CLCNKA and CLCNKB are crucial for the transepithelial transport processes required for sufficient urinary concentrations and sensory mechanoelectrical transduction in the cochlea. Loss-of-function alleles in these channels are associated with various clinical phenotypes, ranging from hypokalemic alkalosis to sensorineural hearing loss (SNHL) accompanied by severe renal conditions, i.e., Bartter’s syndrome. Using a stepwise genetic approach encompassing whole-genome sequencing (WGS), we identified one family with compound heterozygous variants in the ClC-K channels, specifically a truncating variant in CLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB. Breakpoint PCR and Sanger sequencing elucidated the breakpoint junctions derived from WGS, and allele-specific droplet digital PCR confirmed one copy loss of the CLCNKA_CLCNKB contiguous deletion. The proband that harbors the CLCNKA_CLCNKB variants is characterized by SNHL without hypokalemic alkalosis and renal anomalies, suggesting a distinct phenotype in the ClC-K channels in whom SNHL predominantly occurs. These results expanded genotypes and phenotypes associated with ClC-K channels, including the disease entities associated with non-syndromic hearing loss. Repeated identification of deletions across various extents of CLCNKA_CLCNKB suggests a mutational hotspot allele, highlighting the need for an in-depth analysis of the CLCNKA_CLCNKB intergenic region, especially in undiagnosed SNHL patients with a single hit in CLCNKA.The ClC-K channels  CLCNKA and CLCNKB, which are both ClC-K channels, play a crucial role in the transepithelial transport processes essential for water resorption and sufficient urinary concentration [CLCNKA is primarily expressed in the thin ascending limb of Henle and is involved in passive NaCl transport [CLCNKB, which is primarily expressed in the thick ascending limb of Henle, contributes to transcellular Cl− reabsorption and ion exchange balance in cooperation with NKCC1 and Na+/K+-ATPase [CLCNKB is essential for sodium chloride reabsorption in the kidney’s outer medulla and cortical region [The ClC-K channels, which constitute a subgroup of ClC channels, are expressed in the loop of Henle in the kidneys and stria vascularis of the inner ear . CLCNKA ntration ,3,4. CLC+-ATPase . CLCNKB l region .CLCNKA and CLCNKB are expressed in basolateral membranes of marginal cells of the cochlear stria vascularis, where they play a role in the secretion of K+ into the endolymph via NKCC1 and Na+/K+-ATPase. The stria vascularis is responsible for creating the high K+ concentration of 140 mM and the positive potential of +100 mV in the endolymph that fills the scala media [+ recycling within the cochlea and maintaining the balance of chloride, salt, and water in both the scala media and the stria vascularis. Understanding the transport processes needed to uphold this balance is crucial [Additionally, la media . It is i crucial . This baCLCNKB was identified as an underlying disease gene for type III BS [CLCNKA and CLCNKB), as a digenic trait, leads to severe renal salt wasting and sensorineural deafness, which is classified as type IV BS [BSND, which encodes barttin [Loss-of-function alleles in ClC-K channels and barttin (a beta-subunit of ClC-K channels) are associated with a range of clinical phenotypes, including Bartter syndrome (BS) ,8,9. BS e III BS . Moreovepe IV BS . This is barttin , despite barttin ,15.+ recycling, coupled with maintaining the balance of chloride, salt, and water, in both the scala media and the stria vascularis are crucial. The inner-ear-specific disruption of Bsnd in mice led to a reduced driving force for potassium entry through MET channels into sensory hair cells, resulting in the degeneration of the organ of corti and profound hearing loss [Although the exact mechanism remains poorly understood, dysfunction in either ClC-K or barttin channels can hinder the countercurrent mechanism in the loop of Henle, thereby affecting water reabsorption into the collecting duct . Additioing loss . Consequing loss .CLCNKA in trans with contiguous deletion of CLCNKA and CLCNKB, and refines the genotype–phenotype correlations in the context of ClC-K channels. Expanding our knowledge of phenotypes associated with ClC-K channels beyond BS aids in predicting the natural course of these conditions, ending the diagnostic odyssey, and applying suitable clinical management strategies [While the expression of ClC-K channels in the stria vascularis and their role in hearing have been characterized, hearing loss phenotypes resulting from defects in these ClC-K channels and their associated genotype–phenotype correlations remain poorly understood. This study presents the first report of the non-syndromic hearing loss caused by compound heterozygous variants, specifically a truncating variant in rategies ,17.CLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB. The genetic contribution of ClC-K channels to SNHL has been identified to be approximately 0.23%. The proband  was a sporadic case showing signs of hearing impairment with post-lingual onset  was found to harbor compound heterozygous variants, specifically a truncating variant in al onset a. In thial onset b. The aual onset c. Additial onset d. Consisal onset , precludMPZL2 c.220C>T:p.Gln74*, NOTCH1 c.1945C>T:p.Pro649Ser, APAF1 c.2887C>T:p.Gln963*, and CLCNKA c.778C>T:p.Gln206*, which were inconclusive at this stage. The CLCNKA heterozygous nonsense variant has been reported previously [CLCNKA p.Gln206* is considered “pathogenic” based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines [Exome sequencing and bioinformatic analysis were used to prioritize candidate variants for hearing loss . This steviously , and theidelines .CLCNKA and CLCNKB deletion spanning g.16032251 to g.16046349 (the 5′-breakpoint at exon 17 of CLCNKA and the 3′-breakpoint at intron 3 of CLCNKB) with 1 bp of microhomology was identified  was reported with an allele frequency of 2.75 × 10−4. However, the dataset, which encompasses 10,847 samples, did not document any identical structural variation reported herein.To investigate whether there were regions and genetic variants not technically covered by exome sequencing, such as structure variations (SVs) and genomic rearrangements, familiar WGS, which included the patient’s father and mother, was performed. Importantly, a heterozygous contiguous entified a. ResultCLCNKA (Primer F1) and a pair of reverse primers (Primer R1 at intron 18 of CLCNKA and Primer R2 at intron 3 of CLCNKB) were designed. The breakpoints detected by WGS were verified by amplifying the 1222 bp product using primers F1 and R2 across the 5′ and 3′ breakpoints; the 1222 bp amplicon was observed in the proband and father, but not in the control or mother . In the previously reported case, the patient displayed bilateral moderately severe SNHL with a flat configuration (250 Hz to 9 kHz). Conversely, our case showed bilateral moderately severe SNHL with a cookie-bite audiogram configuration. To elucidate our understanding of such audiological phenotypes in BS, access to numerous variants of the implicated gene and profound audiologic data are essential. Therefore, as presented herein, a detailed analysis of both the audiological data and the genotypes in these rare instances is imperative. With this case, we expand the genotypes and phenotypes associated with ClC-K channels and refine the genotype–phenotype correlation in the context of ClC-K channels, expanding our understanding of the disease entities associated with non-syndromic hearing loss.In this study, we report the first identification of non-syndromic hearing loss caused by compound heterozygous variants, specifically a truncating variant in d CLCNKB . In the ants see . HoweverCLCNKA_CLCNKB deletion in undiagnosed cases, even after exome sequencing. In this study, WGS precisely indicates the 5- and 3-end breakpoints, and the breakpoints were accurately validated by breakpoint PCR and Sanger sequencing. In accordance with this approach, the breakpoints of cryptic genomic copy number variations (CNVs) identified through WGS were reassessed using Sanger sequencing or breakpoint PCR, following a stepwise methodology [CLCNKA and CLCNKB. In the literature, there is a lack of detection schemes for the effective identification of cryptic genomic CNVs. Our study suggests that the application of stepwise genomic pipelines could offer a framework for the detection and confirmation of the SVs, including those with a heterozygous contiguous CLCNKA and CLCNKB deletion.The stepwise genetic testing pipeline used in this study, which combines trio-based WGS with breakpoint PCR and Sanger sequencing, is an effective approach for deciphering a contiguous hodology . In thisCLCNKA_CLCNKB deletion has been previously detected in patients with type III and IV BS [CLCNKA_CLCNKB deletion was reported in the Genome Aggregation Database (gnomAD) SV v2.1 (DEL_1_1318) dataset with an allele frequency of 2.8 × 10−4, albeit with deletions across various extents of CLCNKA_CLCNKB. The deletion likely occurs through non-allelic homologous recombination-mediated deletion, since the two breakpoints exhibit high homology [CLCNKA_CLCNKB deletion in undiagnosed patients with SNHL, especially when one pathogenic allele in the CLCNKA is present.The large nd IV BS ,9,23, suhomology . The proCLCNKA and CLCNKB. WGS theoretically has a higher ability to identify a more diverse spectrum of variant profiles, including deep intronic, regulatory, andSVs, compared to exome sequencing [Exome sequencing is effective in identifying point mutations in the coding region. However, it has a limitation in detecting most SVs as the breakpoints of SVs are mostly located in non-coding regions. Although copy-number changes in exome sequencing can be used to infer deletions, it can be challenging to draw conclusions, especially in homologous regions like quencing . Accordiquencing . Given tquencing . NeverthCLCNKA and CLCNKB, which are part of the human ClC channel family, facilitate both inward and outward currents. This dual functionality supports transcellular chloride fluxes in numerous tissues, notably in the inner ear and kidney [CLCNKB in addition to this large deletion [CLCNKA variant in trans, with the large deletion spanning CLCNKA and CLCNKB. The distinct presentation of non-syndromic hearing loss in the case involving a single copy loss of CLCNKB along with biallelic loss-of-function alleles in CLCNKA might be understood through the differential roles that these genes have in the inner ear and kidney. Perhaps, CLCNKA appears to be less susceptible in terms of maintaining the ion balance necessary for proper signal transduction in inner hair cells when mutated. When compounded with heterozygous variants of CLCNKB, the dysfunction of CLCNKA can eventually disrupt the ion transport balance necessary for maintaining the endocochlear potential, leading to SNHL. Conversely, while CLCNKB is also involved in ion transport in the inner ear, its impact on hearing might be less pronounced than CLCNKA. This is evident from clinical observations where CLCNKB mutations predominantly affect renal function with minimal or no impact on hearing . Alternatively, this is likely due to the presence of other compensatory mechanisms in the kidney and inner ear. Similarly, one BSND variant  causes non-syndromic hearing loss (DFNB73) without affecting renal function, which supports the differential susceptibility of inner ear and kidney function to the dysfunction of the CIC-K/barttin channels [ClC channels and transporters, expressed across a wide range of tissues, fulfill diverse functions in paly. Specifically, d kidney . The typdeletion . In compchannels .CLCNKA. However, research on CLCNKA knockout mice has revealed the presence of a mild form of diabetes insipidus [CLCNKA mutants can be attributed to compensatory mechanisms between the two homologous ClC-K channels. The current case suggests that an additional copy gene defect in CLCNKB, in combination with biallelic loss-of-function alleles in CLCNKA, contributed to the development of the hearing loss phenotype. The expression of ClC-K channels in the inner ear is crucial for maintaining proper hearing function [BSND, it is possible that the additional CLCNKB single-copy defect disrupts protein stability and reduces the membrane expression level below the reference threshold necessary for maintaining inner ear function, despite the compensatory roles of the ClC-K channels. The mechanism through which additional CLCNKB single-copy defects result in hearing loss in cases where CLCNKA is non-functional remains to be elucidated.There have been no reports on human diseases caused by variants solely in nsipidus . This mifunction , and thefunction . Given tCLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB, remains elusive. The limitation stemming from reporting on a single family, despite ongoing efforts to recruit additional families via gene-matcher [CLCNKB polymorphisms and conditions such as hypertension and reduced hearing thresholds [CLCNKA have been associated with elevated arterial pressure [At this point, the precise mechanism underlying the non-syndromic hearing loss caused by compound heterozygous variants, specifically a truncating variant in -matcher , cannot resholds ,32. On tpressure . DespiteCLCNKA_CLCNKB deletion in patients with SNHL who remain undiagnosed, particularly when one pathogenic allele in CLCNKA is identified.Nevertheless, these findings contribute to the growing body of knowledge regarding genotype–phenotype correlations in the context of ClC-K channels, enhancing our understanding of the disease entity associated with non-syndromic hearing loss. Moreover, our observations suggest the significance of the in-depth genomic analysis of the All procedures in this study were approved by the Institutional Review Boards of Seoul National University Hospital (IRB-H-0905-041-281). This study utilized a retrospective design and focused on participants attending the Hereditary Hearing Loss Clinic within the Otorhinolaryngology division at the Center for Rare Diseases, Seoul National University Hospital, Korea, from March 2021 to May 2023. Of total, we decomposed 428 unrelated SNHL families who underwent a stepwise approach encompassing exome sequencing and WGS. The demographics of 428 probands were summarized in https://gnomad.broadinstitute.org/) (accessed on 1 May 2023), and ethnically-matched controls, including Korean Variant Archive, for a reference database of genetic variations in the Korean population (KOVA2) (https://www.kobic.re.kr/kova/search_detail_view_hg19) (accessed on 1 May 2023). (iii) The pathogenic potential of each variant was determined using in silico tools (Combined Annotation Dependent Depletion (CADD), https://cadd.gs.washington.edu/ and Rare Exome Variant Ensemble Learner (REVEL), https://sites.google.com/site/revelgenomics/) (accessed on 1 May 2023). In addition, we used the GERP ++ score from the UCSC Genome Browser (http://genome.ucsc.edu/) (accessed on 1 May 2023) to estimate the evolutionary conservation of the amino acid sequences. (iv) Furthermore, compatibility with inheritance patterns and audiological/clinical phenotypes was evaluated. Additionally, the ClinVar and HGMD databases were screened to check whether candidate variants had been previously identified in other patients. (v) The candidate variants were confirmed through Sanger sequencing, and a segregation study was performed using parental DNA samples. Resultantly, we classified the pathogenicity of candidate variants according to the ACMG-AMP guidelines for SNHL [Genomic DNA was extracted from peripheral blood samples utilizing the Chemagic 360 instrument . The exome sequencing was performed through SureSelectXT Human All Exon V5 . We prepared a library that was paired-end sequenced using a NovaSeq 6000 system  with an average coverage depth of 100×. The sequence reads were aligned to the human reference genome GRCh38) and processed following the guidelines of the Genome Analysis Toolkit (GATK)  to ident and procfor SNHL .TM . The sequenced reads were mapped to the GRCh38 using the BWA-MEME algorithm, and duplicated reads were removed using SAMBLASTER [DNA libraries for WGS were generated using the TruSeq DNA PCR-Free kit  from 1 µg of genomic DNA. WGS was conducted on the NovaSeq platform 6000 (Illumina) to generate 151 bp paired-end reads. The raw sequencing data analysis and downstream interpretation were performed using RareVisionMBLASTER . Mean coMBLASTER  and StreMBLASTER . GenomicMBLASTER , and theTM ddPCR system. Procedures followed the manufacturer’s protocol with minor modifications. Briefly, the master mix for ddPCR consisted of 100 ng of genomic DNA, 1×ddPCR supermix for probes , 1.0 μM primer, and 0.25 μM probe . The samples were thoroughly combined before being put into a Bio-Rad QX100TM/QX200 droplet generator’s DG8 cartridge. The cartridge was then put into the QX200 Droplet GeneratorTM (Bio-Rad) and added Droplet Generation Oil. After droplet formation, the droplets were carefully moved to a twin-tec semi-skirted 96-well PCR plate . PCR was then carried out in a C1000 Touch thermal cycler (Bio-Rad) for the two-step running program of 94 °C for 30 s and 56 °C for 1 min for 40 cycles with initial and final incubation at 95 °C and 98 °C, respectively. Signal in each droplet was read by a droplet reader and analyzed using QuantaSoft program (Version 1.7) (Bio-Rad). For breakpoint PCR, primers were designed . Additionally, Cystatin C levels were evaluated using immunoturbidimetric methods. Furthermore, patient’s peripheral blood sample was also collected in a gel EDTA mixed tube for the assessment of renin activity-basal and aldosterone-basal. Reference values were documented based on age and gender.A total of 5.0 mL of the patient’s peripheral blood sample was collected in a serum separator tube (SST) for analysis of a renal panel, which included measurements of Calcium, Phosphorus, Uric Acid, Blood Urea Nitrogen (BUN), Creatinine, Sodium, Potassium, Chloride, and Total Carbon Dioxide (TCOCLCNKA in trans with a contiguous deletion of CLCNKA and CLCNKB, through a stepwise genomic approach encompassing WGS. These results provide an additional basis of genotype–phenotype correlations in the context of ClC-K channels, expanding our understanding of the disease entities associated with non-syndromic hearing loss.This study presents the first report of non-syndromic hearing loss caused by compound heterozygous variants, specifically a truncating variant in


The AVP-induced Ca2+ mobilization in inner medullary collecting ducts is essential for apical exocytosis and is mediated by the exchange protein directly activated by cyclic adenosine monophosphate (Epac). Murine principal kidney cortical collecting duct cells (mpkCCD) is the cell model used for transcriptomic and phosphoproteomic studies of AVP signaling in kidney collecting duct. The present study examined the characteristics of Ca2+ mobilization in mpkCCD cells, and utilized mpkCCD as a model to investigate the Epac-induced intracellular and intra-organellar Ca2+ mobilization. Ca2+ mobilization in cytosol, endoplasmic reticulum lumen, and mitochondrial matrix were monitored with a Ca2+ sensitive fluorescent probe and site-specific Ca2+ sensitive biosensors. Fluorescence images of mpkCCD cells and isolated perfused inner medullary duct were collected with confocal microscopy. Cell permeant ligands of ryanodine receptors (RyRs) and inositol 1,4,5 trisphosphate receptors (IP3Rs) both triggered increase of [Ca2+]i and Ca2+ oscillations in mpkCCD cells as reported previously in IMCD. The cell permeant Epac-specific cAMP analog Me-cAMP/AM also caused a robust Ca2+ mobilization and oscillations in mpkCCD cells. Using biosensors to monitor endoplasmic reticulum (ER) luminal Ca2+ and mitochondrial matrix Ca2+, Me-cAMP/AM not only triggered Ca2+ release from ER into cytoplasm, but also shuttled Ca2+ from ER into mitochondria. The Epac-agonist induced synchronized Ca2+ spikes in cytosol and mitochondrial matrix, with concomitant declines in ER luminal Ca2+. Me-cAMP/AM also effectively triggered store-operated Ca2+ entry (SOCE), suggesting that Epac-agonist is capable of depleting ER Ca2+ stores. These Epac-induced intracellular and inter-organelle Ca2+ signals were mimicked by the RyR agonist 4-CMC, but they were distinctly different from IP3R activation. The present study hence demonstrated that mpkCCD cells retain all reported features of Ca2+ mobilization observed in isolated perfused IMCD. It further revealed information on the dynamics of Epac-induced RyR-dependent Ca2+ signaling and ER-mitochondrial Ca2+ transfer. ER-mitochondrial Ca2+ coupling may play a key role in the regulation of ATP and reactive oxygen species (ROS) production in the mitochondria along the nephron. Our data suggest that mpkCCD cells can serve as a renal cell model to address novel questions of how mitochondrial Ca2+ regulates cytosolic Ca2+ signals, inter-organellar Ca2+ signaling, and renal tubular functions.Arginine vasopressin (AVP) induces an increase in intracellular Ca It was concluded that AVP-induced Ca2+ oscillation in IMCD is mediated by an Epac-dependent mechanism through the interplay of Ca2+ release from ryanodine receptors and a Ca2+ influx mechanism involving SOCE -dependent phosphorylation of RYRs  induces intracellular Cat (IMCD) . ConfocaCD cells , the latCD cells . It has sed IMCD . Moreoveresponse . Previoufficking , and thefficking . AVP couing SOCE . Epac-in of RYRs , leadingrhythmia . Epac-intivation .2+ mobilization without activation of cAMP/PKA signal pathway  and mitochondria. Our results demonstrate that mpkCCD cells display similar characteristics of intracellular Ca2+ mobilization observed in intact cells of collecting duct, and that the Epac agonist triggered intracellular Ca2+ mobilization and oscillation are mediated by RyR-gated Ca2+ release and SOCE associated with reciprocal decrease of Ca2+ content in the ER. Moreover, the Epac agonist can effectively shuttle ER luminal Ca2+ to both the cytosol and mitochondrial matrix.Murine principal kidney cortical collecting duct (mpkCCD) cells are commonly used cell model used for transcriptomic and phosphoproteomic studies of AVP-signaling in kidney collecting ducts . It is a pathway . These onsipidus . HoweverC14, kindly provided by Dr. Douglas Eaton, Emory University) grown in AVP-free culture medium. Cells were maintained in a 1:1 mixture of DMEM/Ham’s F12 medium with phenol red (Gibco), supplemented with dexamethasone (50 nM), triiodothyronine (1 nM), selenium (60 nM), insulin (5 μg/mL), mouse EGF (10 ng/mL), transferrin (5 μg/mL), and 2% fetal calf serum in a humidified atmosphere with 5% CO2 at 37°C. mpkCCD cells between 20 and 30 passages were grown on collagen coated glass bottom dish prior to the experiments.Experiments were performed on a male mouse CCD principal cell line  in phenol red free medium  for 30 min at 37°C, followed by 20 min for de-esterification. Fluorescent images were collected with a Leica TCS SP5 confocal imaging system using water immersion objective lens  equipped with environmental chamber. Cal-520 was excited at 488 nm, and the emission was collected with a spectral window of 495–530 nm at 1 Hz. The spatial and temporal variations of [Ca2+]i in individual cells were measured from the stored images with Leica Application Suite Advanced Fluorescence software as reported previously (2+ in the extracellular buffering solution.mpkCCD cells grown on collagen coated glass bottom dish (MatTek) were loaded with cell permeant Caeviously . Store-o2+] ([Ca2+]ER) or mitochondrial [Ca2+] ([Ca2+]MITO) simultaneously with cytosolic [Ca2+]i, mpkCCD cells were transfected with either ER Ca2+ biosensor R-CEPIA1er  or mitochondrial Ca2+ biosensor mito-RCaMP1h  (4 cells/cm2 on collagen coated glass bottom dishes for 24 h before transfection. Cells were transfected with Lipofectamine (0.5 µg DNA/1 × 105 cells) for 24 h according to manufacturer’s instruction. Studies were performed in transfected cells from 48 to 72 h after transfection. Cytosolic [Ca2+]i was monitored simultaneously with cell permeant Ca2+ sensitive fluorescence probe  in the transfected cells incubating with phenol red free medium . To monitor ER-mitochondrial Ca2+ transfer in mpkCCD cells, cells were co-transfected with the ER Ca2+ biosensor  and mitochondrial biosensor mito-RCaMP1h. Fluorescent images were collected with the respective laser lines for excitation and spectral windows for emission using the Lecia TCS SP5 imaging system.To monitor ER [Ca–600 nm) . Cells w2+]i in perfused IMCD was monitored with fluo-4/AM  in individual IMCD cells. Confocal fluorescent images of IMCD were collected and analyzed as reported previously  and was approved by the University of South Florida Institutional Animal Care and Use Committee (PROTOCOL #R3982). IMCDs were isolated from male Sprague-Dawley rats and perfused as described previously . Cytosoleviously .3-InsP3/AM was purchased from SiChem. ATP, 4-CMC (4-Chloro-m-cresol), H-89, and thapsigargin were from Sigma-Aldrich .6-Bnz-cAMP-AM, Me-cAMP-AM (8-pCPT-2′-O-Me-cAMP-AM), and ESI-09 were purchased from Biolog (Germany). Ryanodine, SKF-96365, and Xestospongin C were purchased from MilliporeSigma . Btt tests for paired or unpaired data and considered significant when p < 0.05.Time series of fluorescence emission variations in individual mpkCCD cells were extracted and normalized with respective to the base line from stored XYT images. Time series of Cal-520 emission from individual cells were sampled at 1 Hz for spectral analysis. Each time series was subjected to linear trend removal. 512 or 1,024 data points were used to calculate the power spectrum with an algorithm based on Fast Fourier Transform . Results3Rs) are the predominant Ca2+ release channels of the ER in non-excitable cells. We have previously shown that endogenous agonist of RyRs and IP3Rs triggered Ca2+ oscillations in individual cells of perfused IMCD, indicating both functional RyR-gated and IP3R-gated intracellular Ca2+ stores are present in IMCD cells  or Bt3-InsP3/AM (a cell permeant agonist of IP3R). Ryanodine was used as blocker of RyRs, and Xestospongin C was used for IP3Rs. Both agonists triggered robust intracellular Ca2+ mobilization and oscillations in mpkCCD cells P3/AM triggered Ca2+ response (∼100–150 s). The 4-CMC-induced Ca2+ transient was completed at 800 s, whereas the IP3-triggered response was more sustained. The delayed IP3–induced Ca2+ response in the Ca2+ responses were possibly related to the rate of membrane permeation and de-esterification of Bt3-InsP3/AM. The differences in the kinetic profiles triggered by the two stores could be due to differences in the potency of the agonists, inactivation kinetics of the receptors, and the depletion or replenishment of the SR Ca2+ stores. Both 4-CMC and Bt3-InsP3/AM-induced Ca2+ responses were completely abolished in the presence of their respective receptor blockers. These observations confirmed that mpkCCD cells possess both functional RyR- and IP3R-gated Ca2+ stores as in IMCD.RyRs are mainly expressed in the sarcoplasmic reticulum of skeletal, cardiac and smooth muscle cells, whereas inositol 1,4,5 trisphosphate receptors P3/AM, were shown in 3/AM-induced Ca2+ oscillations were similar, except Me-cAMP/AM induced Ca2+ oscillation had more power at the higher frequencies (0.03–0.1 Hz frequency range).To examine the Epac-dependent Caillation , mpkCCD  and IP3 . The Ca2CD cells . In contillation . The bri2+ stores in mpkCCD cells, the temporal variations of cytosolic Ca2+ and ER luminal Ca2+ were monitored simultaneously with the Ca2+ sensitive-fluorescence probe Cal-520/AM and the ER luminal Ca2+ biosensor R-CEPIA1er, respectively. Me-cAMP/AM triggered an increase of cytosolic Ca2+ which was associated with a synchronized decrease in ER luminal Ca2+ content . RyR-agonist 4-CMC triggered similar response in mpkCCD cells P3/AM only triggered decrease in ER luminal Ca2+ without concomitant increase of mitochondria Ca2+ in mpkCCD cells  in the absence of extracellular Ca2+. Reintroduction of 2 mM Ca2+ in the external solution elicited robust Ca2+ entry, which was inhibited by the SOCE inhibitor SKF96365 (50 µM)  . Incubatsigargin . Robust pongin C . MoreoveSKF96365 . These r2+ mobilization was associated with AVP-induced apical exocytosis in perfused IMCD. mpkCCD cells have been used as the cell model for transcriptomic and phosphoproteomic studies of AVP-signaling in the collecting duct  triggered long lasting Ca2+ oscillations. Moreover, long-term regulation of AQP2 by AVP in mpkCCD cells is mediated by Epac but not by PKA   . An inhi3 (NHE3) . A recen3 (NHE3) , suggest2+ oscillations resemble those activated by a RyR agonist in mpkCCD cells, suggesting that Epac-agonist mobilizes ER Ca2+ in mpkCCD cells via RyRs. Organelle-specific Ca2+-sensitive biosensors expressed in mpkCCD cells showed that the Epac agonist triggered synchronized Ca2+ spikes in cytosol and mitochondrial matrix, which are temporally correlated with reciprocal changes in ER luminal Ca2+. These observations indicated that agonist-induced Epac activation not only triggered release of Ca2+ into cytosol, but also transferred Ca2+ from ER to mitochondria.Our data also show that Epac-agonist triggered Ca2+] of mitochondrial matrix is comparable to the resting cytosolic [Ca2+]. Mitochondrial Ca2+ uptake takes place at specialized microdomains where cytosolic [Ca2+] are high. Such microdomains are localized in the mitochondria-associated membranes (MAMs), where the endoplasmic reticulum membrane is within 10–30 nm from the outer mitochondrial membrane (2+ released from ER enters the intermembrane space through voltage-dependent anion channels (VDACs) localized in the outer membrane, and then enters mitochondrial matrix through the mitochondrial Ca2+ uniporter (MCU) of the inner membrane P3/AM, which effectively activated cytosolic Ca2+ oscillation, only triggered a decrease in ER luminal Ca2+ but not concomitantly increased mitochondria Ca2+ in mpkCCD cells. It is possible that IP3Rs are less efficacious in facilitating ER-mitochondrial Ca2+ transfer in mpkCCD cells; or the cell permeant Bt3-InsP3/AM, had a poor access to the IP3Rs in MAMs. To test the latter hypothesis, the native agonist ATP was applied to increase endogenous IP3 abundance in mpkCCD cells. ATP triggered synchronized increase in cytosolic [Ca2+] with concomitant elevation in mitochondrial matrix [Ca2+] and depletion ER luminal Ca2+. ATP-induced Ca2+ transfer between ER and mitochondria was also visualized in cells co-expressed with ER and mitochondrial Ca2+ biosensors. These observations indicated that IP3Rs is capable of mediating Ca2+ transfer between ER and mitochondria in mpkCCD cells. However, the kinetics of ATP-induced cytosolic, endoplasmic, and mitochondrial Ca2+ responses are distinctly different from those induced by Epac or RyR agonists, distinguishing the Ca2+ signals activated by the two different agonists induced signaling pathways. Our studies also support that this mechanism is at play in vivo and it is noteworthy that 5 µM ATP also triggered Ca2+ mobilization and oscillations in isolated perfused IMCD (2+ mobilization is present in both intact IMCD cells and mpkCCD cells. Consistent with this, studies in acutely isolated connecting tubule/collecting duct of mice support that an acute increase in cytosolic [Ca2+] inhibits ENaC activity (2 receptor activation (The resting [Camembrane . Ca2+ remembrane . RyR ago in MAMs , but thesed IMCD , confirmactivity  mediatedtivation .2+dynamics plays important roles in intracellular Ca2+signaling, cell metabolism, cell survival, and other cell-type specific functions (2+ oscillations, SOCE and Ca2+-dependent gene expression in response to receptor-mediated stimulation (2+ shuttling via MCU sustains the cytosolic Ca2+ signal by preventing Ca2+-dependent inactivation of IP3Rs and store-operated CRAC channels (2+ transfer also stimulates Ca2+-sensitive dehydrogenases and respiratory chain components to promote oxidative phosphorylation, ATP, and ROS production (2+ transfer in the regulation of cellular functions in renal tubular cells remain to be determined.Mitochondrial Caunctions . As descmulation . It has channels . ER-mitooduction . The phy2+ mobilization observed in isolated perfused IMCD. The temporal relationship between cytosolic Ca2+, ER luminal Ca2+, and mitochondrial matrix Ca2+ activated by Epac and RyR-agonists are highly compatible, but is distinctly different from those induced by IP3R stimulation. Furthermore, we have provided the first characterization of ER-mitochondrial Ca2+ transfer in mpkCCD cell, which can be used as a renal cell model to address novel questions of how mitochondrial Ca2+ regulates cytosolic Ca2+ signals, inter-organellar Ca2+ signaling, and other renal tubular functions.In conclusion, mpkCCD cells retained all reported features of Epac-induced Ca


Infectious complications reflect a major challenge in the treatment of patients with acute myeloid leukemia (AML). Both induction chemotherapy and epigenetic treatment with hypomethylating agents (HMA) are associated with severe infections, while neutropenia represents a common risk factor. Here, 220 consecutive and newly diagnosed AML patients were analyzed with respect to infectious complications dependent on treatment intensity and antifungal prophylaxis applied to these patients.n = 102) was 61 years (25–76 years). Patients receiving palliative AML treatment (n = 118) had a median age of 75 years (53–91 years). We assessed the occurrence of infectious complication including the classification of pulmonary invasive fungal disease (IFD) according to the EORTC/MSG criteria at diagnosis and until day 100 after initiation of AML treatment. Furthermore, admission to intensive care unit (ICU) and subsequent outcome was analyzed for both groups of AML patients, respectively.We retrospectively analyzed 220 patients with newly diagnosed AML at a tertiary care hospital between August 2016 and December 2020. The median age of AML patients undergoing induction chemotherapy (P < 0.001) including a higher probability of atypical pneumonia . Furthermore, urinary tract infections are more frequent in the palliative subgroup at the time of AML diagnosis . Surprisingly, the incidence of pulmonary IFD is significantly lower after initiation of palliative AML treatment compared to the occurrence after induction chemotherapy  despite only few patients of the palliative treatment group received Aspergillus spp.-directed antifungal prophylaxis. The overall risk for infectious complications at AML diagnosis is significantly higher for palliative AML patients at diagnosis while patients undergoing induction chemotherapy have a significantly higher risk of infections after initiation of AML treatment. In addition, there is a strong correlation between the occurrence of pneumonia including atypical pneumonia and pulmonary IFD and the ECOG performance status at diagnosis in the palliative AML patient group. Analysis of intensive care unit (ICU) treatment (e.g. in case of sepsis or pneumonia) for both subgroups reveals a positive outcome in 10 of 15 patients (66.7%) with palliative AML treatment and in 15 of 18 patients (83.3%) receiving induction chemotherapy. Importantly, the presence of infections and the ECOG performance status at diagnosis significantly correlate with the overall survival (OS) of palliative AML patients  in this intent-to-treat analysis.AML patients subsequently allocated to palliative AML treatment have a significantly higher risk of pneumonia at diagnosis compared to patients undergoing induction chemotherapy (37.3% vs. 13.7%, The risk and the pattern of infectious complications at diagnosis and after initiation of AML therapy depends on age, ECOG performance status and subsequent treatment intensity. A comprehensive diagnostic work-up for identification of pulmonary IFD is indispensable for effective treatment of pneumonia in AML patients. The presence of infectious complications at diagnosis contributes to an inferior outcome in elderly AML patients.The online version contains supplementary material available at 10.1007/s00432-022-03995-2. Infectious complications play a pivotal role in patients with acute myeloid leukemia (AML) and contribute to morbidity and mortality of AML patients significantly. There is a broad spectrum of infectious complications ranging from blood stream infections (BSI) potentially associated with severe sepsis to fungal infections regularly presenting as pneumonia. Treatment-related mortality (TRM) following induction chemotherapy with cytarabine in combination with daunorubicin occurs in about 5% of AML patients, while the major cause of fatal outcome is associated with BSI  is classified according to the EORTC/MSG recommendations and have a high impact on morbidity and prognosis in AML patients undergoing intensive chemotherapy  at diagnosis is significantly associated with duration of neutropenia, bloodstream infections and subsequently early death following induction chemotherapy of AML   on the frequency and severity of infectious complications.A total of 220 consecutive AML patients were identified at the Department of Hematology and Oncology, University Hospital Jena, Germany. Diagnosis and start of induction chemotherapy, epigenetic treatment or cytoreductive chemotherapy were between August 2016 and December 2020. The patient cohort considered all patients with newly diagnosed AML during this period including those AML patients treated with cytoreductive chemotherapy combined with best supportive care. Patients´ characteristics are indicated in Table Performance status and co-morbidity index have been documented according to the criteria published by the Eastern Cooperative Oncology Group (ECOG) and by application of a modified version of the Charlson co-morbidity index, respectively (Kelly and Shahrokni All patients were included in the SAL registry (Study Alliance Leukemia). Patients gave their written informed consent for data acquisition after pseudonymization and analysis in the SAL registry. The participation in the AML registry of the SAL study group and the retrospective analysis presented here have been approved separately by the Ethical review committee of the University Hospital Jena and was performed in accordance with the provisions of the Declaration of Helsinki.Induction chemotherapy was applied according to the 7 + 3 regimen or according to the OSHO protocol. In detail, patients underwent induction chemotherapy with cytarabine (100 mg/sqm as continuous 24-h infusion days 1–7) and daunorubicin (60 mg/sqm day 3–5) after inclusion in the DaunoDouble trial of the SAL study group  applied 5-Azacytidine (75 mg/sqm day 1–7 of a 28-day cycle) or decitabine (20 mg/sqm day 1–5 of a 28-day cycle) administered intravenously while low-dosed cytarabine (LODAC) was given s.c. (e.g. 40 mg day 1–7 every 4 weeks).Best supportive care (BSC) regimens included infectious prophylaxis, treatment of infections and transfusion of blood products if indicated.Standard regimens consisted of acyclovir (400 mg BID) and cotrimoxazole (960 mg BID twice a week or 960 mg three times a week). Antibacterial prophylaxis with ciprofloxacin (500 mg BID) and antifungal prophylaxis with either fluconazole (200 mg BID) or posaconazole (300 mg each day after a loading dose) was initiated in case of ANC below 1000/µL at diagnosis or under treatment, respectively.Screening for relevant nosocomial pathogens (e.g. 3MRGN or VRE) was performed regularly. In case of fever, diagnostic work-up included physical examination, X-ray or mostly computed tomography of the chest and analysis of blood cultures, screening for urinary tract infections (UTI) and clinical evaluation of central venous catheters.Standard therapy in patients with neutropenic fever consisted of immediate initiation of intravenous antibiotic treatment with piperacillin/tazobactam. In those patients previously screened positive of 3MRGN or in case of hypotonic RR values despite rapid application of intravenous fluids with the necessity of vasopressors, antibiotic treatment was escalated to meropenem.Neutropenia was defined as absolute neutrophil count (ANC) below 1000/µL and ANC was further categorized accordingly when ANC was below 500/µL or below 100/µL. Neutropenic fever was characterized by a body temperature of higher than 38.3 °C or of at least 38.0 °C sustaining for 1 h in the presence of absolute neutrophil counts less than 1000/µL.By means of clinical and microbiological investigation, sites of infection were classified, e.g. as blood stream infection, catheter-related infection or gastrointestinal (GI) tract infection. The following infectious complications analyzed in this study are described in more detail.Fever of unknown origin (FUO) was defined as fever according to the above defined criteria in the absence of an identifiable focus of infection provided that during the current fever episode no other site of infection could be defined later.5 colony-forming units (cfu) per ml in microbiological analyses or as “probable UTI” in symptomatic patients and at least 104 cfu/ ml but not exceeding 105 cfu/ ml.Urinary tract infections (UTI) were documented as “proven UTI” in case of at least 10Blood cultures were routinely collected in case of fever and positive samples were monitored for antimicrobial susceptibility according to local laboratory protocols and standards. Positivity concerning skin pathogens was defined when peripheral and central venous line (CVL) blood cultures were tested positive. An infection of CVL in case of positive CVL and peripheral blood cultures  was documented when time-to-positivity (TTP) was at least 2 h shorter for CVL blood culture compared to peripheral blood culture unless peripheral samples were tested negative. The clinical event systemic inflammatory response syndrome (SIRS) and sepsis were defined according to the guidelines published by the German Sepsis Society in 2020  in combination with at least two of the following criteria: cough, sputum production, temperature > 38 °C or < 35 °C, hemoptysis, thoracic pain or auscultatory findings consistent with pneumonia. Pneumonia was classified as atypical by radiographic criteria and pulmonary IFD was diagnosed based upon the criteria reported by the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) in  enabling a reliable prognostic stratification according to the European LeukemiaNet (ELN) 2017 classification  test. P values of < 0.05 were considered as statistically significant. Statistical analyses were performed using GraphPad Prism 8.0.2 (GraphPad Inc.).To test the statistical significance of differences in categorical data we applied the Chi-square test while for continuous variables the Student's This analysis comprises 220 consecutive AML patients including 118 Patients who received palliative AML treatment and 102 AML patients who underwent induction chemotherapy (Table P 0.025).There was no significant difference between both AML subgroups concerning AML history, previous therapy with hypomethylating agents (HMA) or distribution of AML subtypes according to the French-American-British (FAB) classification. Stratification according to the ELN 2017 classification revealed a significantly higher percentage of AML patients (25.5%) that could not be classified as favorable compared to the subgroup of AML patients receiving palliative treatment  at AML primary diagnosis.First-line treatment of both AML subgroups is summarized in the supplemental Table S1. In detail, the majority of palliative AML patients (82.2%) received HMA-based therapy either as single treatment or as combined treatment . Of patients receiving intensive AML therapy, 83 of 102 (81.4%) were treated with single induction chemotherapy according to the 7 + 3 schedule or the age-adapted OSHO protocol.A detailed overview of AML patients receiving palliative treatment or undergoing induction chemotherapy, respectively, is provided in the CONSORT diagrams in the Supplement , respectively.A comprehensive analysis of all infections that were documented at the time of diagnosis and admission to the hospital was performed. Table Patients allocated to palliative AML treatment presented a significantly higher probability of infections at diagnosis. In detail, 64 of 102 (62.7%) patients consecutively undergoing induction chemotherapy had no clinical signs of infections while only 47 of 118 (39.8%) of AML patients with palliative treatment presented without infections at diagnosis. In contrast, there was no difference concerning FUO, bloodstream infections and sepsis while more patients in the subgroup with subsequent palliative AML treatment had urinary tract infections (UTI) at diagnosis.Detailed analysis of type of pneumonia especially considered the classification of potential invasive fungal disease (IFD) for all AML patients for this study. Significantly more patients attributed to palliative AML treatment did present with pneumonia at diagnosis of AML. Importantly, this observation does also translate in a significantly higher percentage of atypical pneumonia in these elderly AML patients. In detail, 37.3% of patients subsequently receiving palliative AML treatment presented with pneumonia including 22.0% of these patients with atypical pneumonia compared to 13.7% with pneumonia and 10.8% with atypical pneumonia, respectively, in the subgroup of AML patients allocated to induction chemotherapy.P 0.0049) for patients with infections prior to start of palliative treatment in this intent-to-treat analysis  of palliative AML patients. In detail, the OS of elderly AML patients presenting without infectious complications at diagnosis was 315 days compared to only 69 days .We next analyzed the spectrum of infectious complications after initiation of AML therapy to evaluate the impact of treatment intensity on the occurrence of severe infections in both AML treatment groups. The observation period was defined as follows: until day 100 after initiation of palliative AML treatment and until start of subsequent therapy  following induction chemotherapy. 10 out of 118 AML patients of the palliative subgroup were lost of follow-up, so that the analysis of consecutive infectious complications included 108 patients of this AML subgroup. Figure n = 11).Surprisingly, after successful treatment of infectious complications presenting at AML diagnosis the majority of patients receiving palliative AML treatment (63.9%) did not show further infectious complications until day 100 after initiation of AML therapy. This was significantly different compared to those AML patients with induction chemotherapy and does inversely correlate with the occurrence of both FUO and bloodstream infections. In detail, 60 out of 102 patients presented with at least one episode of FUO after initiation of induction chemotherapy and additional 35 patients had a bloodstream infection or fulfilled the criteria of sepsis .P < 0.001) and 49.0% versus 11.1% (P < 0.001), respectively. Of note, the percentage of pulmonary IFD in the subgroup of atypical pneumonia is nearly identical between both patient subgroups at diagnosis—14 of 26 patients (53.8%) versus 6 of 11 patients (54.5%), respectively–and does also reveal a comparable distribution after treatment initiation: 9 of 12 patients (75.0%) versus 34 of 50 patients (68.0%), respectively. There is a clinically relevant and significantly higher risk of pulmonary IFD in AML patients undergoing induction chemotherapy. In detail, we could observe a total of 34 patients with pulmonary IFD including 1 proven IFD (Rhizomucor spp.) in this treatment group , while only 9 Patients developed pulmonary IFD after initiation of palliative AML treatment .The occurrence of pneumonia after initiation of AML therapy dependent on the applied treatment intensity was also studied. Figure Candida glabrata and one patient with sinusitis due to a suspected fungal infection.The analysis of additional infections revealed no significant difference for such infectious complications as UTI or soft tissue infections. Because all patients undergoing AML induction chemotherapy had a CVL catheter in contrast to only few palliative AML patients, the comparison of CVL infections between both subgroups is not applicable. Importantly, there was no venous thrombosis in case of CVL infection. Of note, there were three non-pulmonary IFD including the already described bloodstream infections with Supplement.An overview of most relevant infectious complications dependent on the time point of detection and on treatment intensity is presented in Figure S4 of the n = 7) or severe pneumonia (n = 4). In AML patients treated with induction chemotherapy, indication for ICU treatment was documented in 18 of 102 (17.6%) patients including 7 patients with mechanical ventilation support due to severe pneumonia. Within the whole cohort of AML patients, 3 of 220 patients underwent leukapheresis. In 10 out of 15 (66.7%) palliative AML patients and 15 of 18 (83.3%) intensively treated AML patients, the acute clinical situation could be managed by ICU treatment enabling continuation of AML therapy.We next analyzed the necessity of intensive care unit (ICU) treatment for both AML treatment groups. An overview of distinct ICU indications  for AML patients either receiving palliative therapy or undergoing induction chemotherapy is given in Table The early death rate defined as the mortality until day 60 after initiation of either palliative AML treatment or induction chemotherapy significantly differed between both subgroups Table . In patiIn contrast, 34 of 108 (31.5%) evaluable AML patients starting palliative therapy died between treatment initiation  and day 60. As expected, there was a broad spectrum of causes of death in the palliative subgroup of AML patients with a potential overlap between clinical complications and AML progression in three patients and co-occurrence of sepsis and pneumonia in another AML patient. The main cause of death was pneumonia (44.1%) or sepsis (23.5%), respectively. Of note, we observed a very low rate of IFD with respect to early mortality in elderly AML patients .P < 0.001). In contrast, stratifying the cohort of elderly AML patients with respect to co-morbidities (mCCI < 1 vs. mCCI > 2) revealed only a trend in favor of patients with a low mCCI score with respect to the observed difference in OS: 165 days (range 6–1165 days) vs. 112 days (range 2–1219 days), respectively  was detected. In detail, median OS for patients with ECOG 0 or 1 was 353 days (range 17–1165 days) compared to 50 days (range 2–1219 days) in patients presenting with ECOG 2 or higher, respectively .Treatment of AML has been improved by recent supportive care concepts and implementation of new targeted therapies of AML. However, AML therapy remains challenging in both younger and elderly AML patients. Treatment-related mortality (TRM) following induction chemotherapy of AML could be substantially reduced during the last 2 decades, e.g., by improvement of antifungal prophylaxis with posaconazole  . Because there was no significant difference between our AML patient cohorts with respect to the percentage of de novo AML or hematological malignancies prior to AML diagnosis, respectively, we did not perform subgroup analysis focusing on this potential factor ; 3rd, the treatment-associated toxicity of conventional chemotherapy.In detail, despite there was no significant difference concerning ANC at diagnosis between both AML patient groups, subsequent reduction of neutrophil counts can be expected to be much more pronounced after application of induction chemotherapy as compared to palliative AML treatment (e.g. with hypomethylating agents). Furthermore, the duration of usually undetectable neutrophils following induction chemotherapy depends on such important factors as age, antecedent hematological malignancy and especially on the response to the first induction cycle  at low levels in the bronchoalveolar system in combination with chemotherapy-induced pulmonary tissue damage might contribute to the high rate of atypical pneumonia  in case of pulmonary infiltrates. Both diagnostic strategies led to a high detection rate especially of possible IFD being classified by characteristic infiltrates or a combination with distinct clinical symptoms, while the cumulative incidence of proven and probable IFD was well comparable with recently published studies  has been observed . Despite the low rate of possible IFD diagnosed in the cohort of elderly AML patients, this represents another potential limitation of our retrospective analysis.Taken together, pneumonia including atypical pneumonia can be significantly more often detected at diagnosis in elderly AML patients subsequently allocated to palliative treatment. In contrast, after initiation of AML therapy the rate of infectious complications and especially bloodstream infections, fever of unknown origin or fungal infections is significantly higher following induction chemotherapy. Importantly, in elderly AML patients the ECOG performance status and the presence of infections at diagnosis represent clinically relevant prognostic factors concerning the outcome of patients undergoing palliative treatment.Figure S1: CONSORT diagram of AML patients (n=118) with palliative treatment. Illustration of antecedent hematological disease or specific therapy prior to diagnosis of AML and distribution of first-line AML therapy in patients allocated to palliative AML treatment. (PPTX 44 kb)Figure S2: CONSORT diagram of AML patients (n=102) with induction chemotherapy. Overview of antecedent hematological disease or specific therapy prior to diagnosis of AML, distribution of hematological response and subsequent treatment of patients undergoing induction chemotherapy as first-line treatment of AML. (PPTX 195 kb)Figure S3: Spectrum of pathogens obtained from blood culture samples dependent on AML treatment intensity. Distribution of positive blood culture specimens indicating the spectrum of isolated bacteria in febrile patients after palliative AML treatment (A) or following induction chemotherapy (B). (PPTX 44 kb)Figure S4: Comparison of most relevant infectious complications at diagnosis and after initiation of AML treatment for both patient subgroups. Percentage of most relevant infectious complications at diagnosis of AML or after initiation of AML therapy for patients receiving palliative AML treatment or induction chemotherapy. (PPTX 54 kb)Supplementary file5 (DOCX 16 kb)Below is the link to the electronic supplementary material.


Key pore residues, responsible for Ca2+ binding, have been recently identified. There is also a substantial progress in the mechanistic and structural understanding of the plant TPC1 gating by membrane voltage and cytosolic and luminal Ca2+. Collectively, these gating factors at resting conditions strongly reduce the potentially lethal Ca2+ leak from the vacuole. Such tight control is impressive, bearing in mind high unitary conductance of the TPC1 and its abundance, with thousands of active channel copies per vacuole. But it remains a mystery how this high threshold is overcome upon signaling, and what type of signal is emitted by TPC1, whether it is Ca2+ or electrical one, or a transduction via protein conformational change, independent on ion conductance. Here we discuss non-exclusive scenarios for the TPC1 integration into Ca2+, ROS and electrical signaling.Two-pore cation channel, TPC1, is ubiquitous in the vacuolar membrane of terrestrial plants and mediates the long distance signaling upon biotic and abiotic stresses. It possesses a wide pore, which transports small mono- and divalent cations. K Originaa includes TPC1 from angiosperms, ferns and bryophytes. It is characterized by highly conserved binding sites for cytosolic and vacuolar Ca2+, which are not conserved in the clade b (mosses and liverworts)  and Na+ can be accumulated to even higher concentrations under a severe salt stress. On the background of 100 mM K+ in the vacuole, 2 mM vacuolar Ca2+ or Mg2+ approximately twice reduced the TPC1 current, mainly mediated by K+  TPC1-mediated current by patch-clamp and Ca2+ flux by a fluorescent dye yielded the fractional Ca2+ current of 5–10% of the total one  pore entry. Recent in silico study specified two residues in the extracytosolic part of the AtTPC1 pore, just next to the SF, whose mutation to Ala primarily affected (handicapped) Ca2+ conductance but to a much lesser extent affected the conductance of monovalent cations ; these residues do not participate in the binding of a short diamine putrescine, Put. PAs are natural blockers of the TPC1 channels. The concentration of PAs is differentially modulated by stresses. For instance, K+ deficiency is associated with a high Put level, whereas salt stress is normally associated with a high Spm  sensitivity of the mutants to high salinity. And, using these mutants, one can elucidate whether Ca2+ conductance of the TPC1 is physiologically important. Recent cryo-EM structural study revealed additional residues, which coordinated the external  cation-binding site, although it could not be discerned whether observed bound cation was Ca2+ or Na+ due to their very similar diameter  luminal Ca2+ binding as compared to its affinity in the intermediate and open channel conformations; Mg2+ binding was by more than an order of magnitude weaker , in analogy to CICR mediated by ryanodine receptor channels of the endoplasmic reticulum in animal cells  cytosolic Ca2+  decreased their affinity to Ca2+  bears several positively charged residues   state to the open one  cytosolic Ca2+ and high positive voltages, all unphysiological. Such experiments demonstrate enormous potential of the TPC1 channels, which are capable to generate huge (nA) currents per vacuole. At the same time, a rather sensitive non-invasive MIFE technique is required instead of patch-clamp to measure tiny ion fluxes, when it comes to physiological conditions. Yet, even at elevated (2 μM) cytosolic Ca2+ the cytosol directed net Ca2+ flux was equivalent to just 1.5 pA per vacuole , whose duration was strongly dependent on the stimulus strength, reaching few seconds at current injections in the range of hundred pA. Plateau disappeared in lack-of-function TPC1 mutants and upon introduction of physiological (1 mM) vacuolar Ca2+. The role of K+ release via K+-selective tonoplast channels was proven by a substantial decrease of the post-stimulus plateau in TPK knock-out mutants. Whereas TPC1 is readily permeable for Cs+, with a conductance of about half of that for K+ /CBL-interacting protein kinase cascade  function of members of voltage-dependent channels superfamily, including various Shaker K+ (Kv), Nav and Cav channels  whereas mutations, increasing the voltage activation threshold, were inefficient. It has been concluded that the cytosolic signaling domain, part of which is formed by certain scaffold protein, senses the conformational changes, associated with the VSD movement in Kv1.3 (Cidad et al. 2+ should be required anymore.When modeling the plateau duration on the stimulus strength a very unexpected theoretical result was obtained: the activity of the TPK channels has to follow/ to be coupled with a voltage-dependent gating of the TPC1 ones, otherwise the simulated post-stimulus depolarization duration became independent on the stimulus strength, in contrast to experimental observations (Jaślan et al. 2+ and voltage (Ye et al. 2+ and voltage conditions. This prediction works fine for resting conditions and explains the experimentally observed low channel probability. Yet, there is a gap in understanding how the TPC1 works upon signaling. Here we considered possible mechanisms of the TPC1 integration into Ca2+, ROS and electrical signaling processes. To overcome the high threshold for the TPC1 activation we assume the generation of high Ca2+ microdomains, or, alternatively, a direct sensing of the TPC1 conformational changes, which occur before cytosolic Ca2+ binding and channel opening. In both cases, the integration of the TPC1 into the long-distance signaling is paradoxically relied on nanolocal interactions. We have critically discussed different and non-exclusive modes of the TPC1 integration, which are dependent on its Ca2+ or K+ conductance, or ion flux independent voltage sensing. To address these issues experimentally, specific channel mutations should be tested, which include those affecting voltage gating or pore conductance and selectivity. Some relevant mutants, such as for instance a Na+ vs Ca2+selective one, already exist (Guo et al. + and K+ vs Ca2+ permeability, are pinpointed (Navarro-Retamal et al. There is a solid body of evidence for functional roles of the TPC1 channels in stress signaling (Table


Respiratory infectious diseases (RIDs) pose threats to people’s health, some of which are serious public health problems. The aim of our study was to explore epidemic situations regarding notifiable RIDs and the epidemiological characteristics of the six most common RIDs in mainland China. We first collected the surveillance data of all 12 statutory notifiable RIDs for 31 provinces in mainland China that reported between 2010 and 2018, and then the six most prevalent RIDs were selected to analyze their temporal, seasonal, spatiotemporal and population distribution characteristics. From 2010 to 2018, there were 13,985,040 notifiable cases and 25,548 deaths from RIDs in mainland China. The incidence rate of RIDs increased from 109.85/100,000 in 2010 to 140.85/100,000 in 2018. The mortality from RIDs ranged from 0.18/100,000 to 0.24/100,000. The most common RIDs in class B were pulmonary tuberculosis (PTB), pertussis, and measles, while those in class C were seasonal influenza, mumps and rubella. From 2010 to 2018, the incidence rate of PTB and rubella decreased; however, pertussis and seasonal influenza increased, with irregular changes in measles and mumps. The mortality from PTB increased from 2015 to 2018, and the mortality from seasonal influenza changed irregularly. PTB was mainly prevalent among people over 15 years old, while the other five common RIDs mostly occurred among people younger than 15 years old. The incidence of the six common RIDs mostly occurred in winter and spring, and they were spatiotemporally clustered in different areas and periods. In conclusion, PTB, seasonal influenza and mumps remain as public health problems in China, suggesting that continuous government input, more precise interventions, and a high-tech digital/intelligent surveillance and warning system are required to rapidly identify emerging events and timely response. Respiratory infectious diseases (RIDs) pose threats to people’s health and lives, some of which are serious public health problems. RIDs are known to spread mainly through droplets and short-distance contact , and peoMany studies have explored the epidemiological characteristics of infectious diseases in China ,5,6,7. Fhttp://www.phsciencedata.cn, accessed on 10 March 2022) of the Chinese Center for Disease Control and Prevention [http://bzdt.ch.mnr.gov.cn/, accessed on 20 February 2023) of the Ministry of Natural Resources of the People’s Republic of China.Data between 2010 and 2018 were collected from the monthly and regional reports from the China Public Health Science Statistics Center , H7N9 influenza and diphtheria) and four RIDs from class C , mumps and rubella). Influenza A (H1N1) was classified into class C and combined with seasonal influenza in 2013 .Thirty-one provinces in mainland China are divided into seven areas according to geographic location, population, environment, and other factors: northern China , northeastern China , eastern China , central China , southern China , southwestern China , and northwestern China .We first analyzed the overall epidemic situation of 12 RIDs from 2010 to 2018, then focused on the top three RIDs from class B  and the top three RIDs from class C  with the highest incidence rates from 2010 to 2018 in mainland China, to analyze age-specific rates, temporal trends, and seasonal and spatiotemporal clustering characteristics. The deaths caused by most RIDs are sporadic, so we only analyzed the deaths related to PTB and seasonal influenza, which caused higher mortality.Descriptive analysis was used to present the population distribution characteristics of RIDs, and the figure drawing was conducted with OriginPro .p < 0.05) and to decrease if APC < 0 (p < 0.05), otherwise rates were considered stable [The temporal trends of incidence rate and mortality were characterized by annual percentage changes (APCs) and were estimated with Joinpoint regression software . The grid search method (GSM) was the default modeling method, and the Monte Carlo permutation test was the default optimization method of the model. The Bayesian information criterion (BIC) was used to correct the statistically significant level. The most appropriate joinpoints of the interval piecewise function were finally selected by the above methods . The aged stable . The figSeasonal decomposition of time series by the LOESS method for extracting components was used to assess the seasonal pattern of the data. The decomposition breaks the time series of RIDs incidences into three components: Yt = Tt + St + Rt ,12. HereSaTScan  was used to identify the space–time clusters of RIDs in different areas and during different periods based on the Poisson probability model ,14. The From 2010 to 2018, there were 13,985,040 notifiable cases of RIDs in mainland China. The incidence rate of RIDs increased from 109.85/100,000 in 2010 to 140.85/100,000 in 2018. PTB was the most prevalent RIDs and accounted for 42.08–67.62% of notifiable cases between 2010 and 2018. During the period 2010 to 2018, PTB had the highest average annual incidence rate , followed by mumps , seasonal influenza , scarlet fever , measles , rubella , pertussis  and epidemic cerebrospinal meningitis . The incidence of HPAI and H7N9 were extremely low: there were only 14 notifiable cases of HPAI during 2010–2015 and 1385 notifiable cases of H7N9 seasonal influenza, which mainly occurred from 2013 to 2017. No diphtheria cases were reported .During the 9 years, 25,548 deaths for 12 RIDs were reported, and the range of mortality was from 0.18/100,000 to 0.24/100,000. PTB accounted for 89.70–94.96% of RIDs death cases, ranking as first, followed by seasonal influenza (0.26~4.61%). There were 919 reported deaths from the other ten RIDs during the 9 years, including H7N9 influenza (560 deaths), epidemic cerebrospinal meningitis (167 deaths), measles (153 deaths), pertussis (13 deaths), HPAI (10 deaths), mumps (seven deaths), scarlet fever (six deaths), and rubella (three deaths).The age-specific incidence rates of the six RIDs are shown in Due to the relatively higher burden posed by PTB and seasonal influenza among the six selected RIDs, we only analyzed the age-specific mortalities of these two RIDs . We founp < 0.001) (p < 0.001) (p = 0.828) (p = 0.076) (p = 0.002) (p = 0.006) from 2010 to 2016, and increased with APC of 58.35%  from 2016 to 2018  A, while < 0.001) B; for me= 0.828) C and −8.= 0.076) E, respec= 0.002) F. For se to 2018 D.p = 0.001) and increased by 9.20%  from 2015 to 2018 (p = 0.066) from 2010 to 2015 and 81.88%  from 2015 to 2018 . The other four clusters of PTB were before 2014 (p < 0.001) (p < 0.001) (p < 0.001) (p < 0.001) (p < 0.001)  B. For me< 0.001) C. The th< 0.001) A. The th< 0.001) B. The th< 0.001) C.p < 0.001)  A. The otInfectious diseases are serious public health problems affecting a large portion of the population all over the world. The Chinese government has attached great importance to and is strongly committing to the control of infectious diseases. National surveillance for the selected infectious diseases in China started from a paper-based reporting system in the 1950s, moving to an electronic file-based system in 1985 and to a web-based reporting system (CISDCP) since 2003 . By analOur results showed that some kinds of RIDs were already at low incidence levels  or had even basically been eliminated . However, given increased economic globalization, urbanization, mobility and the emergence of drug-resistance, the incidences of some RIDs  showed dumps, or were still at high levels in some areas of China. In the present study, we particularly analyzed the epidemiological characteristics of the six most common RIDs in mainland China.The present study showed that the morbidity for PTB in mainland China has been decreasing yearly, which is similar to the global trend  and may With the development of the health service, especially the great improvement in diagnostic ability and surveillance systems, the number of notifiable cases of seasonal influenza has increased year by year. In our study, the incidence of seasonal influenza mostly occurred in the population aged less than 15 years old. Previous studies showed that students and primary and secondary schools are high-risk groups for seasonal influenza, and specific places are prone to occur cluster epidemics ,23,24. APertussis, measles, rubella and mumps are all vaccine-preventable diseases and mainly attack people under 15 years old. The peak-incidence months for measles, rubella, mumps and pertussis appear in sequence in spring and summer. However, the duration of peak period and temporal trends for the four RIDs were inconsistent. The incidence of pertussis in China has been decreasing due to the expanded program of immunization (EPI) , though The incidence rate of measles and rubella remained at a low level in mainland China. The low incidence rates for measles and rubella reflected comprehensive preventative measures, including China’s Measles Elimination Action Plan , high seThe present study demonstrated the achievement and challenges in the prevention and control of RIDs in mainland China, and called for continuous and increased efforts to prevent and control RIDs. We recommend more attention to projects or actions associated with strengthening the core capacity of public health, including enhancing government leadership and multiple sectors’ cooperation, expanding investment in prevention programs, enhancing and integrating digital/intelligent surveillance and warning systems, and strengthening pandemic preparedness . MoreoveThe incidence rate of RIDs increased from 2010 to 2018, and PTB, seasonal influenza and mumps were still at high levels. Some RIDs have been at low incidence levels  or almost eliminated . The study indicated that the incidence rate of PTB and rubella had decreased, but pertussis and seasonal influenza had increased, and there were irregular changes in measles and mumps. The mortality for PTB increased from 2015 to 2018, and the mortality for seasonal influenza changed irregularly. PTB was mainly prevalent among people over 15 years old, and the other five main RIDs mostly occurred in populations less than 15 years old. The incidence of the six RIDs mostly occurred in winter and spring. As for spatiotemporal clustering, the incidence and mortality of PTB were relatively higher in northwestern China during 2014–2018, while that of seasonal influenza clustered in most provinces during 2017–2018. The incidence rate of mumps clustered in most provinces, mainly occurring before 2013. In all, we still face big challenges and arduous tasks ahead for RIDs control and prevention in China and should take more precise and effective intervention, as well as establish new and effective high-tech digital/intelligent surveillance and warning platforms, to rapidly identify emergent events and instigate timely response.


Cytological approaches have long been used in the diagnosis, prognosis, and management of acute myeloid leukemia (AML) and myelodysplastic neoplasms. Technological advances in molecular biology, in particular next-generation sequencing (NGS), have made it possible to establish a molecular list of several gene mutations in AML and MDS, within a matter of days. The combination of cytological approaches and NGS makes it possible to pinpoint genetic mutations and cancer cell survival and proliferation pathways, enabling better clinical management and improved prognosis of patients with acute leukemia and myelodysplastic neoplasms.CBFbeta fusion with MYH11 and PML-RAR fusion in the previous cases. In these two examples, the molecular abnormality allows us to better define the pathophysiology of leukemia, to adapt certain treatments , and to follow up the residual disease of strong prognostic value beyond the simple threshold of less than 5% of marrow blasts, signaling the complete remission. However, the new sequencing techniques of the next generation open up broader perspectives by being able to analyze several dozens of molecular abnormalities, improving all levels of management, from diagnosis to prognosis and treatment, even if it means that morphological aspects are increasingly relegated to the background.For decades, the diagnosis, prognosis and thus, the treatment of acute myeloblastic leukemias and myelodysplastic neoplasms has been mainly based on morphological aspects, as evidenced by the French-American-British classification. The morphological aspects correspond quite well, in a certain number of particular cases, to particular evolutionary properties, such as acute myelomonoblastic leukemias with eosinophils or acute promyelocytic leukemias. Advances in biology, particularly “classical” cytogenetics (karyotype) and molecular cytogenetics (in situ hybridization), have made it possible to associate certain morphological features with particular molecular abnormalities, such as the pericentric inversion of chromosome 16 and translocation t in the two preceding examples. Polymerase chain reaction techniques have made it possible to go further in these analyses by associating these karyotype abnormalities with their molecular causes,  The management of acute myeloid leukemia (AML) and myelodysplastic neoplasia (MDS) has been based, for a long time, on a cytological approach, which was already based on undiscovered molecular bases. We can mention the eosinophilia-associated features of AML4eo, for which an association with inv(16) or an equivalent was often found, without forgetting the clinical and cytological features of acute promyelocytic leukemia (APL) linked to t or an equivalent. Among the major technological evolutions, the progress in deoxyribonucleic acid (DNA) sequencing and/or ribonucleic acid (RNA) amplification by a polymerase chain reaction (PCR) has allowed us to refine our classifications of MDS/AML in order to better understand the mechanisms of leukemogenesis, and thus, to be able to better adapt the treatments. However, until now, these tools were more or less used gene-by-gene and target-by-target. The technological evolution of next-generation sequencing (NGS) now allows us, with a delay of a few days compatible with patient management, to obtain a molecular identity card of MDS/AML involving the analysis of mutations in several dozen genes at the same time. To illustrate the interest of this new approach, the new classifications of MDS and AML, largely based on molecular data , should allow us, in the near future, to offer precision medicine.RBM15), myocardin-related transcription factor-A (MRTFA) fusion, or other fusion genes than Myeloid/Lymphoid or mixed-lineage Leukemia, translocated to 3 (MLLT3) associated to lysine N-methyltransferase 2A (KTM2A) in the ICC classification, which, in contrast, is absent in the WHO classification. The most important change is that the 20% blast cutoff is not required in both classifications to define AML, except for AML with the Breakpoint Cluster Region (BCR): ABL Proto-Oncogene 1, Non-Receptor Tyrosine Kinase (ABL1) in both classifications, for CEBPA mutations in the WHO classification, AML-Myelodysplasia-related (MR) and AML-NOS defined by differentiation. Nonetheless, while the ICC has defined a cut-off at 10% for the AML with defining genetic abnormalities, the WHO has not assigned an arbitrary lower bone marrow blast cut-off if the molecular data correlates with the morphologic findings. Regarding the defining mutations of AML-MR, eight mutations are common to both classifications. The WHO does not retain the Runt-related transcription factor 1 (RUNX1), in contrast to the ICC. Some differences are also present regarding the AML-MR defining cytogenetic abnormalities .For the European Leukemia Net (ELN), the International Consensus Classification (ICC) and the World Health Organization (WHO) ,2,3, molSince these molecular anomalies do not have the same prognostic values and therapeutic consequences, a hierarchy in the diagnostic classification was proposed in order to allow a clinical use of these data . The mosRAR α), KMT2A, or MDS1 and EVI1 complex locus (MECOM)). The presence of these anomalies has another consequence, i.e., the decrease in the diagnostic threshold of AML, which is, in these cases, reduced to only ≥10% of the blasts. Regarding the cytological diagnosis, myeloblasts, monoblasts and megakaryoblasts are considered as blasts, while monoblasts and promonocytes  are also classified as blasts in AML subtypes with myelomonocytic or monocytic differentiation. In the setting of APL, promyelocytes are also counted as blasts.In first position are the recurrent anomalies (Tumor Protein (TP)53 mutations , which define a new type of pathology intermediate between acute leukemia and myelodysplastic syndrome (MDS/AML) if blasts are between 10 and 19%, and AML if blasts are ≥20%.In the second hierarchical position are In the third position, we find  the mutations related to myelodysplastic syndromes.In the fourth position, we only find complex karyotypes and/or certain abnormalities related to myelodysplastic syndromes . This downgrading shows the predominant role taken by molecular analysis  in the classification.In the fifth position are the leukemias presenting none of the previous characteristics. In addition to the creation of the MDS/AML category, it is important to note the disappearance of the categories of AML with myelodysplasia-related changes (AML-MRC) and treatment-related AML. Indeed, it has been shown that it is the presence of the above-mentioned molecular abnormalities/mutations that impacts the response to treatment, as well as the prognosis, more than these clinical features. This concept is, in fact, not new since it was already known that in AML-MRC or treatment-related categories, karyotypic abnormalities were preponderant in the prognosis.PML-RAR translocation, which confers, even for high-risk APL, an excellent prognosis with a 2-year DFS and OS of 94% and 100% in an intention-to-treat analysis  polymerase 1 (PARP1) that decreases MECOM(EVI1) expression, providing an interesting strategy for these AMLs , ASXL1 confers a poor prognosis. Of note, this mutation can orientate the therapeutic intervention since some experiments suggest that ASXL1 mutants confer sensitivity to BET bromodomain inhibitors (BETis) and resistance to Histone DeACetylase inhibitors (HDACi) [ (HDACi) .■BCOR gene encodes for a subunit of the Polycomb Repressive Complex (PRC) 1.1, one of the six complexes that constitute the PRC1. By pairing the analysis of 6162 patient samples with cell line experiments, Schaefer et al. [BCOR mutation inactivates the repressive activity of PCR1.1, leading to aberrant oncogenic signaling, suggesting that specific inhibitors of the RAS/MAPK pathway could be of interest in this AML subset.The r et al.  have sho■The Enhancer of Zeste Homolog 2 (EZH2) mutation corresponds to the loss of function of a histone methyltransferase activity that participates in the Polycomb Repressive Complex 2 (PRC2), whose role is to silence genes involved in stem cell renewal, including leukemic stem cells (LSC). Interestingly, the identification of EZH2 mutations was correlated, in most cases, with the loss of EZH2 protein expression, as evaluated by immunohistochemical analysis, thus directly linking the sequencing data to the AML phenotype [henotype .■RUNX1 gene, as previously seen, is involved/translocated in some recurrent genetic abnormalities in AMLs (of good prognosis), although it can also be mutated in 10% of MDS or AMLs. In this case, a loss of function is the common consequence of the mutation (missense/deletion truncation), and mtRUNX1 confers a poor prognosis. The mutation of RUNX1 impairs ribosome biogenesis (RiBi), leading to the hypothesis that drugs that perturb RiBi could be of interest in that AML subset. In accordance, AMLs with mtRUNX1 show an important sensitivity to homoharringtonine and BET inhibitors, with a synergistic effect of the B-cell lymphoma 2 (BCL2) inhibitor venetoclax, suggesting a specific chemotherapy regimen for these AMLs [The ese AMLs .■Splicing Factor 3B subunit 1 (SF3B1) mutation corresponds to most “sideroblastic anemias” of the previous classification and is considered MDS-related. This mutation is not restricted to the myeloid lineage since it is found in CLL, T–ALL and solid tumors, such as melanoma, pancreatic adenocarcinoma or breast cancer. The main function of SF3B1 is the assembly of the spliceosome, so many inhibitors of the spliceosome are under investigation in AML or MDS [The L or MDS .■Serine and Arginine Rich Splicing Factor 2 (SRSF2) is a splicing factor in which mutations occur in up to 20% of AML/MDS and is frequently associated with mtRUNX1, conferring a particularly poor prognosis [BRIP1, NABP1, TBRG4 and AKAP8L). This example demonstrates the usefulness of a comprehensive and simultaneous analysis of all these mutations in order to take account of their possible cooperative effects on AML pathophysiology.The rognosis . The rearognosis . Very in■STromal AntiGen 2 (STAG2) is one of the core components of the cohesin complex, a global protective structure around DNA molecules, whose mutation usually leads to a loss of function. The mutation of STAG2 induces a switch of the cohesion complex to use STAG1, which is rarely mutated in AML/MDS [ AML/MDS . This sw■U2 small RNA nuclear auxiliary factor 1 (U2AF1) plays a central role in the alternative splicing of pre-mRNA and is mutated in 10% of AML/MDS. It is considered a poor prognosis marker, and although no specific drugs target this molecule, splicing modulators may be of interest in this AML subtype [The  subtype .■ZRSR2  is a splicing factor rarely mutated (5% of MDS/AML) and often associated with mutation of the epigenetic regulator TET2. Its various mutations lead to a loss of function. Interestingly, although many abnormalities are detected in ribosome functions, inflammation or cell mobility, a major dysregulation of the MAPK pathway seems to play a central role in leukemogenesis [The ogenesis , suggestogenesis .The fourth hierarchical level of ICC classification corresponds to MDS/AMLs with a complex karyotype, with a more or less classical poor prognosis abnormality, such as del(7q) or −7. Of note, before the NGS era, cytogenetic data were clearly positioned at the first hierarchical level of AML classification, further underlining the current central role of NGS in MDS/AML management.Finally, few MDS/AMLs will correspond to the fifth hierarchical level of MDA/AML, not otherwise specified in the exhaustive molecular and cytogenetic analysis, which are mandatory for myeloid malignancies management.TP53multihit mutations, KMT2A (MLL) partial tandem duplication (PTD), and FLT3-ITD correlated with the worse prognosis, while ASXL1, BCOR, EZH2, NRAS, RUNX1, STAG2 and U2AF1 mutations were also associated with adverse risks. The good prognosis of SF3B1 was modified by the presence of co-mutations. Clearly, the analysis of prognosis is not directly feasible by the clinician since it requires taking into account all the mutations and their co-occurrence. Consequently, a useful M-IPSS Web calculator, easily available, has been developed  that also includes missing values and is both applicable for primary and secondary/therapy-related MDS.Among the most interesting impacts of the availability of NGS data is the possibility to refine the prognosis for each patient while evaluating both the classical hematological data , cytogenetic data and the large mutational analysis data. This led to the elaboration of the Molecular International Prognostic Scoring System (M-IPSS) . The cliFLT3/NPM1, KIT/NRAS/KRAS/PTPN11, GZTA2/CEBPA/WT, TP53/IDH1/IDH2/RUNX1, or DNMT3A/ASXl1/TET, among others. Regarding the 247 instances with both MPFC and NGS MRD tests, there were 197 MPFC+/NGS+ instances, 3 MPFC−/NGS− instances, 44 MPFC−/NGS+ instances and 3 MPFC+/NGS− instances; thus, there was a 19% (47 out of 247) discrepancy between the two methods. Of note, these discrepant results were mainly observed with mutations not associated with adverse outcomes , mutations of unknown significance (14 tests), mutations that were likely pathogenic (3 tests) and 1 patient without mutations. If we exclude these tests, regarding the remaining 131 pairs, 12 are discrepant with 2 MPFC+/NGS− and 10 MPFC−/NGS+. Although the study sample size is limited, these data suggest that NGS may avoid missing low-volume MRD. Nonetheless, the clinical impact of such findings is to be evaluated in controlled trials so that NGS cannot directly impact the treatment that still relies on MPFC and RT-qPCR MRD evaluation. Another impact of NGS is the level of sensitivity of the method used for MRD assessment. The predictive value of NGS vs. MPFC MRD was analyzed and compared in the study by Tsai et al. [DNMT3A, TET2 and ASXL1) was not informative for MRD assessment. Then, this study showed that the MRD assessment at time 1 by NGS was not predictive of relapse if MPFC MRD was negative but had a poor prognosis value at time 2 even if MPFC was still negative. Due to the rise of specifically targeted therapy, the specific assessment of druggable mutations is of pivotal interest. One of the most common pathogenic mutations detected in AML is FLT3-ITD (25% of patients), with various targeted drugs, such as midostaurin or gilteritinib. The detection of FLT3-ITD MRD is challenging and is mainly done via fragment analysis (FA) with a detection sensitivity of 2%. Recently, the development of novel NGS analysis methods has resulted in a 0.001% sensitivity that was superior to FA analysis [FLT3-ITD MRD detection by NGS is an important prognosis factor that can precisely define the relapse risk.The evaluation of MRD can be done with various techniques, mainly using multiparameter flow cytometry (MPFC), real-time polymerase chain reaction (RT-PCR) or NGS . The impi et al. . This stanalysis . The clianalysis . The MRDhttps://mds-risk-model.com, accessed on 27 October 2022). Some pivotal advances in MDS/AML management have been voluntarily not discussed in this article, more particularly concerning druggable mutations, such as FLT3, IDH1 or IDH2, since they have been extensively reviewed. Of note, data from NGS are useful for diagnosis in order to select the best therapeutic options for patients .  review, .DNMT3A or NPM1 mutations or MLL translocations). In 2016, a different approach was used, relying not on mutation detection as in the previous paper but on the identification of a 17-gene stemness transcriptional signature that predicted a poor prognosis even when allogeneic transplantation was performed [Finally, it is useful to have a historical view of NGS in AML via some milestone publications. In 2012, Patel et al.  publisheerformed . Nonetheerformed .BCR::ABL1 fusion gene, FLT3 or IDH1/2 mutations) is not the “magic bullet” since leukemia heterogeneity will allow tumor escape via clonal drift under chemotherapy pressure. Probably the next step of the NGS approach in AML should require single-cell RNA or DNA analysis, thus requiring more and more bioinformatics resources to allow their routine clinical use. Advances in sequencing speed and steadily decreasing costs suggest that this approach could become part of routine AML assessment in the future. In combination with this highly targeted approach, the use of drugs aimed at targeting pathways central to the survival of leukemia cells, and leukemic stem cells in particular [The next challenge now will be in the elaboration of predictive models to be able to account for all the molecular markers, i.e., fusion gene transcripts, mutations, and gene expression. Nonetheless, all these efforts have a sense only if they are able to develop precision medicine, allowing the introduction of therapy that reaches a good target. More precisely, targeting a single mutation (as tyrosine kinase inhibitors for rticular , will he