR) of root divergence  was appFinally, a regression model was then created considering the only variable type of treatment. Using a multivariable logistic regression model as response variable “yes” was considered the success variable, and as independent variables gender, age, treatment type and, for each one of the results, one of the 3 interradicular conformation variables. Statistically significant associations were not found. The exception was the sex variable in the model that considers the variable RFW. In all 3 logistic regression models considered, the variables RFW, IFW, and MR were express in quintiles.To establish how much the different factors are involved in the regenerative outcomes, a factor influence ranking was used. The associations between covariates and outcomes were investigated with a univariate analysis.The variables associated with the success full cases were, in order of strength of association: center effect, horizontal FI, vertical FI, treatment, and sex . Therefore, the center was the variable strongly associated with the success of the regenerative outcomes of mandibular molars with FI. Age, RFW, and IFW were not found instead be associated with the outcome success.It has been suggested that the interradicular anatomy is associated with the outcomes of periodontal regeneration –18. SeveOur finding was that the outcome of periodontal regeneration of furcation defects was not affected by the interradicular anatomy. The lack of association was observed when the interradicular anatomy was assessed using the conventional method and when the assessment was performed with the use of the two ratios. On the other hand, the analysis showed that the outcome of regenerative therapy of furcation defects was affected by the technique, center effect, type of horizontal/vertical FI, and sex. In addition, regenerative therapies utilizing EMD achieved the highest CAL gain, FI improvement, and, overall, the highest probability of treatment success.The effectiveness of EMD has been evaluated by several clinical studies over the last 20 years , 20. EMDThe grade of the horizontal and/or vertical component of the furcation defect affects the prognosis of molars with FI and is a key parameter when evaluating the outcomes of periodontal regeneration , 23. TonThe IFW and RFW values calculated from the 138 radiographs were consistent with those of the population sample examined in a recent study from our group . Given tFinally, the statistical analysis was able to highlight, using a factor influence ranking, that the strongest effect on the treatment outcomes was obtained by the center effect, followed by the type of FI, treatment, and sex. The possible influence of furcation morphology on the effectiveness of the treatment was analyzed by Meyle et al. , with reWithin the limitations of the present study, it should be mentioned that some of the radiographs may not have been performed in the ideal manner with the correct inclination between the X-ray beam and the film, although the paralleling technique could reduce the amount of distortion in the images. In addition, both the small sample size and the heterogenous nature of the clinical centers (university versus private practice) could have affected our findings. Future prospective clinical studies evaluating the influence on the interradicular anatomy assessed with conventional and novel methods are encouraged.Within the limitations of the present study, it can be concluded that interradicular anatomy does not seem to affect the outcomes of periodontal regenerative therapy. EMD was found to be the most effective treatment approach in terms of CAL gain and reduction of the horizontal and/or vertical FI. Center effect, horizontal/vertical FI at baseline, and sex were also significantly associated with the surgical outcomes.


Cutaneous angiomyolipoma is a rare mesenchymal tumor that is demographically, clinically, and immunohistochemically distinct from its renal and extrarenal counterparts. We present a case of cutaneous angiomyolipoma in the right retroauricular area of a 35-year-old male patient and provide a broad systematic review of the literature and the largest compilation of cutaneous angiomyolipomas reported to date. According to the findings presented in this review, we conclude that cutaneous angiomyolipoma should be completely separated from renal and extrarenal angiomyolipomas and therefore be considered a distinct entity in the classification of skin tumors. Cutaneous soft tissue tumors are a heterogeneous group of neoplasms arising from different dermal and subcutaneous tissue components. Benign tumors vastly outnumber sarcomas .Cutaneous angiomyolipoma (hereinafter described as “cutaneous AML”) is a benign tumor composed of varying proportions of thick-walled blood vessels, mature adipose tissue, and smooth muscle cells arranged in bundles, histologically identical to renal and extrarenal angiomyolipoma (hereinafter described as “classic AML”). Cutaneous AML is extremely rare and is not included in the latest 2018 World Health Organization (WHO) classification of skin tumors [A total of 43 cases have been reported in English and Spanish literature to date; we present a new cutaneous AML in a 35-year-old male, which would represent the 44th case. We present the largest compilation of cutaneous AMLs, describe their clinical and morphological features, and contrast them with classic AMLs.Our findings reveal that although they share similar histopathologic features, classic and cutaneous AML should be considered separate entities owing to their distinct demographic, clinical, and immunohistochemical features. Immunostains for melanocytic markers  are crucial in differentiating these 2 entities, being positive in classic AML  and nega2 laser.A 35-year-old male patient presented with a mass on his right ear, which progressively increased in size and became painful to touch after local trauma. He was otherwise in good health and had no clinical signs or familiar history of tuberous sclerosis complex (TSC) or classic AML. Physical examination revealed a nodular, erythematous, soft, mobile, subcutaneous mass in the right retroauricular area, which had a diameter of 1.7 cm . ClinicaThe excisional skin biopsy showed a subcutaneous nodular mass covered by a rugged grayish-tan epidermal surface. At the cut surface, a well-circumscribed, subepidermal, whitish-yellow, heterogeneous soft mass was present, measuring 1.3 × 0.6 cm .Hematoxylin-eosin–stained sections revealed a well-circumscribed nodule, a surrounding fibrous pseudocapsule , small oMasson’s trichrome staining revealed smooth muscle bundles (red), muscular blood vessels (red), stromal connective tissue (blue), and the fibrous pseudocapsule (blue) .Immunohistochemical analysis using the Ventana BenchMark ULTRA platform with the UltraView detection system revealed positive staining for smooth muscle actin   and negaBased on the findings, the case was diagnosed as a completely excised cutaneous AML. The patient had no recurrence at 1 month follow-up.Soft tissue cutaneous tumors are a heterogeneous group of neoplasms originating from distinct dermal and subcutaneous tissue components. The most common benign mesenchymal tumors are lipomas, dermatofibromas (fibrous histiocytomas), vascular or smooth muscle lesions, and nerve sheath tumors. These tumors are usually superficial and small, measuring less than 5 cm, and present clinically as painless plaques or nodules with variable growth rates. Benign tumors are generally successfully treated with complete excision and rarely recur locally .cutaneous angiomyolipoma and cutaneous angiolipoleiomyoma without date restrictions, 43 patients with cutaneous AML have been reported to date  family, which also includes lymphangioleiomyomatosis ,70-73, cAlthough all these tumors have distinct histologic features, they all originate from perivascular epithelioid cells, which have the peculiarity of coexpressing both melanocytic and myogenic markers. Therefore, these tumors probably originate from a cell with myomelanocytic differentiation, although no normal counterpart for this cell has been described ,86.TSC1  and TSC2  [TSC2 mutations [The majority of classic AMLs are sporadic 80%). In comparison, up to 20% of them are associated with TSC ,88—a rar0%. In co16p13.3) ,90-92 vi16p13.3)  cause mTutations ,93. TSC-utations .Classic AML can also be associated with adult polycystic kidney disease, neurofibromatosis type 1 (NF1), and von Hippel-Lindau syndrome .Classic AML accounts for less than 1% of renal tumors; however, it is the most common renal mesenchymal tumor ,87. SporMost classic renal AMLs are asymptomatic and incidentally detected through imaging, surgery, or autopsy . HoweverClassic renal AML is easily diagnosed with uncontrasted computed tomography (CT) or magnetic resonance imaging (MRI) because of its abundant fat tissue. In 2016, Song et al  establisClassic AML is a yellow-white, smoothly rounded tumor with well-circumscribed, nonencapsulated borders. Its appearance varies depending on the proportion of adipose, vascular, and muscular components present -8,41. TuClassic AML comprises the characteristic triad of thick-walled blood vessels devoid of lamina elastica, mature adipose tissue, and bundles of spindled or epithelioid smooth muscle cells ,41,42,87There are several histologic variants, including microscopic AML  ,105, intEAMLs (5%-7% of classic AML) require more than 80% of epithelioid morphology ,104, conClassic AML is typically positive for melanocytic markers (95%) such as HMB-45 (expressed in a patchy pattern), Melan-A, Micropthalmia transcription factor, and Tyrosinase ,8,40,114Surgical management is recommended in AMLs with a tumor size greater than 1 cm, symptomatic patients, or those with a high risk of tumor bleeding or rupture. Some tumors have been treated with embolization. In some cases, medical therapy with mTORC1 inhibitors, such as sirolimus, has shown a positive clinical response and prevented renal failure ,118,119.Asymptomatic patients with AMLs smaller than 1 cm and those with significant comorbidities with AMLs smaller than 3 cm should be followed up periodically with CT or MRI .Recurrence in classic AML is rare; however, approximately 25% of cases of EAML with atypia can recur, metastasize, and cause cancer-related death ,114. In Cutaneous AML is demographically, clinically, and immunohistochemically distinct from its classic counterpart  and 2. CUnlike its classic counterpart, cutaneous AML occurs predominantly in males (70%). The age range is wide (2-77 years), with a peak incidence between the age of 30-50 (median 48) years.This tumor occurs predominantly in the head (76%) but has also presented in the limbs (22%) and abdomen (2%). Of the head tumors, the ear was the most frequent location in 62% of cases, followed by the nose in 19%, and, less frequently, in the forehead, chin, and eyelid (19%).Most patients are asymptomatic, presenting only with a visible or palpable nodular lesion with slow growth, ranging from 2 months to 40 years (median 5 years). Some patients experience tumor size fluctuation over time or that associated with environmental temperature changes  ,28, painIn the majority of cases, cutaneous AMLs are clinically misdiagnosed. The most common clinical diagnoses are cystic lesions , lipomas (28%), and benign vascular tumors 17%; , the latNo cases of cutaneous AML have been associated with TSC to date. Only one case of AML in the skin in a patient with TSC has been reported ; howeverOwing to its superficial location and easily accessible surgical approach, imaging studies are usually unnecessary for diagnosis. In the few cases reported, CT and MRI confirmed adipose and vascular components , similarCutaneous AMLs are well-circumscribed, whitish-gray dermal tumors, measuring 0.4-5 (median 1.5) cm, generally smaller than their classic counterpart (median 9 cm).Histologically, most cases are well-circumscribed, with an admixture of small to medium, thick-walled, muscular blood vessels (some dilated and containing thrombi), mature adipose tissue, and smooth muscle bundles in variable proportions, identical to classic AML.Half of the cutaneous AMLs are surrounded by a fibrous pseudocapsule, probably as a stromal response to tumor growth. Some cases present epidermal changes such as atrophy or hyperplasia. Faint chronic inflammatory infiltrate was also present in some cases ,22.Unlike classic AML, there is no epithelioid variant in cutaneous AMLs; consequently, they do not display cellular atypia, necrosis, or mitosis. Only one case had pleomorphic and bizarre nuclear changes in the smooth muscle component ; howeverWhen requested, Masson’s trichrome staining revealed smooth muscle cells in red and collagen fibers (present in the stroma and fibrous pseudocapsule) in blue. Elastic fiber staining shows an absent or defective lamina elastica in some vessels.Cutaneous AML is characteristically positive for smooth muscle markers such as SMA, Calponin, and Desmin. However, unlike classic AML, all cutaneous AMLs are negative for melanocytic markers such as HMB-45, Melan-A, MART-1, and SOX-10. Other frequently positive markers include S-100, Factor VIII, CD31, CD34, and FLI1.Complete surgical excision is the diagnostic and therapeutic procedure indicated for cutaneous AML; these tumors are usually easily “shelled out” ,12,23. CIn the skin, some tumors are composed of one or more of the AML components. Angiolipoma is composed of mature fat cells and clusters of thin-walled capillaries and lacks smooth muscle bundles. Although angioleiomyoma is also characterized by thick-walled blood vessels (as in AML), its smooth muscle cells are arranged concentrically around blood vessels, and it lacks adipose tissue. Arteriovenous malformation is composed of large-caliber arteries, arterioles, capillaries, venules, and thick-walled veins; however, it lacks smooth muscle bundles and adipose tissue .The most important differential diagnosis is classic AML in the skin  since thOwing to the rarity of cutaneous AML, it is currently not included in the 2018 WHO classification of skin tumors [Our review strongly suggests that cutaneous and classic AMLs must be considered separate entities. In summary, the main differences reside in the following aspects:Clinical: predominantly in males, more frequent in or around the ear, and presenting exclusively as a solitary lesion.Etiopathogenesis: without any reported association with TSC.Morphology: lacking aggressive variants such as EAML, necrosis, and atypical mitoses.Immunohistochemistry: absent melanocytic markers.Prognosis: benign behavior with lack of recurrence following complete surgical excision.The immunohistochemical findings discard PECs or any other cell with melanocytic differentiation as a possible origin for cutaneous AML; hence, unlike classic AML, this tumor does not belong to the PEComa family. It is reasonable to consider cutaneous AML as a true and pure “angio-myo-lipoma.”Future updates of the WHO classification of skin tumors should consider including cutaneous AML as a separate entity. Finally, physicians should be aware of the possibility of a cutaneous AML when presented with a nodular mass in the ear, as appropriate treatment can provide patients with complete clinical resolution.