MDMX, a crucial zinc-containing negative regulator of p53, has been found to be amplified or overexpressed in various cancers and implicated in the cancer initiation and progression. We report here that zinc depletion by the ion chelator TPEN or Chelex resin results in MDMX protein degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Restoration of zinc led to recovery of cellular levels of MDMX. Further, TPEN treatment inhibits growth of the MCF-7 breast cancer cell line, which is partially rescued by overexpression of MDMX. Moreover, in a mass-spectrometry-based proteomics analysis, we identified TRPM7, a zinc-permeable ion channel, as a novel MDMX-interacting protein. TRPM7 stabilizes and induces the appearance of faster migrating species of MDMX on SDS-PAGE. Depletion of TRPM7 attenuates, while TRPM7 overexpression facilitates, the recovery of MDMX levels upon adding back zinc to TPEN-treated cells. Importantly, we found that TRPM7 inhibition, like TPEN treatment, decreases breast cancer cell MCF-7 proliferation and migration. The inhibitory effect on cell migration upon TRPM7 inhibition is also partially rescued by overexpression of MDMX. Together, our data indicate that TRPM7 regulates cellular levels of MDMX in part by modulating the intracellular Zn Zinc is the second most abundant transition metal in living organisms . It playp53, a zinc-containing transcription factor, is the most frequently mutated tumor suppressor protein in human cancer . The rol2+, Mg2+, and Ca2+ as well as environmentally toxic metals such as Ni2+, Cd2+, and Ba2+ with particularly high permeation of Zn2+  is a bifunctional protein with both ion channel and α-type protein kinase feature . TRPM7 i of Zn2+ . The C-t of Zn2+ , 26. It patterns , 27.Here, we report that zinc depletion results in MDMX degradation in a ubiquitination-independent and 20S proteasome-dependent manner. Adding back zinc recovers the cellular levels of MDMX while depletion of TRPM7 attenuates the effects of zinc. In addition, we show that TRPM7 interacts with and induces changed migration of the MDMX polypeptide upon denaturing polyacrylamide gel electrophoresis (SDS-PAGE) that requires the TRPM7 channel function. Moreover, TRPM7 inhibition decreases MCF-7 cancer cell growth and migration, while MCF-7 cells with MDMX overexpression are more resistant to TRPM7 inhibition-mediated cell migration suppression. Together, our results indicate that TRPM7 regulates cellular levels of MDMX in part by modulating intracellular zinc concentration, which in turn may provide a new therapeutic target for combinational cancer treatment.A). We found that TPEN treatment induces p53 as previously reported . Similarly, when we treated MCF-7 cells with bispicen, another zinc chelator , we observed induction of p53 and MDM2 levels but reduction of MDMX levels . MDMX reduction was also observed in cells grown in zinc-deficient DMEM medium in comparison to the cells that were grown in zinc-deficient DMEM supplemented with 5 μM ZnSO4 or regular DMEM medium . Note that under this condition, no p53 or MDM2 induction was observed. This may be due to the fact that TPEN and bispicen are membrane-permeable agents, which can deplete intracellular zinc more efficiently than the extracellular zinc-deficient medium . Furthermore, we found that the levels of ectopically expressed MDMX proteins in HEK293T cells also decrease upon TPEN treatment .In order to investigate the effects of zinc on the p53 signaling pathway, MCF-7 breast cancer cells were treated with 5 μM TPEN, an intracellular membrane-permeable zinc chelator .To show that the effects of TPEN on cellular levels of MDM2, MDMX, and p53 are due to zinc depletion but not other divalent cations, we pretreated MCF-7 cells with 5 μM TPEN and added back 25 μM ZnSO and p53 H.I, TPEN treatment led to reduced cellular levels of MDMX but increased levels of MDM2 and p53 in LNCaP prostate cancer cells. In PC3 cells, another p53-null prostate cancer cell line, we also observed TPEN-induced MDMX degradation. Together, our data demonstrate that zinc depletion has distinct effects on the three key components of the p53 signaling pathway. Most importantly, our results show that zinc depletion leads to MDMX degradation.The effect of TPEN was not limited to MCF-7 cells. As shown in Figure 1A). Similarly, in LNCaP cells, MG-132 but not chloroquine or bafilomycin A1 cotreatment reversed the effect of TPEN on cellular levels of MDMX . Note that both chloroquine and bafilomycin A1 inhibited LC3B-II degradation (A and B), indicating their effectiveness under the experimental condition.TPEN-mediated zinc chelation can cause oxidative DNA damage and chromosome breaks  and actiFig. 2C). Similarly, Z-VAD-FMK had no effect on TPEN-induced MDMX reduction but inhibited TPEN-induced PARP cleavage in LNCaP cells (B). Additionally, we observed that TPEN treatment reduces the cellular levels of ectopically expressed wild-type MDMX as well as the mutant MDMX (D361A), a mutant that is resistant to caspase cleavage (D). Note that we detected a slight increase in ROS (Reactive Oxygen Species) levels in TPEN-treated cells in comparison to the DMSO-treated cells, but to a much lesser extent when compared with cells treated with pyocyanin, a cell-permeable compound capable of redox cycling . Similarly, ubiquitination of p53 reduced upon TPEN treatment. It was previously reported that zinc chelation by TPEN abolishes the E3 ligase function of MDM2 (A), confirming that MDM2-mediated MDMX ubiquitination does not account for TPEN-induced MDMX degradation.MDMX degradation is controlled in part by MDM2-mediated ubiquitination and proteasome degradation . To examFig. 3 of MDM2 . As MDMX of MDM2  and comp of MDM2 ]. We fou of MDM2 A, confirB). Intriguingly, at higher concentrations, CZM treatment induced a similar effect as TPEN and both drugs displayed a synergistic effect on MDMX and p53 expression. Note that CZM is a quinoline-8-thiol derivative and its inhibition of Rpn11 activity could be through binding to the catalytic Zn2+ ion in the active site of Rpn11 (C and D). Note that siRpn2-2-treated cells had higher basal levels of MDMX than siRpn2-1 or control siRNA-treated cells. The inconsistent effect might due to the off-target effect of one of the siRNAs. Alternatively, the knockdown of Rpn2 might need to reach a certain threshold level to block the 26S proteasome function toward MDMX as we noticed that siRpn2-2 had a higher efficiency to knockdown Rpn2. We would like to point out that cellular levels of p53 were elevated upon inhibition of 26S proteasome function as previously reported . Similarly, TPEN induced MDMX degradation to a similar extent in MCF-7 cells with or without pretreatment of MD-224, an MDM2 PROTAC degrader (B). Note that MD-224 failed to degrade MDM2 but induced cellular levels of p53 in MCF-7 cells. In 22RV1 cells, a prostate cancer cell line that MD-224 did degrade MDM2 under our experimental condition, and TPEN induced MDMX degradation with or without MD-224 treatment (B). In addition, we constructed two mutant forms of MDMX containing a mutation (C463A) that is known to be defective in MDM2 interaction (C), further supporting our conclusion that MDM2 may not be required for TPEN-mediated MDMX degradation. Moreover, we transfected a construct expressing human MDMX into mouse embryo fibroblasts (MEFs) that lack MDM2 and p53 expression, 2KO   (D). Importantly, when we examined endogenously expressed MDMX (Mdm4) protein in 2KO  MEF cells, we found that it too is decreased to TPEN treatment (E). Note that TPEN treatment also reduced the cellular levels of a previously reported  MEF cells (E). Based on several lines of evidence, we conclude that MDM2 is not likely to be required for TPEN-mediated MDMX degradation.As MDM2 has been found to associate with several subunits of the 19S proteasome regulatory particle and target p53, p21, and Rb for proteasome-dependent but ubiquitination-independent degradation, we assumed that the accumulated MDM2 upon TPEN treatment would still somehow facilitate the degradation of MDMX. Accordingly, we transfected MCF-7 cells with control siRNA or two different siRNAs, which target MDM2. Remarkably, TPEN induced MDMX degradation in cells treated with the MDM2 siRNAs to a similar extent as in cells treated with control siRNA A. Similadegrader  Fig. S2A. Similareatment B. In adderaction , 41: (i)eraction C, furthemdm2−/−)  or fibromdmx−/−) . In bothmdmx−/−) D. Importreatment E. Note treported  truncateEF cells E. Based A, the mutant MDMX proteins were as sensitive to TPEN treatment as wild-type MDMX. Moreover, the MDMX protein carrying mutation at all these three residues was more resistant to etoposide or doxorubicin than TPEN treatment (B). Furthermore, using U2OS cell clones that express mutant MDMX (S367L) from the endogenous MDMX locus, we found that endogenous MDMX S367L is more resistant to etoposide than TPEN treatment (C). In line with these results, TPEN treatment induced cellular levels of phosphorylated H2AX, a marker for DNA double-strand breaks, although to a much lesser extent in comparison to etoposide, a topoisomerase II inhibitor, and chemotherapeutic agent . Using coimmunoprecipitation assay with ectopically expressed proteins, we confirmed its interaction with MDMX (D). Intriguingly, when we ectopically expressed TRPM7 with MDMX in the presence or the absence of MDM2, we found that TRPM7 coexpression stabilized MDMX and induced the appearance of a more rapidly migrating MDMX polypeptide on SDS-PAGE (A). Since TRPM7 is a bifunctional protein with both ion channel and α-type kinase function . Together, our results reveal that the channel function of TRPM7 is critical for TRPM7-induced MDMX stabilization and the appearance of faster moving species of MDMX on SDS-PAGE.In a mass-spectrometry-based proteomics analysis, we identified TRPM7, an ion channel protein, as a potential MDM2/MDMX heterodimer interacting protein , A–C. Usith MDMX D. IntrigSDS-PAGE A. Since function , we intrfunction  or a kinfunction  into TRPnhibitor C. Togeth4 was added. As shown in Figure 6D, Zn2+-mediated recovery of MDMX in the presence of TPEN is attenuated upon the depletion of TRPM7.To further test if TRPM7 is involved in TPEN-mediated MDMX degradation, MCF-7 cells were transfected with control siRNA or two different siRNAs that target TRPM7. Forty-eight hours later, cells were pretreated with 5 μM TPEN and then 5 μM ZnSO2+ on TPEN-induced MDMX degradation (E). On the other hand, we found that in TPEN-treated HEK293T cells, overexpression of TRPM7 facilitates the Zn2+-mediated recovery of MDMX (F). Note that the observed effects of TRPM7 knockdown, inhibitor treatment, or overexpression on MDMX most likely happened at a posttranscriptional level as the mRNA levels of MDMX remained fairly constant under the experimental condition  and Fig. 6G). Together, our data indicated that TRPM7 is involved in Zn2+-mediated recovery of MDMX in TPEN-treated cells, which is most likely through its channel function.In line with the data obtained with TRPM7 knockdown, NS8593 attenuated the effect of Znradation E. On the of MDMX F. Note tondition . In addiondition . As showA). TPEN treatment also repressed MCF-7 cell growth in a cell proliferation assay (B). NSC207895, an MDMX inhibitor that blocks the MDMX promoter and inhibits the MDMX expression (B). Similar results were obtained using MCF-7 cells treated with control siRNA or siRNA targeting MDMX in the absence or presence of TPEN (C). On the other hand, using MCF-7 cells with tetracycline-inducible MDMX overexpression, we observed that the cells growing in the presence of tetracycline (a.k.a. with MDMX overexpression) were more resistant to the TPEN treatment in the colony formation assay (D). Together, these results indicated that MDMX protects MCF-7 cells from zinc depletion-mediated cell growth inhibition.TPEN treatment inhibited MCF-7 cell growth on a colony formation assay, whereas the addition of zinc completely abolished the inhibitory effect of TPEN A. TPEN ton assay B. NSC207pression , enhancepression B. Simila of TPEN C. On theon assay D. TogethE). However, using MCF-7 cells with tetracycline-inducible MDMX overexpression inhibition of TRPM7 with either siRNAs  or NS8593 , we did not observe a cell growth difference for the cells with or without MDMX overexpression. Similarly, no cell growth difference was observed between MCF-7-Myc-MDMX cells with stably expressed Myc-MDMX and MCF-7-pcDNA3 control cells in response to NS8593 treatment . Therefore, under our experimental conditions, MDMX overexpression does not rescue TRPM7 inhibition-mediated growth of MCF-7 cells.Previously it was reported that downregulation of TRPM7 in human cancer cells impairs cell proliferation, migration, and invasion . As our Fig. 7A) or MCF-7 cells with tetracycline-induced MDMX overexpression  were partially resistant to this inhibitory effect in comparison to control cells. Similarly, depletion of TRPM7 using siRNA inhibited MCF-7 cell migration while MCF-7 cells with stably expressed MDMX  or MCF-7 cells with tetracycline-induced MDMX overexpression  were partially resistant to this inhibitory effect in comparison to control cells. Together, our data indicated that TRPM7 may function through its regulation of MDMX to affect cell migration during tumorigenesis.Since MCF-7 is a noninvasive line naturally, it is not suitable for a transwell migration assay. We then assessed MCF-7 cell migration with a wound-scratch assay. As shown in Fig. 8via p21 through a p53-independent pathway or may involve the p53 family member p73  mediating the zinc influx so that MDMX can coordinate zinc for a proper structure to avoid degradation; (ii) interacting with MDMX and protecting it from degradation. The two mechanisms may function in concert as TRPM7 may increase the local zinc concentration around MDMX through its interaction with MDMX, allowing a more efficient recovery of MDMX folding. The recovery of cellular levels of MDMX may have a slower kinetics due to TRPM7 knockdown. In addition, the recovered MDMX may be targeted for degradation by MDM2. Furthermore, we demonstrated that suppression of MCF-7 cell migration upon the inhibition of TRPM7 can be partially rescued by MDMX overexpression. Based on these findings, we speculate that overexpression of TRPM7 in breast cancer cells raises their intracellular zinc concentration, which, in turn, increases cellular levels of MDMX to promote cancer cell migration. As the cleaved C-terminal kinase domain of TRPM7 has other activities in the nucleus , it is vTRPM7 is not unique among proteins that are relevant to zinc biology in regard to breast cancer. Other zinc-permeable ion channels, zinc transporters, as well as metallothioneins may employ similar mechanisms to influence breast cancer development by modulating the cellular levels of zinc and MDMX levels. Indeed, when we knocked down ZIP7, a zinc transporter that is overexpressed in breast cancer, we observed similarly attenuated MDMX recovery in TPEN-treated MCF-7 cells upon adding back zinc . Therefop53−/−, mdm2−/−), and MEF 3KO  cells were cultured in DMEM medium with 10% FBS  and 1% penicillin-streptomycin (Gibco) at 37 °C, 5% CO2. MEFs were provided by Dr Guillermina Lozano (MD Anderson Cancer Center). LNCaP cells were cultured in RPMI medium with 10% FBS  and 1% penicillin streptomycin (Gibco) at 37 °C, 5% CO2. For zinc-deficient DMEM medium, FBS was incubated overnight with 5% Chelex 100 (Bio-Rad) before supplemented (10%) into DMEM medium. MCF-7-pcDNA3 and MCF-7-Myc-MDMX cell lines were constructed by transfecting pcDNA3 or Myc-MDMX plasmid into MCF-7 cells and then selecting G418 (1 mg/ml) resistant clones for 2 weeks. Pooled cell clones were maintained in DMEM with 250 μg/ml G418. MCF-7 with tetracycline-inducible MDMX cell line was obtained from Dr Xinbin Chen  and was maintained in DMEM with 4 μg/ml Blasticidin and 25 μg/ml Zeocin. U2OS MDMX S367L mutant cells were generated using CRISPR/Cas9 genome-editing technology , actin , Flag , HA , TRPM7 , Parp , LC3B , Rpn1 , Rpn2 , and GFP . Mouse monoclonal antibodies against human p53 (DO-1) and MDM2 , MDMX  were used as supernatants from hybridoma cultures. Reagents used in this study were as follows: TPEN  - 1,2-ethanediamine), MG132, CaClPlasmid transfections were performed using PEI  for HEK293T cells and Lipofectamine 2000 (Thermo Fisher Scientific) for MCF-7 cells. siRNA transfections in MCF-7 cells were performed using Dharmafect 1 (Dharmacon) using 50 nM of control siRNA , siMDMX , siRpn1 , or siRNA targeting TRPM7, MDM2 or Rpn2. The target sequences are: 5′-TTAATGTATCTACCGTCAGGG-3′ (siTRPM7_1), 5′-GAGTATTTCATGGCAAGAC-3′ (siTRMP7_2), 5′-AAGCCATTGCTTTTGAAGTTA-3′ (siMDM2_1), 5′- AAGGAATAAGCCCTGCCCA-3′ (siMDM2_2). 5′-GTCTAGATGATCACAAGTA-3′ (siRpn2_1) and 5′-GGGTGTAATTCATAAGGGT-3′ (siRpn2_2).RNA was extracted using a Qiagen RNeasy mini-kit, and cDNA was synthesized with the QuantiTect reverse transcription kit (Qiagen). Samples were analyzed by quantitative real-time PCR on a Bio-Rad CFX 96 using PowerUp SYBR Green (Thermo Fisher Scientific). RNA expression was normalized to RPL32 mRNA expression. Relative levels were calculated by the comparative Ct method (ΔΔCT method). The results are expressed as means ± sd of four experiments. Primer sequences are: RPL32, 5′-TTCCTGGTCCACAACGTCAAG-3′(Forward) and 5′-TGTGAGCGATCTCGGCAC-3′ (Reverse); MDMX, 5′-GCAAGAAATTTAACTCTCCAAGCAA-3′ (Forward) and 5′-CTTTGAACAATCTGAATACCAATCCTT-3′ (Reverse).In vivo ubiquitination assays were carried out using a His-Ub construct. After cells were harvested, a Ni-NTA pull-down assay was carried out as previously described (escribed . Eluted 5 (for HEK293T cells) or 3 × 105 (for MCF-7 cells) cells/ml. In total, 100 μl of diluted cells was aliquoted into 96-well plates. For HEK293T cells, 100 μl of DMEM containing DMSO, TPEN (10 μM), or TPEN plus ZnSO4 (10 μM each) was added in triplicates into the cell-containing wells. For MCF-7 cells, 100 μl of DMEM containing DMSO, NS8593 (100 μM), TPEN (10 μM), or ZnSO4 (10 μM) was added in triplicates alone or in combination as indicated into the cell-containing wells. The fluorescence intensity was measured on a TECAN SPARK Microplate Reader (Tecan) after the plates were incubated for 30 min at 37 °C. The cells were excited at a wavelength of 488 nm (bandwidth of 20 nm), and the emission was measured at a wavelength of 535 nm (bandwidth of 20 nm). The fluorescence intensity measurements were obtained by subtracting the readings from 200 μl of DMEM without Zinpyr-1 loaded cells. The relative zinc levels were calculated by normalizing to the measurements of DMSO-treated samples. The results were expressed as means ± sd of three independent experiments.Zinpyr-1 (Santa Cruz Biotechnology) was used to detect free intracellular in live cells. Cells were incubated with 5 μM Zinpyr-1 in DMEM for 30 min at 37 °C. Then the cells were washed twice with 1× PBS, trypsinized, and resuspended. The cell numbers were counted, and the cell suspension was adjusted to a final concentration of 4 × 105 MCF-7 cells per well were plated in 96-well plates. The cells in triplicates were then treated as indicated, and the plates were read on a TECAN SPARK Microplate Reader (Tecan) at a wavelength of 490 nm. The results were expressed as means ± sd of three independent experiments.Cell proliferation was measured using the CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) following manufacturer's instruction. 1 × 104 MCF-7 cells (on 35 mm dishes), MCF-7 with tetracycline-inducible MDMX overexpression (on 6-well plate with or without 2.25 μg/ml of tetracycline), or MCF-7 transfected with siRNA targeting TRPM7 (on 6-well plate 6 h after transfection) were plated. The cells were treated with indicated drugs for 24 h, and then fresh DMEM medium was added and replaced every other day. Five to seven days after treatment, the cells were fixed and stained with crystal violet (0.05% in 20% ethanol). The crystal violet stained cells were scanned using LI-COR Odyssey CLx imaging system. The intensity of crystal violet signal in each well was quantified to represent the cell numbers in the well.5 × 106 cells MCF-7 were plated  to reach ∼90% confluence, the cells were starved with DMEM plus 0.5% FBS for 24 h. Then the cells were washed twice with 1× PBS, scratched with a 200-μl pipette tip to produce a straight cell-free “wound,” and washed again with 1× PBS to remove debris followed by addition of fresh DMEM plus 0.5% FBS DMEM with DMSO or NS8593 (30 μM) as indicated. Images were taken at 0 or 24 h after scratch using a phase-contrast microscope  with 10× magnification. The healing areas were measured using MRI Wound Healing Tool  in Image J. Three different scratched areas for each dish/well were imaged and measured. Percentage of healed wound area at 24 h related to 0 h time point was graphed as percentage of healed area = (area 0 h − area 24 h)/area 0 h × 100%.About 2 × 10All data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


BRAF mutant protein expression was evident in the epithelial component only, while β-catenin was negative in both the components. The histopathological diagnosis of papillary thyroid carcinoma with nodular fasciitis-like stroma was offered. Sanger sequencing revealed a BRAFV600E  mutation. Post-operatively, no residual tumor was detected on ultrasound and radioiodine scans. The patient was doing well at follow-up of 9 months. PTC-NFS/DTF is a histological variant of PTC with a favorable prognosis. Our index case was associated with the BRAF mutation, which was restricted to the epithelial component. Thorough sampling of the excised specimen is essential in order not to miss the epithelial component, which, in most reported cases (including ours) appears to be small.Papillary thyroid carcinoma with desmoid-type fibromatosis (PTC-DTF) or nodular fasciitis-like stroma (PTC-NFS) is a rare morphological variant of PTC with a favorable prognosis. There is a paucity of molecular data regarding this entity. We present the case of a 20-year-old female who presented with a palpable mass over the anterior aspect of the neck for the past 3–4 months, which was diagnosed as PTC-NFS. Ultrasonogram of the neck revealed a bulky left lobe of thyroid that contained a well-defined heterogenous lesion measuring around 24 × 26 × 36 mm with involvement of the adjacent isthmus. She underwent a total thyroidectomy with central compartment lymph node dissection. Histological examination revealed a biphasic tumor with epithelial and stromal components resembling nodular fasciitis. Two dissected lymph nodes showed metastasis of the epithelial component only. On immunohistochemistry,  WHO Classification of Tumors of the Endocrine Organs (2017 edition).Papillary thyroid carcinoma with desmoid-type fibromatosis (PTC-DTF)—or nodular fasciitis-like stroma (PTC-NFS)—is a rare morphological variant of PTC, characterized as a biphasic tumor comprising extensive proliferating myofibroblastic stroma and PTC component in varying proportions.Organs 207 editionA 20-year-old young female presented with palpable swelling over the anterior aspect of the neck for the past 3–4 months, without any associated pain, dysphagia, or change of voice. On neck examination, the swelling was felt as a firm-to-hard, mobile nodule that was arising from the left lobe of the thyroid. An ultrasonogram (USG) of the neck revealed a bulky left lobe of thyroid that showed a well-defined heterogenous lesion measuring approximately 24 × 26 × 36 mm with involvement of the adjacent isthmus. Mild vascularity within the lesion was seen on color flow imaging. Fine needle aspiration (FNA) confirmed the diagnosis of PTC. She underwent total thyroidectomy with central compartment lymph node dissection in an outside hospital and was referred to our institute for pathology review and further management.The pathology review of the total thyroidectomy specimen revealed a well-circumscribed, grayish-white nodule measuring 36 × 35 × 34 mm in the left lobe. The isthmus also showed two tiny grayish-white nodules measuring 3 × 2 mm and 2 × 2 mm. On microscopic examination, a non-encapsulated and well-circumscribed tumor was noted, which showed a biphasic growth pattern. The predominant component was formed by a stromal element (65%–70%) with intervening epithelial component (30%–35%) . The epiBRAF V600E mutant protein  (BRAF gene (exon 15) sequencing, which revealed the BRAFV600E  mutation. Two lymph nodes in the central quadrant showed evidence of metastasis. However, in the metastatic site, only the epithelial component was detected, showing features of classical papillary carcinoma without the presence of a stromal component  was made.No significant atypia, mitotic activity, or necrosis was evident within the stroma. Two nodules in the isthmus region displayed features of nodular hyperplasia; no evidence of papillary carcinoma was noted. On immunohistochemistry (IHC) assessment , the epiVentana) , beta-caVentana) , PAX8 (FVentana) , pan cytomponent . Based oPostoperatively, two USG scans (3 months apart) were performed, which did not reveal any residual lesion. Stimulated serum thyroglobulin (4.06 ng/mL) and anti-thyroglobulin (106 IU/mL) were also within the normal range. Subsequently, the low-dose whole-body radioiodine scan performed was also negative. The patient was doing well at follow-up of 9 months.WHO Classification of Tumors of Endocrine Organs-Many histological variants of PTC are recognized and most of them carry an excellent prognosis owing to their indolent biological behavior. The recent 2017 edition of CTNNB1 mutations.BRAF and CTNNB1 mutations were restricted to the epithelial and stromal components, respectively.,CTNNB1 gene encodes for β-catenin that drives the Wnt/β-catenin signaling pathway for cell growth and/or survival. The cases, without β-catenin expression or CTNNB1 mutation, were considered as PTC-NFS, which is in concordance with our index case. However, both PTC-DTF and PTC-NFS follow a generally favorable course when managed in a similar way to conventional papillary thyroid carcinoma.MYH9-USP6 rearrangement is commonly seen in 90% of soft tissue nodular fasciitis; however, its presence has not been established in PTC-NFS.BRAF mutation in PTC-DTF found that the activation of the BRAF Val600Glu mutation is the most common genetic alteration seen in the epithelial component.,BRAF V600E mutation. IHC showed BRAF mutant protein expression only in the epithelial component, indicating that the epithelial component is neoplastic while the stromal component is likely reactive. Interestingly, in the index case at the metastatic site, the tumor showed only the epithelial component. This further indicates that the stromal component is probably reactive. However, we did not perform a CTNNB1 gene mutation or next generation sequencing. In the English literature, approximately 50% of cases reported so far showed lymph node metastasis. Although the number of reported cases is small and there is insufficient follow-up in most of the reports, it is suggested that the PTC/NFS and/or PTC/DTF seems to carry a similar prognosis to conventional PTC, irrespective of the lymph node status.PTC-DTF is differentiated from PTC-NFS by β-catenin nuclear immunoexpression in the stromal component as well as activating FNA is commonly used for the diagnosis of thyroid neoplasms. The presence of only the stromal component makes the diagnosis of PTC-NFS more difficult and challenging in FNA. A literature search revealed that FNA cytology was used to diagnose PTC-NFS or PTC-DTF in 13 cases, out of which both epithelial and stromal components were identified (7 cases), while in 5 cases, only the epithelial component was identified. However, in 1 case, the authors identified only the stromal component, which was misdiagnosed as a sarcoma on FNA.BRAF mutation, which was restricted to the epithelial component. Thorough sampling of the excised specimen is essential in order not to miss the epithelial component, which, in most reported cases—including ours—appears to be small.PTC-NFS/DTF is a histological variant of PTC with a favorable prognosis. Our index case was associated with the


Among those responses, heat-induced muscle contractions are highlighted. It is also demonstrated that the short-term heating hyperactivates molecular motors to exceed their maximal activities at optimal temperatures. We discuss future prospects for opto-thermal manipulation of cellular functions and contributions to obtain a deeper understanding of the mechanisms of cellular temperature-sensing systems.Could enzymatic activities and their cooperative functions act as cellular temperature-sensing systems? This review introduces recent opto-thermal technologies for microscopic analyses of various types of cellular temperature-sensing system. Optical microheating technologies have been developed for local and rapid temperature manipulations at the cellular level. Advanced luminescent thermometers visualize the dynamics of cellular local temperature in space and time during microheating. An optical heater and thermometer can be combined into one smart nanomaterial that demonstrates hybrid function. These technologies have revealed a variety of cellular responses to spatial and temporal changes in temperature. Spatial temperature gradients cause asymmetric deformations during mitosis and neurite outgrowth. Rapid changes in temperature causes imbalance of intracellular Ca Sensing the temperature is an essential activity for life. Humans sense a variety of temperatures of air, water, and food in their daily lives, such as sauna (~ 100 °C), hot tea (~ 60 °C), comfortable shower (~ 40 °C), iced coffee (~ 4 °C), and ice cream (~ − 10 °C). We also sense our own internal temperature to maintain our body temperature. If the temperature-sensing system is dysfunctional, we cannot predict threats such as thermal injury and hypo- and hyperthermia.Q10 values of the TRP channels, which describe the rate of change in the current amplitude when the temperature is elevated by 10 °C, exceed 7  channels, which are the temperature-sensitive ion channels  methods are frequently used in studies of protein thermodynamics. For instance, a water-soluble triphenylmethane dye crystal violet was heated by a 532-nm laser pulse to induce the unfolding of RNase A  with 1064-nm laser light were reported to directly heat membranes of CHO cells with heating efficiency of ~ 1.15 °C per 100 mW  and freezing front (0 °C) Ishizaka  or estimThe development of microscopic luminescence thermometry has constituted progress in fluorescence microscopy Fig. . TemperaLuminescence properties of the temperature probes can also be affected by non-thermal environmental parameters such as pH. This issue can be resolved by a probe that has perfect robustness. For example, temperature-sensitive dye can be covered by another material that functions as a coating to protect the dye from environmental changes. We demonstrated this strategy experimentally for the first time, where Eu-TTA dye was enclosed in a glass micropipette  along the spindle of anaphase spermatocytes accelerates the development of the aster at the warmer side, moves the spindle toward the cooler side, and induces asymmetric division Ishizaka . Nicklas2+ influx was demonstrated. We proposed the mechanism based on heat-accelerated polymerization of actin filaments and microtubules and their sliding by molecular motors.Cellular response to the temperature gradient is also the subject of studies examining the mechanism in the optical guidance of neurite outgrowth. Ebbesen and Bruus calculated the temperature gradient during the optical guidance using an infrared (IR) laser light and proposed that the heat activation of TRP channels mediates the neuronal guidance  increases in various types of cell. For instance, rapid cooling from 36.5 °C to a temperature below 18 °C induces guinea pig cardiac muscle contractures, named rapid cooling contractures (RCC) (Kurihara and Sakai 2+ release from intracellular Ca2+ store sarcoplasmic reticulum (SR) mainly through ryanodine receptors (RyRs)   . The electrochemical potential of Ca2+ across the ER membrane is enhanced. (ii) At the end of heating, the net Ca2+ flow is quickly reversed as the activities of SERCA and IP3R immediately return to the pre-heating states. (iii) The Ca2+ leak that follows the enhanced Ca2+ gradient serves as the initial Ca2+ for a large Ca2+ leak known as the Ca2+-induced Ca2+ release of IP3R. In summary, the Ca2+ response observed here is a result of the asymmetry between the temperature sensitivities of Ca2+ pumps and Ca2+ release channels and their thermal perturbation.Interestingly, similar [CaC. elegans  and concluded that the major intracellular Ca2+ sources were mitochondria  applied at 2.5 Hz  sarcomeric oscillations termed hyperthermal sarcomeric oscillations (HSOs) ] at the sarcolemma. (ii) The Ca2+ influx from extracellular space triggers intracellular Ca2+ release from SR. (iii) The Ca2+ influx and release increase [Ca2+]i, which promotes Ca2+ binding to troponin C (TnC) on thin filaments. (iv) The Ca2+ binding to TnC shifts the state of thin filaments to “on,” which allows the interaction of actomyosin. (v) Sarcomere shortening is initiated. (vi) As [Ca2+]i decreases mainly due to Ca2+ uptake into SR by SERCA and Ca2+ efflux by sarcolemmal Na+/Ca2+ exchanger, Ca2+ dissociates from TnC and the state of thin filaments shifts to “off,” which blocks actomyosin interaction.What is the mechanism of these heat-induced Caows Bers   at physiological temperature for rapid and efficient contraction in systole.Heat-induced contraction without [Ca2+-independent contraction was also demonstrated using NIR laser light and the photothermal property of gold nanoshells that were internalized in myotubes differentiated from the skeletal muscle model C2C12 cell line , cellular morphology, gene and protein expression, and cellular functions such as migration differ from those on soft biomaterials that mimic soft tissues in vivo (1–1000 kPa) (van Helvert et al. −4 to 10−5 °C. For interested readers of this issue, we recommend Baffou et al.’s commentary (Baffou et al. Finally, there remains substantial room for the development of advanced optical heaters and thermometers. Intracellular thermometry reported 1 °C or greater local temperature gradients in both stimulated and non-stimulated cells at the organelle level, such as in nuclei (Okabe et al. In this review, we introduced opto-thermal technologies and their applications for investigating cellular temperature-sensing. They have already been widely applied in the field of human, animal, and plant studies, and the number of studies is increasing. Ongoing advances of these technologies will reveal novel temperature-sensing systems and physiological significance of thermogenesis in cells and lead us to the development of advanced thermal therapies.


Pesticides have been shown to alter Ca2+ homeostasis, mainly by increasing its intracellular concentration above physiological levels. The 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 endoplasmic reticulum. It has also been observed that intracellular increases in the 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 review, we also discuss some proposed strategies to counteract the detrimental effects of pesticides on Ca2+ homeostasis.Pesticides of different chemical classes exert their toxic effects on the nervous system by acting on the different regulatory mechanisms of calcium (Ca This ion 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 i can cause to the nervous system, which put the survival of non-target organisms at risk. Such is the case of the insecticide flubendiamide, whose application induced strong Ca2+ transients in the antennal neurons of honeybees i induced by some organophosphates were substantially different. On the one hand, the team of Qian et al. [2+]i in CCF human astrocytoma cells, an effect that was not observed for the DFP insecticide. In line with these results, in the research by Vatanparast et al. i, while repeated treatment with a reduced dose of pesticide (0.05 mM) decreased the levels of the ion within the differentiated cells. These data suggest that paraoxon could exert a different effect on the [Ca2+]i in a dose-dependent manner.However, the changes in the [Can et al.  demonstrt et al.  it was ft et al.  found tht et al.  observed2+]i caused by depolarization. This result suggests that the inhibition of Ca2+ influx evoked by depolarization could be specific to organophosphate since this effect was not observed after exposure to carbaryl, a carbamate insecticide [Meijer et al.  also fouecticide .2+]i is by its influx through channels present in the plasma membrane. There is a wide variety of channels that allow Ca2+ influx, including VGCCs, SOCs and ROCs, with their open or closed state markedly influencing the [Ca2+]i in neurons.The effects observed on the functioning of membrane channels and pumps are shown in 2+ influx necessary to initiate synaptic transmission at conventional synapses i induced by rotenone, dichlorvos and methamidophos depend on their action on L-type VGCCs i i . Therefol. [2+]i , the metl. [2+]i . Thus, t2+]i increase elicited by depolarization. These non-additive effects contrast with previous observations of other classes of pesticides, in which mixtures of organochlorine insecticides or azole fungicides exerted additive inhibition of VGCCs i-dependent K+ currents, which contribute to repolarization and after-potential hyperpolarization i in virtually all cells. SOCs are not voltage-dependent but are activated by depletion of ER Ca2+ stores with the goal of allowing faster return of the ion to the intracellular stores i induced by both malathion and the pyrethroid lambda-cyhalothrin i in substantia nigra pars compacta neurons was dependent on TRPM2 activation.Another type of channel present in the neuronal membrane is the transient receptor potential channel (TRP channel), a type of non-selective cation channel that allows Caignaling . Within ignaling . The finignaling  suggest  the CNS . This ch species . In line species  found th2+ homeostasis could be the activation of some ROCs present in the cell membrane, both ionotropic and metabotropic. Thus, it has been shown that muscarinic (mAChRs) and nicotinic (nAChRs) acetylcholine receptors could be one of the molecular targets of organophosphates pesticides. The neurotoxicity exerted by organophosphates is mainly due to the irreversible inhibition of acetylcholinesterase activity, resulting in the accumulation of acetylcholine in the synaptic cleft and the subsequent activation of their respective receptors [2+]i, since the activation of nAChRs induces the direct entry of Ca2+, while the stimulation of mAChRs can trigger signal transduction cascades leading to phosphorylation/dephosphorylation of Ca2+ channels and/or mobilization of Ca2+ stored in intracellular stores i.The effects of exposure to different pesticides on intracellular Caeostasis ,103,104.diseases ,106. Undpoptosis ,108. Thecultures ,45. The 2+ signaling. Specifically, there are specialized structures called mitochondria-associated membranes, which are responsible for the intimate association between these two organelles and are involved in the control of Ca2+ homeostasis i and buffer severe overloads of this ion i is above the threshold, mitochondria accumulate Ca2+ rapidly and release it slowly [2+]i elevations induced by certain pesticides. Specifically, the findings of the in vivo study by Kaur et al. i increase. These results agree with those obtained in previous studies, in which neuronal damage resulting from organophosphate exposure was associated with an increase in IP3 levels i i ,131. Someostasis . In lineeostasis  demonstrt et al.  also obs2+]i induced by certain pesticides can lead to the activation of Ca2+-activated proteases, mainly calpain. Calpain is an evolutionarily conserved Ca2+-dependent cysteine protease that influences a wide range of physiological events, including development, differentiation, cell proliferation, and apoptosis, among others i can also influence the activity of Ca2+/calmodulin-dependent protein kinases II (CaMKIIs), which are key regulators of Ca2+ signaling in eukaryotic cells [2+]i and phosphorylates target proteins involved in processes such as ion channel modulation and regulation of gene expression, among others [2+]i leads to CaMKII activation, resulting in inhibition of mammalian or mechanistic target of rapamycin (mTOR) signaling and induction of neuronal apoptosis. Since the protein kinase mTOR acts as a central controller for neuronal survival, suppression of this pathway can trigger apoptosis i and consequent neurotoxicity in early developmental stages.Deltamethrin exposure has been shown to cause positive up-regulation of S1002 and 15 . This po neurons . Therefo2+ chelators and antioxidant substances on the pesticide-induced neuronal toxicity are shown in 2+]i. Consequently, one of the strategies that showed the greatest efficacy in reversing pesticide-induced alterations has been the use of Ca2+ chelators. Such chelators mainly include 1,2-bis-(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) and ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA). Treatment with BAPTA-AM, a selective intracellular Ca2+ chelator, showed protective potential to prevent cytotoxicity evoked by malathion, rotenone and lambda-cyhalothrin i. In contrast, the study by Vatanparast et al. i by inducing its release from intracellular stores and/or its entry from the extracellular medium i and ROS generation induced by deltamethrin. Tert-butylhydroquinone is a food additive known for its antioxidant properties i i ,167. The2+]i dysregulation induced by acute rotenone exposure. Creatine is an endogenous compound that is predominantly stored in skeletal muscles but is also present in other parts of the body, such as the liver, kidneys, or brain  in neurons documented in the in vivo studies provide sufficient evidence about the ability of these substances to cross the blood–brain barrier and enter the CNS, where they can produce significant neurotoxic effects.The data described in all the studies included in this systematic review coincide in pointing out the ability of different classes of pesticides to induce alterations in the Ca2+ acts as a second messenger in many neuronal signaling pathways, where it directly or indirectly influences many cellular functions. For this reason, cells possess multiple mechanisms aimed at maintaining Ca2+ homeostasis, since an alteration in its levels can have significant harmful effects. During nervous system development, Ca2+ is responsible for orchestrating a wide range of biochemical cascades and gene expression programs critical for neurodevelopment i of approximately 100 nM, by storing it in intracellular organelles and expelling it into the extracellular space [2+]i by perturbing the mechanisms involved in the Ca2+ homeostasis and increasing its intracellular levels. 2+ homeostasis.Under physiological conditions, cells maintain a basal [Caar space . The stu2+]i can occur due to the influx of the ion from the extracellular medium through plasma membrane channels and/or due to a Ca2+ release from internal stores. Ca2+ influx from the extracellular space may occur mainly through VGCCs or ROCs. It has been demonstrated that some organophosphate pesticides can activate VGCCs, especially L- and T-type VGCCs, thereby enhancing Ca2+ influx into the cellular interior. There is also evidence about the influence of this class of pesticides in the activation of nAChRs and mAChRs, whose activity is related to the Ca2+ influx or its release from intracellular stores, respectively. However, the action of organophosphates on these receptors appears to vary, and they may act as agonists or inhibitors depending on the particular organophosphate, receptor subtype, and cell type. Pesticides also appear to induce the opening of other channels present in the plasma membrane, such as TRPs, non-selective cation channels that allow Ca2+ influx into certain cell types.As previously mentioned, an increase in the [Ca2+]i levels occurs, various types of proteins are activated to expel the ion against the gradient by means of ATP hydrolysis (PMCAs) or by using the energy stored in the electrochemical Na+ gradient (NCXs) i are rapidly eliminated to return to basal levels, an alteration in Ca2+ extrusion systems by pesticides will increase the permanence of the ion inside the cell.When an increase in the i increases. However, it is possible that pesticides may induce the activation of this pathway by different ways: (1) by activating G-protein-coupled receptors on the plasma membrane (mainly muscarinic receptors); (2) by directly interacting with PLC; (3) by directly interacting with IP3 receptors.One of the main pathways involved in Ca2+ stores induced by pesticides can lead to the depletion of the ion. To protect itself from the deleterious effects of Ca2+ store depletion, the cell has a store-operated gating mechanism, the SOCs, present in the plasma membrane i induced by some pesticides were mediated by ion entry through the SOCs. Furthermore, Ca2+ influx through these channels could be also regulated by PKCs, a large family of proteins that plays an important role in modulating the activity of direct Ca2+ regulators such as Ca2+ channels i can also trigger the activation of different proteins such as calpain, a Ca2+-activated cysteine protease involved in a wide range of physiological events i i ,187,188. [Ca2+]i . The evi [Ca2+]i .β protein was another change caused by pesticides. This multifunctional protein could exert a dual neurotoxic effect on neurons. On the one hand, extracellular S100β could increase ROS levels inside neurons by binding to the membrane receptors for advanced glycation end products (RAGEs) [β protein found inside neurons could potentiate the increase in [Ca2+]i i .2+]i was not observed in all of the studies analyzed, with organophosphates being the class of pesticides that exerted the most imprecise effects on the Ca2+ levels. Therefore, although most of the evidence included here suggests that pesticides induce an increase in Ca2+ levels, further research is needed to confirm whether the differences found between studies could be due to differences between species, pesticide class, dose and/or exposure characteristics used in each study.However, the increase in i homeostasis in neurons. Most of the pesticides analyzed seem to induce a significant increase in [Ca2+]i by activating membrane Ca2+ channels and inhibiting Ca2+ extrusion mechanisms; at the same time, they favor the Ca2+ release from intracellular stores, especially from the ER. An excessive decrease in Ca2+ stored in the ER can induce the response to stress in this organelle and prompt the mitochondria to assume the storage of large amounts of Ca2+ coming from both the ER and the cytosol. Ca2+ overload in mitochondria would induce a significant impairment in the functioning of the mitochondrial respiratory chain, severely disturbing ATP synthesis and increasing ROS generation. Generally, under these oxidative stress conditions, oxidants activate Ca2+ channels, inhibit pumps, and can reverse Na+/Ca2+ exchangers, thus potentiating the increase in [Ca2+]i. Likewise, ROS cause deterioration of membrane lipids, favoring Ca2+ influx from the extracellular medium, in addition to damaging the membranes of organelles that stores Ca2+, potentiating the leakage of the ion into the cytosol. As a result of oxidative stress and intracellular Ca2+ overload in the mitochondria and nucleus, a cascade of biochemical processes is activated, including calpain activation and CAMKII-dependent mTOR inhibition, leading ultimately to neuronal death by apoptosis.The findings described in the present review provide ample evidence for the ability of different classes of pesticides to alter [Ca2+]i or even showed contradictory results, especially organophosphate compounds. Therefore, the discrepancy between some findings points to the need for further studies to identify how factors such as the type of pesticide, the dose, or the mode of exposure can modify the effects of these compounds on Ca2+ homeostasis in the CNS.However, some pesticides did not increase the [Ca


It is shown that the PO-6 can effectively bind to the CD123+ AML cells and the micellar formulation mPO-6 increases the dissolution stability and the specific binding capacity. When injected intravenously, mPO-6 significantly prolongs the survival of the refractory AML mice by interfering CD123/IL-3 axis, evidenced by the down regulation of phosphorylation of STAT5 and PI3K/AKT and the inhibition of activated NF-κB in the nucleus, as well as by the analysis results of next generation RNA-sequencing (RNA-seq) with the bone marrow of the AML mice. The antagonistic effect leads to the significantly reduction of AML cells infiltration in the bone marrow of the AML mice. In conclusion, mPO-6 could provide a potent antagonistic therapeutic approach for targeted treatment of AML.Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival rate due to the refractory feature and high rate of relapse. CD123 is highly expressed on multiple types of AML cells, especially leukemia stem cells, and closely associated with the poor prognosis of AML. Aiming to meet the urgent demand to targeted therapeutics for the refractory AML patients, herein we synthesize a CD123 antagonistic peptide (PO-6) loaded in nanomicelles (The online version contains supplementary material available at 10.1186/s13045-021-01206-y. Acute myeloid leukemia (AML) is a common malignant heterogeneous hematopoietic disease with very low average 5-year survival of 25% due to the refractory feature and high rate of relapse , 2. CD12+ AML cell line MOLM-13 cells in a concentration-dependent manner while weakly bind to the CD123− AML cell line HL-60 and chronic myeloid leukemia cell line K562 cells  with the average diameter of 38 nm  has been obtained based on screening of the cell culture from a group of de novo designed peptides targeting to various segments of the CD123 protein   of the AML mice post three intravenous administrations of ted Fig. A, and thted Fig. B. Furtheted Fig. C. Wester BM Fig. D, which  BM Fig. E observe BM Fig. F detecte BM Fig. G of the In summary, we report a novel and chemically synthesized peptide with antagonistic function towards CD123. The peptide in its micellar formulation displayed significant anti-leukemia activities in the refractory AML mice, providing an effective and safe therapeutics to the refractory AML treatments.Additional file 1. Supplementary material and methods.  Fig. S1. Affinity of PO-6 and mPO-6 to different leukemia cells. Fig.S2. The TEM image of mPO-6. Fig. S3. Confocal microscopy observation of PO-6 binding with MOLM-13 cells. Fig. S4.mPO-6 inhibited the antibodies binding to CD123. Fig. S5. Affinity of mPO-6 to the AML blasts. Fig. S6. CD123 expression on GFP+ cells in BM of AE & CKITD816V mice. Fig. S7. Therapeutic efficacy of Ara-C or HHT in AE & CKITD816V mice. Fig. S8. The fluorescent representative imaging of main organs at designated time points after mPO-6-FITC injection in healthy mice. Fig. S9. Acute toxicity evaluation of mPO-6 in healthy mice. Table S1. Human and mouse CD123 alignment of the extracellular amino acid sequences.Additional file 2. Materials and Methods.


Owing to their diverse functions, TMEM16 proteins have been implicated in various human diseases, including asthma, cancer, bleeding disorders, muscular dystrophy, arthritis, epilepsy, dystonia, ataxia, and viral infection. To understand TMEM16 proteins in health and disease, it is critical to decipher their molecular mechanisms of activation gating and regulation. Structural, biophysical, and computational characterizations over the past decade have greatly advanced the molecular understanding of TMEM16 proteins. In this review, we summarize major structural features of the TMEM16 proteins with a focus on regulatory mechanisms and gating.The transmembrane protein 16 (TMEM16) family consists of Ca As passive phospholipid transporters, TMEM16 CaPLSases can efficiently translocate phospholipids at high speed -bisphosphate [or PIP2], and pH regulate TMEM16 Ca2+-dependent gating. This is by no means a comprehensive review of TMEM16 structure and function. The readers are encouraged to refer to the excellent reviews of the biophysics  topology, we now know that each TMEM16 monomer consists of 10 TM segments preceded by a long N-terminal cytosolic domain (NCD) and followed by a short C-terminal extension of TM10 . TM7 andng sites , 2. The ng sites . Notablyng sites . This pulippases . This molippases . For dualippases . In supplippases . The liplippases . By contr lipids ,B. The r2+-dependent activation and PIP2-dependent regulation (see in the next sections), we recently proposed a modular model of TMEM16 proteins to simplify the complex TMEM16 architecture P2 binding are structurally and functionally segregated, the PGD of TMEM16A can be further divided into two modules (2+ module consists of TMs 6–8 and is responsible for binding Ca2+ and initiating Ca2+-dependent activation. The regulatory module (TMs 3–5) forms the other half of the PGD, which works synergistically with the Ca2+ module to facilitate TMEM16 gating and permeation. In TMEM16A, PIP2 binding to the regulatory module stabilizes the open state and prevents the PGD from collapsing and entering the desensitized state. TMs 1, 2, 9, and 10, on the other hand, form the supporting domain. As will be discussed below, the supporting domain contains a conserved third Ca2+ binding site that is allosterically coupled to the PGD  assay  assay , it is e binding . Unlike 2+ binding residues, including E650 on TM6, E698 and E701 on TM7, E730 and D734 on TM8  as additional Ca2+ coordinates  delay after establishing the whole-cell patch clamp configuration -induced cytotoxicity, suggesting that basal Ca2+ activity is sufficient to open the inner activation gate. Remarkably, F518K and Y563K result in constitutively active TMEM16F scramblases even when the primary Ca2+ binding sites are destroyed. More strikingly, the TMEM16A L543K mutation, equivalent to TMEM16F F518K, converts the TMEM16A CaCC into a GOF phospholipid scramblase  and the main-chain carbonyl group of an isoleucine (I857 in mTMEM16F and I613 in hTMEM16K). The main-chain carbonyl of S854 in TMEM16F (A610 in hTMEM6K) also appears to provide a coordination for the bound Ca2+. Interestingly, there is a conserved lysine , which apparently forms a stabilizing electrostatic interaction with the aspartate in TM10.In addition to the extensively studied primary Ca TMs 6–8 , recent g domain . This th2+ binding site in TM2 and TM10 was conducted -mediated bridging of substituted cysteines at E425 in TM2 and D879 in TM10 strongly inhibits channel activation in a manner independent of the primary Ca2+ sites. These results could also explain previous studies implicating the functional importance of TM10’, the extended alpha helix following TM10. In fact, replacing or truncating the C-terminal region following TM10 markedly altered the Ca2+ sensitivity of TMEM16A  patient with unknown pathophysiology P2 markedly reduces its activity. However, it is worth noting that several approaches used to alter PIP2, namely PLC activation or inhibition of PI4K, could also affect other intracellular signaling events that may lead to changes in intracellular Ca2+. One possibility is activation of PLC, while reducing PIP2 also leads to additional Ca2+ release from internal stores, thereby enhancing TMEM16A activation. In fact, numerous subsequent studies from several laboratories all suggested that PIP2 serves as a positive regulator of TMEM16A P2 -bisphosphate [or PIPsporters . PIry cells . Reducin TMEM16A  and para TMEM16A . DepletiIP2 . A hallmtivation ; exogeno patches .2-dependent regulation of TMEM16A, unbiased mutagenesis screens were conducted to identify basic residues that play important roles in desensitization in TMEM16A P2 binding could directly affect Ca2+-dependent channel gating. Finally, site C/4 is situated on TM2-3 loop and is defined by R461 (R457), K480 (K476), and R484 P2 binding sites led to further dilation of the permeation pathway P TMEM16A . On one EM16A P2 hydrolysis following membrane depolarization-induced activation of DrVSP led to reduced TMEM16A activity. Interestingly, the TMEM16A(ac) isoform is more sensitive toward PIP2 depletion than the TMEM16A(a) isoform, which lacks the EAVK segment in the TM2-3 loop. Consistent with the proposed PIP2 binding site reported by Le et al., Ko et al. also identified R482 (R486 in the TMEM16A(ac) isoform) in TM2-3 loop as the most critical residue for PIP2 binding P2 degradation by DrVSP. Pharmacological inhibition of CaMKII promotes TMEM16A opening due to augmented single channel conductance. Notably, S669 (S673) at the cytosolic end of TM6 is likely the substrate for CaMKII-mediated phosphorylation, as the phosphomimetic mutation S669D reduces, whereas the S669A mutation enhances the PIP2 sensitivity of TMEM16A. These results hint at an allosteric mechanism involving PIP2 binding and CaMKII-dependent phosphorylation in controlling TMEM16A channel activity.More recently, Ko et al. reported that TMEM16A exhibits isoform-specific PIPsitivity . By co-e binding . Mutatio2 to the proposed binding site in TM3-5 consistently leads to spontaneous pore opening, which is wide enough to allow Cl− permeation P2, whereas its N-terminus (towards the outer leaflet) moves in the opposite direction, thereby separating from TM6 and widening the central constriction site. The “pivot” movement of TM4 is endowed by the helix–helix packing between TM4 and TM5 on the intracellular side. This proposed PIP2-dependent gating in TMEM16A is reminiscent of the TMEM16 scramblases in which disruption of the TM4 and TM6 interaction leads to opening of the lipid pathway Prmeation . This po pathway .2+ sensitivity and pronounced current rundown during prolonged Ca2+ stimulation, both of which were shown to be a result of the rapid dissociation and/or hydrolysis of endogenous membrane-bound PIP2 P2 synthesis or depletion of PIP2 markedly attenuated TMEM16F-mediated Cl− current in Caco-2 cells (2 may interact with TMEM16F at two adjacent sites (or KR motifs) at the N-terminus formed by two clusters of basic residues: one proximal site formed by K281-K290 and one distal site formed by K87-R98 P2 binding, underscoring the functional importance of the proximal KR motif in PIP2 binding. Paradoxically, electrophysiological studies by 2+ sensitivity as well as the ability of exogenous PIP2 to rescue TMEM16F current after rundown. By contrast, neutralization of the basic residues in the proximal KR motif  had no effect on TMEM16F Ca2+ sensitivity P2 on TMEM16A P2-dependent regulation.Ion channel activity of TMEM16F also exhibits a reduced CaIP2 . Interes-2 cells . It was sitivity . While t TMEM16A . So far, TMEM16A , have be2 in regulating TMEM16F scrambling P2 supplementation allows it to undergo a pronounced upward movement toward the membrane to widen the intracellular vestibule without changing the ion permeation pore, especially the upper constriction region P2-dependent conformational changes affect TMEM16F channel activity.A recent structural study revealed the potential structural role of PIPrambling . In the n region . The resn region . Future i) suppresses endogenous Ca2+-activated Cl− channels (CaCCs) from the human colon carcinoma cell line T84 and lacrimal gland acinar cells  activation of TMEM16A. The authors further demonstrated that double mutation of Ca2+ binding residues in TM6-8, including N650A/E654Q  isoform), E702Q/E705Q (TM7), and E734Q/D738N (TM8) abolished this proton-mediated inhibition. Based on this evidence, the authors proposed that protons may inhibit TMEM16A channel activation by competing with Ca2+ binding to Ca2+ binding sites in TM6-8.Previous studies showed that low intracellular pH  assay  assay . ConsistF) assay  and in nF) assay , low pHi results . Second,g fusion .i on TMEM16A, low extracellular pH enhances TMEM16A channel opening without altering the apparent Ca2+ sensitivity  isoform) of TM6 and R515 (R511) together constitute the equivalent SE site proposed by In contrast to the effects by pHsitivity . This su2+ site is allosterically coupled to the PGD and how all three Ca2+ bindings sites synergistically control TMEM16 activation under physiological conditions. Second, future investigations are needed to dissect how Ca2+ and voltage synergistically operate TMEM16 gating. The answer to this question is critical to uncover the physiological functions of TMEM16 proteins in excitable cells such as neurons and muscles. Together, we have started to understand the molecular mechanisms of TMEM16 ion and lipid permeation and identified several molecular determinants that define whether a TMEM16 protein is a sole ion channel or a dual function scramblase/ion channel. Comprehensive studies are needed to demonstrate how ion and phospholipid permeation are dynamically controlled by Ca2+- and voltage-induced conformational changes in the PGD. Substantial progress has been made on deciphering how pH and PIP2 regulate TMEM16 proteins. Identifying other physiological regulatory factors, such as post-translational modifications, are needed to further reveal how TMEM16 protein activities are fine-tuned under physiological conditions. Additionally, the Ca2+-bound TMEM16A and TMEM16F structures were captured in non-conductive states. Future structural studies are needed to capture the open conformations, which will enhance our understanding of TMEM16 gating transitions in response to Ca2+ and voltage stimulation. Apart from the ER-resident TMEM16K, the other mammalian TMEM16 proteins expressed in intracellular organelles are largely uncharacterized. Functional and structural characterization of these TMEM16 proteins will help us better evaluate their biological functions in health and disease. Finally, the evolutionary relationships between TMEM16, OSCA/TMEM63, and TMC proteins within the transmembrane channel-scramblase (TCS) superfamily are intriguing. A combination of structural, functional, and computational approaches is needed to unveil the molecular underpinnings of how this superfamily of membrane ion channels and scramblases posses different permeation, activation, and gating properties. In this review, we summarize the collective efforts from the TMEM16 field over the past decade. We propose a “modular design” model for TMEM16 assembly, and the “clam-shell” and “pore-dilation” gating/permeation models for TMEM16 scramblases and channels, respectively. We hope these simplified models serve as a steppingstone for answering the aforementioned questions, driving the field forward.Structural, functional, and computational studies in the past decade have greatly advanced our understanding of TMEM16 proteins at the molecular level. In the next phase, the answers to the following questions will further advance our understanding of these enigmatic proteins. First, it will be important to demonstrate how the third CaSCL, PL, AJL, and HY wrote the manuscript. SCL plotted all the figures. All authors contributed to the article and approved the submitted version.This work was supported by NIH grant DP2-GM126898 (to HY) and American Heart Association Pre-doctoral Fellowship 19PRE34380456 (to SCL).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.


Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.Spontaneous AP  firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca SR Ca2+ content and LCRs were measured with a confocal microscope  in the line-scan mode at 35 ± 0.5 °C, as previously described i in response to Na+/K+ pump inhibition by the digitalis glycoside (digoxigenin) perturbs SANC automaticity via impacting numerous coupled-clock mechanisms by producing changes in Na+ and Ca2+ electrochemical gradients [2+ pumping by phosphorylation of PLB that modulates SERCA2A Ca2+ pumping, phosphorylation of phospholemman modulates Na+/K+ pump activity [+ and Ca2+ electrochemical gradients.PPs can regulate pacemaker activity directly and indirectly via impacting other clock functions. We have previously demonstrated that even a small increase in [Naradients . A subseradients  validateactivity ,47. Of nactivity . In othe2+ and M clocks [2+ release in the context of increases in the SR Ca2+ pumping rate and ICaL, mimicking the experimental result of increased protein phosphorylation and increased ICaL amplitude by CyA  [f conductance [Importantly, the observed AP firing rate increase is fully quantitatively explained only when protein phosphorylation of both clocks is increased, i.e., when the Ichanism)  or via aductance .s), simulating possible effects of RyR phosphorylation has only a minor effect on the AP firing rate , the AP firing rate slows down. In other words, a stronger, premature Ca2+ release interferes with SR Ca2+ refilling [2+ pumping by an excessive amount of Ca2+ to be pumped may explain previous observations of a slower rate and arrhythmia caused: (i) by catecholaminergic polymorphic ventricular tachycardia (CPVT) in which RyR mutations result in excessive RyR release [2+ release in the presence of ryanodine [That a major increase in RyR opening rate  that modulate the basal state clock coupling and the spontaneous AP firing rate. The main functional effects of phosphorylation on both M and Ca2+ clock proteins is an increase in the size and an earlier occurrence of spontaneous RyR-generated LCRs  during diastolic depolarization, which shifts the acceleration of diastolic depolarization to earlier times, leading to a reduction in the AP cycle length [2+ homeostasis. The net result maintains the basal AP firing rate well below its maximum, enabling rapid, robust responses to external input aimed at affecting the AP firing rate. In other words, PP and PDE activities comprise a “double braking system”  and the surface membrane proteins (Ie length . The rede length ,53.Thus,e length  act in c system”  on the fvate PDE  and like


GUCA1A gene, is a neuronal calcium sensor protein involved in shaping the photoresponse kinetics in cones and rods. GCAP1 accelerates or slows the cGMP synthesis operated by retinal guanylate cyclase (GC) based on the light-dependent levels of intracellular Ca2+, thereby ensuring a timely regulation of the phototransduction cascade. We found a novel variant of GUCA1A in a patient affected by autosomal dominant cone dystrophy (adCOD), leading to the Asn104His (N104H) amino acid substitution at the protein level. While biochemical analysis of the recombinant protein showed impaired Ca2+ sensitivity of the variant, structural properties investigated by circular dichroism and limited proteolysis excluded major structural rearrangements induced by the mutation. Analytical gel filtration profiles and dynamic light scattering were compatible with a dimeric protein both in the presence of Mg2+ alone and Mg2+ and Ca2+. Enzymatic assays showed that N104H-GCAP1 strongly interacts with the GC, with an affinity that doubles that of the WT. The doubled IC50 value of the novel variant (520 nM for N104H vs. 260 nM for the WT) is compatible with a constitutive activity of GC at physiological levels of Ca2+. The structural region at the interface with the GC may acquire enhanced flexibility under high Ca2+ conditions, as suggested by 2 μs molecular dynamics simulations. The altered interaction with GC would cause hyper-activity of the enzyme at both low and high Ca2+ levels, which would ultimately lead to toxic accumulation of cGMP and Ca2+ in the photoreceptor outer segment, thus triggering cell death.Guanylate cyclase-activating protein 1 (GCAP1), encoded by the  Subtle alterations of their levels in the outer segment during the response to light triggers feedback mechanisms, which permit a timely shutoff of the cascade as well as adaption to specific light or dark conditions  in the <19 nM–1 mM range. The GCAP1 concentration at which GC activation is half-maximal (EC50) was measured by incubation of increasing concentrations of N104H-GCAP1 (0–10 µM) in the presence of <19 nM free Ca2+. The cGMP synthesized during the enzymatic reactions was quantified by means of HPLC using a C18 reverse phase column . Data are reported as the mean ± standard deviation of at least three data sets. The statistical significance of the differences between the maximal and minimal GC activation by WT- and N104H-GCAP1 was assessed by two-tailed t-test (p = 0.001).To test whether the N104H substitution in GCAP1 affected the regulation of GC1 activity, specific enzymatic assays were conducted to monitor cGMP synthesis. HEK293 cells were used to stably express human recombinant ROS-GC1 (GC) as previously described . GC assa2+ or 1 mM Mg2+ and 1 mM Ca2+. After a 5-min incubation at 25 °C, samples were boiled and loaded onto the gel. Electrophoresis was run for 50 min at constant voltage (200 V) and protein bands were revealed by Coomassie blue staining. Limited proteolysis was performed on 20 µM WT and N104H-GCAP1 in the same conditions as for the mobility shift assay, with the addition of 0.3 µM trypsin  to each reaction mix. To evaluate the optimal incubation time with trypsin  containing ~25 µM 5,5′Br2-BAPTA. The absorbance at 263 nm was recorded upon sequential additions of 3 µM Ca2+ to the solution at room temperature until a plateau was reached. Data were fitted to a three-sequential binding site model using CaLigator software [i) and apparent affinity constants (Kdapp = 10-(logK 1+ logK 2+ logK 3)/3) reported in 2+ binding sites:Q] and [P] are the concentrations of 5,5′Br2-BAPTA and GCAP1 variants, respectively, measured at the end of each repetition by Bradford assay, A263 is the absorbance at 263 nm, minA and maxA are the minimal and maximal absorbance values registered.The Caescribed ,33,34. Lsoftware  to estim2+ or 1 mM Mg2+ + 300 µM free Ca2+. Near-UV CD spectra were recorded in a 1 cm quartz cuvette with a protein concentration of ~39 µM after serial additions of 500 µM EGTA, 1 mM Mg2+ and 500 µM free Ca2+. Thermal denaturation profiles were recorded by monitoring the ellipticity at 222 nm in a temperature window spanning from 20 °C to 96 °C (scan rate 90 °C/h). Denaturation data were fitted according to the following model, as in [n and u are the native and unfolded states, b is the baseline value, T is the temperature, k is the slope of the plateaus and ΔnuG is the Gibbs free energy for folded-to-unfolded transition which can also be expressed in terms of change in enthalpy and heat capacity upon denaturation at constant pressure as follows The alterations in thermal stability, secondary and tertiary structure of N104H-GCAP1 upon ion binding were analyzed by means of Circular Dichroism (CD) spectroscopy on a Jasco J-710 spectropolarimeter supplied with a Peltier-type cell holder. Proteins were resuspended in 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT and each recorded spectrum was the average of 5 accumulations. Far-UV CD spectra and thermal denaturation profiles were recorded in a 0.1-cm quartz cuvette with a protein concentration of 15 µM and 10 µM, respectively, in the presence of 300 µM EGTA, 300 µM EGTA + 1 mM Mgl, as in :(3)𝛳222=2+ and Ca2+-saturating conditions. Protein samples (20 µM) were loaded onto a Superose 12 10/300 column , previously equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT + 500 µM EGTA and either 1 mM Mg2+ or + 1 mM Mg2+ and 1 mM Ca2+ at room temperature. Elution profiles were collected at 280 nm and the distribution coefficient (Dc) was calculated as follows:eV is the elution volume, tV represents the total column volume (25 mL) and vV is the void volume (8 mL). Finally, the molecular weight was estimated from the calibration curve of log (MW) vs. cD as previously described [Analytical gel filtration was employed to analyze the apparent molecular weight and the oligomeric state of GCAP1 variants under Mgescribed .To investigate the variations in hydrodynamic diameter, oligomeric state, and aggregation propensity of N104H-GCAP1 in different cation-loading states, the samples from aSEC were directly analyzed in a Zetasizer Nano-S  at 25 °C using previously established settings . The ana2+-loaded myristoylated human GCAP1 (UniProt entry: P43080) was obtained employing the “Advanced Homology Modeling” tool provided by the software Bioluminate , using the Ca2+-loaded myristoylated GCAP1 from G. Gallus (PDB entry: 2R2I [2+-bound) was obtained by deleting the Ca2+ ion bound to EF-4 and substituting the remaining Ca2+ ions in EF-2 and EF-3 with Mg2+ as performed in earlier work [2+-bound and Ca2+-loaded GCAP1 variants underwent energy minimization and equilibration procedures as elsewhere described (2 ns in NVT ensemble with and without position restraints) [The homology model of Cary: 2R2I ) as a teier work . Moleculier work  using thier work  force fitraints)  prior totraints)  based on


Currently, cytogenetic and genetic markers are the most important for risk stratification and treatment of patients with acute myeloid leukemia (AML). Despite the identification of many prognostic factors, relatively few have made their way into clinical practice. Therefore, the identification of new AML biomarkers is useful in the prognosis and monitoring of AML and contributes to a better understanding of the molecular basis of the disease. Homeobox (HOX) genes are transcription factors that lead to cell differentiation blockade and malignant self-renewal. However, the roles of HOX genes in AML are still not fully understood and need further exploration, which may provide new strategies for the prognosis and monitoring of AML.We analyzed the RNA sequencing and clinical data from The Cancer Genome Atlas (TCGA), VIZOME, GSE13159, and GSE9476 cohorts. Analyses were performed with GraphPad 7, the R language, and several online databases. We applied quantitative polymerase chain reaction, Western Blotting, CCK8 cell proliferation assays, and flow cytometry to verify the conclusions of the bioinformatics analysis.in silico protein–protein interaction analysis and 450K TCGA DNA methylation data analysis and found that HOXB5 interacted with two HOX genes (HOXA7 and HOXB4) that were commonly regulated by DNA methylation levels.We identified HOXB5 as the only gene among the HOX family that was not only elevated in AML but also a significant prognostic marker in AML patients. HOXB5 was highly expressed in AML patients with NPM1, FLT3, or DNMT3A mutations and was expressed at the highest level in patients with NPM1-FLT3-DNMT3A triple-mutant AML. Gene Ontology analysis and gene set enrichment analysis revealed that HOXB5 showed a negative correlation with the myeloid cell differentiation signature and that the tumor necrosis factor/nuclear factor κB signaling pathway was involved in the molecular mechanism. Moreover, we performed HOXB5 is associated with the malignant development of AML and may be a treatment target and biomarker for AML prognosis prediction. Acute myeloid leukemia (AML) is a hematopoietic neoplasm characterized by clonal proliferation and differentiation blockade and inhibited apoptosis of malignant stem or progenitor cells . AML is Homeobox (HOX) genes are highly conserved homeodomain-containing transcription factors important for early development, maintaining the stemness of stem cells, and hematopoiesis . The humin silico enrichment analysis showed that HOXB5 was negatively associated with the regulation of myeloid cell differentiation, probably through the tumor necrosis factor (TNF)/nuclear factor κB (NF-κB) pathway. HOXB5 could be a potential biomarker for AML prognosis prediction and treatment targeting.In this study, we first identified four HOX members that were significantly upregulated in AML patients and found HOXB5 to be the only significantly altered HOX gene with prognostic value. Then, we analyzed the relationship between HOXB5 and the malignant phenotypes of AML and found that HOXB5 might participate in the progression of AML. Moreover, 1. Transcriptome sequencing data of 26 AML patients and 10 healthy volunteers were obtained from GSE94762. One hundred seventy-nine AML patients’ clinical information and transcriptome sequencing data of The Cancer Genome Atlas (TCGA) were downloaded from https://xenabrowser.net. The AML mutation data (MAF file), copy number variation (CNV) data, and DNA methylation data were also downloaded from https://xenabrowser.net. Clinical information along with transcriptome sequencing data of VIZOME (451 patients) were downloaded from https://www.vizome.org/ and https://www.cbiopportal.org/. Transcriptome sequencing data of 17 AML cell lines were downloaded from https://www.portals.broadinstitute.org/ccle/data. Drug sensitivity data of 17 AML cell lines were obtained from http://www.cancerrxgene.org/.Transcriptome sequencing data of 542 AML patients, 206 MDS patients, and 74 healthy volunteers were collected from GSE131593 to find possible functions of HOXB5. Gene set enrichment analysis (GSEA) was carried out to verify the AML-related functions and explore the potential signal pathway leading to malignant phenotypes between patients with high and low HOXB5 expression4. Heatmaps were made by R language to express information correlated with HOXB5 expression. HOXB5 protein–protein interaction was detected using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and GeneMANIA datasets. STRING and GeneMANIA are both frequently used datasets, which can provide protein–protein interaction information  enrichment analysis was performed by DAVID 6.8ormation .2.The human AML cell lines THP1, KASUMI-1, HL60, and MOLM14 were purchased from the Chinese Academy of Sciences Cell Bank . The human AML cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C with 5% COTM Reagent , PrimeScriptTM RT Master Mix  , TB Green® Premix Ex TaqTM II (Tli RNaseH Plus) , bortezomib , daunomycin , cytarabine , CCK8 , and PMA .Antibodies used were as follows: HOXB5 antibody , GAPDH antibody , horseradish peroxidase (HRP)–conjugated Affinipure Goat Anti-Mouse immunoglobulin G (IgG) (H + L) , HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) , TNF-α antibody , RELA antibody , p-RELA antibody , IKBα antibody , p-IKBα antibody , and CD11b antibody , CD14 antibody . Reagents were as follows: RIPA Lysis Buffer , TRIzolTotal RNA was isolated from AML cell lines using TRIzol reagent (Invitrogen), according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using Prime-Script RT Master Mix (TaKaRa). Quantitative polymerase chain reaction (qPCR) was performed using SYBR Green Master Mix (TaKaRa). The following conditions were used: one cycle of 95°C for 30 s, followed by 40 cycles of a two-step cycling program . The mRNA expression of target genes was calculated by the 2 ΔΔCt method and normalized to GAPDH mRNA expression.The primers for the HOXB5 gene were as follows: forward: 5′-AACTCCTTCTCGGGGCGTTAT-3′; reverse: 5′-CATCCCAT TGTAATTGTAGCCGT-3′.The primers for the SPI1 gene were as follows: forward: 5′-GACACGGATCTATACCAACGCC-3′; reverse: 5′-CCGTGA AGTTGTTCTCGGCGAA-3′.The primers for the interleukin 1β (IL-1β) gene were as follows: forward: 5′-CCACAGACCTTCCAGGAGAATG-3′; reverse: 5′-GTGCAGTTCAGTGATCGTACAGG-3′.The primers for the IL-6 gene were as follows: forward: 5′-AGACAGCCACTCACCTCTTCAG-3′; reverse: 5′-TTCTG CCAGTGCCTCTTTGCTG-3′.The primers for the HOXA7 gene were as follows: forward: 5′-GCTGAGGCCAATTTCCGCATCT-3′; reverse: 5′-GTAGC GGTTGAAGTGGAACTCC-3′.The primers for the HOXB4 gene were as follows: forward: 5′-CTGGATGCGCAAAGTTCACGTG-3′; reverse: 5′-CGTGT CAGGTAGCGGTTGTAGT-3′.The primers for the GAPDH gene were as follows: forward: 5′-GTCTCCTCTGACTTCAACAGCG-3′; reverse: 5′-ACCAC CCTGTTGCTGTAGCCAA-3′.Total proteins were extracted using whole-cell lysis buffer . Twenty micrograms of protein from each sample was loaded into lanes and electrophoresed using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis followed by transfer to a polyvinylidene difluoride (PVDF) membrane . After blocking with 5% skimmed milk, the PVDF membranes were incubated with the primary antibody overnight at 4°C. Then, the PVDF membranes were incubated with the secondary antibodies at 25°C for 1 h. Protein expression was visualized with a chemiluminescence ECL reagent .4 cells per well) with different drugs and incubated at 37°C in 5% CO2. Then, 10 μL of CCK8 reagent was added directly to the wells, incubating for 2 h and recording the absorbance at 450 nm on an EnVision Plate Reader  at 0, 48, after drug administration. Data analysis was performed using Prism version 7.00 (GraphPad software). Unpaired Student t test was applied as the statistical method.The effects of drugs on cell proliferation were determined by the CCK8 Cell Proliferation Assay (Dojindo) according to the manufacturer’s instructions. Cells were plated in 96-well plates (three replicates per condition at 1 × 105 and 106); 2 μL CD11b antibody (APC channel) and 4 μL CD14 antibody (fluorescein isothiocyanate channel) were added to each tube, and the cells were incubated for 15 min at room temperature in the dark. After that, they were tested on the machine. FACS data were analyzed using FlowJo software.The cells to be tested were taken and washed twice with phosphate-buffered saline (PBS). Then, these were resuspended in 500 μL PBS  and GraphPad Prism 7 were mainly used for statistical analysis and figure drawing. Kaplan–Meier survival analysis and Cox regression analysis were used to indicate prognostic values. χP < 0.01, log2FC > 1) in both databases , which were biomarkers for inferior prognosis  in First, we listed genes positively correlated with HOXB5  had the highest expression level . Then, wThese findings suggest that HOXB5 may play roles in the maintenance of the LSC function and induction of myeloid cell differentiation through the TNF/NF-κB pathway.To explore the clinical significance of HOXB5, we treated THP1 and KASUMI-1 cells with the NF-κB pathway inhibitor bortezomib and found that THP1 cells (with higher HOXB5 expression) were more sensitive to bortezomib than KASUMI-1 cells (with lower HOXB5 expression) . AdditioIn the list of genes closely correlated with HOXB5, we found many other HOX family members. Therefore, the correlated members enriched in the functions of myeloid cell differentiation and definitive hematopoiesis were analyzed, and the results verified the concordance among the selected HOX genes . GSEA inAs HOX genes are clustered in an ordered chromosomal arrangement and recent studies indicated that HOX genes could be regulated by DNA methylation , we furtThese findings suggest that DNA methylation may be a common upstream mechanism regulating the network of HOX family genes, including HOXB5 and its related genes.Currently, chromosomal variations and gene mutations are considered the main factors in the pathogenesis of AML and are combined to assess the cytogenetic risk for prognostic prediction and treatment selection . HoweverPrevious studies have demonstrated that several HOX family members affect the poor prognosis and aggressiveness of AML through transcriptional regulation . HoweverGenetic mutations have significantly contributed to the optimization of not only classification but also risk stratification, residual disease monitoring, and even the development of targeted therapeutics . FLT3-ITAs transcription factors, some HOX genes have been found to activate their own expression or to cross-regulate their family members or their cofactors . We founTaken together, HOXB5 was indicated to be upregulated in patients with AML, which was associated with the progression of AML and inferior prognosis, even in different subgroups. The AML patients with higher HOXB5 expression levels had unique genetic mutation patterns, such as concurrent FLT3-ITD, NPM1, and DNMT3A mutation patterns. In AML patients, higher expression of HOXB5 was closely related to myeloid differentiation and LSC characteristic gene signatures, probably through the TNF/NF-κB pathway. DNA methylation of clustering HOX genes might be one of the regulatory mechanisms that need further investigation. These results suggest that HOXB5 may serve as a novel biomarker for predicting the clinical outcome of patients with AML. However, further investigations are required to determine the biological characteristics of HOXB5 and their potential mechanism involved in leukemogenesis.The original contributions presented in the study are included in the article/MC, YQ, PY, and XY contributed to the study conceptualization. MC contributed to the data curation and took charge of the methodology and the validation. XY contributed to the funding acquisition and supervised the study and took charge of the review and editing. PY contributed to the project administration. MC and YQ contributed to writing the original draft. All authors contributed to the article and approved the submitted version.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.