Ca2+ signaling is typically studied by microscopy in cell lines but can present some limitations to explore human PBMCs, where flow cytometry can be a good alternative.The immune system, composed of organs, tissues, cells, and proteins, is the key to protecting the body from external biological attacks and inflammation. The latter occurs in several pathologies, such as cancers, type 1 diabetes, and human immunodeficiency virus infection. Immunophenotyping by flow cytometry is the method of choice for diagnosing these pathologies. Under inflammatory conditions, the peripheral blood mononuclear cells (PBMCs) are partially activated and generate intracellular pathways involving Ca2+ signaling in PBMCs. We aimed to investigate the structure and evolution of this field in a physio-pathological context, and then we focused our review on flow cytometry analysis of Ca2+ fluxes in PBMCs.In this review, we dived into the research field of inflammation and Ca2+ signaling in PBMCs were published, according to The Clarivate Web of Science (WOS) database used in this review. A bibliometric study was designed for this collection and consisted of a co-citation and bibliographic coupling analysis.From 1984 to 2022, 3865 articles on inflammation and Ca2+ homeostasis, including chemical probe development, identification of the leading players in Ca2+ signaling, and the link with chemokine production in immune cell function. Next, the bibliographic coupling analysis combined 998 articles in 8 clusters. This analysis outlined the mechanisms of PBMC activation, from signal integration to cellular response. Further explorations of the bibliographic coupling network, focusing on flow cytometry, revealed 21 articles measuring cytosolic Ca2+ in PBMCs, with only 5 since 2016. This final query showed that Ca2+ signaling analysis in human PBMCs using flow cytometry is still underdeveloped and investigates mainly the cytosolic Ca2+ compartment.The co-citation analysis was performed on 133 articles: 4 clusters highlighted the global context of Ca2+ signaling in PBMCs, such as reticulum and mitochondria, and presents flow cytometry as a solid option to supplement gold-standard microscopy studies.Our review uncovers remaining knowledge gaps of intracellular players involved in Ca The main components of blood are plasma and leukocytes. The immunophenotyping of leukocytes, notably the peripheral blood mononuclear cells (PBMCs), has emerged as an essential tool for medical research. This diagnostic tool is widespread in hematology, cancerology, and neurology fields.+ T cells in Human Immunodeficiency Virus (HIV) infections is crucial to monitoring disease progression (+ T cell-induced interferon-gamma (IFN-γ) production  . Diagnosgression  and can gression . In factgression , 8), infgression  and degegression  through oduction . Flow cy2+-dependent signaling cascade, starting from the ligand binding on the receptor through intracellular pathways up to the regulation of gene expression. More precisely, membrane receptor activation triggers a transitory Ca2+ release from internal stores, further leading to a store-operated Ca2+ entry (SOCE), raising the intracellular Ca2+ concentration  and Fc receptors for monocytes and macrophages. Their activation through these receptors involves a Cantration , 14, whintration . Thus, Ccordings . So far,hologies , 18. To 22.1The bibliometric analysis is suited when the scope of the review is broad and the dataset is too large for manual review. This analysis presents the intellectual structure and emerging trends of a research topic. The study was designed following the guidelines of Donthu et al. and the methodology of Chalet et al. , 20. The2.22+ signaling in PBMCs in a physio-pathological context. Based on these first results, we further explored the development of Ca2+ analysis by flow cytometry.We aimed to determine the research structure and evolution of inflammatory and Ca2.3The structure and dynamics of the scholar knowledge were assessed and represented using science maps. To provide an overview of our research topic and to explore the emerging trends, co-citation analysis and bibliographic coupling were carried out.- The co-citation analysis determines the relationships among cited publications, highlighting the most influential themes.- The bibliographic coupling forms thematic clusters.2.4© was used to collect data (Copyright Clarivate 2022WoS). It is a selective, structured, and balanced database with complete citation links and enhanced metadata. It can inform about citation indexes representing the connections between scholarly research articles in globally significant journals, books, and proceedings chapters. Exported data included the complete set of references enabling in-depth analysis of the intellectual structure.Clarivate Web of ScienceWe applied the following query to article titles and abstracts:2+” OR calcium] AND [physiological OR inflammat* OR ischem*][pbmc OR “peripheral blood mononuclear cells” OR lymphocyte OR monocyte* OR macrophage*] AND  AND [physiological OR inflammat* OR ischem*] AND [cytometry][pbmc OR “peripheral blood mononuclear cells” OR lymphocyte* OR monocyte* OR macrophage*] AND [“CaWe included all articles, book chapters, and meeting abstracts. The query ranged from 1993 to 2022, and 185 articles were obtained.2.5VOS viewer was used to display bibliometric networks represented through mapping and clustering  to perfo2.6rd quartile.Two articles are co-cited when they appear together in the reference list of another publication. Frequently co-cited documents in a corpus represent its knowledge foundations. This technique highlights influential publications and unveils the structure of the research field. The co-citation was conducted in VOS viewer using reference analysis. The results were displayed as network visualization. The minimum number of co-cited references was fixed to 20 to obtain the most influential articles. 133 co-cited documents were included in our analysis and mapping, and were clustered and displayed on the VOS viewer. Included references grouped by clusters are displayed in 2.72+ and inflammation in PBMCs. Articles from 1990 to 2022 were included, and 998 were grouped in clusters. Cluster constituents are detailed in The bibliographic coupling method considers publications that share common references as an indication of similarities in content. This analysis provides a clustered visualization of the field in themes and includes recent and niche publications. The bibliographic coupling analysis was obtained with VOS viewer, with coupling analysis of references. The results were displayed as network visualization. This bibliographic coupling was carried out to determine the knowledge in the field of Card quartile of the full scores in their cluster were selected for a thorough analysis.The exploration of the clusters was performed on the highest total link strength articles, corresponding to the link of an item with other items in a network. To get an overview of the dominant theme of each cluster, the references with a total link strength equal to or higher than the 32.8The term “inflammat*” covers a large field of research, including several pathologies. Thus, the most influential articles in our “inflammatory” cluster discuss chemokines in HIV. The abbreviation T cells or B cells were not included in the query.33.12+ and inflammation research in immune cells in the current literature. In total, 3865 articles were published between 1984 and 2022. An average of 90 publications per year between 1990 and 2010 were reported. Over the next ten years, publications increased strongly, up to 2-fold from 2010 to 2021. Analysis was done until July 2022, which explains the lowest number of publications for this year , a Ca2+ sensor essential for the SOCE through its binding to ORAI1 . ORAI proteins are STIM-binding partners that form the channel pore in the plasma membrane (2+ influx in T- and B-cells and promote the immune response by partly activating the transcription factor nuclear factor of activated T-cells (NFAT)  , 24, 25.s (NFAT) , appearsFura2-AM –29. Therrd quartile ranging from 1987 to 1991. They refer to calcium-binding proteins (CaBPs), such as the S100 protein. S100 was reported to be associated with specific stages of monocyte differentiation  or chemokine ligand 5 (CCL5) axis in HIV infections. Indeed, RANTES research was achieved at the time of the outbreak emergence of HIV at the beginning of the 1980s, instigating extensive research on this chemokine.The last two clusters, green and blue, are located on the opposite side of the maps and share close positions and content. No temporal evolution was observed in these two clusters, since all the articles were published in the 90s –36. The 2+ and chemokine signaling in the immune cell function, which depends on signal integration, stress, chemical environment, and inflammation, key features of several pathologies. More precisely, all these parameters lead to modulation and cell activation by intracellular pathways, activating CaBPs and partially leading to their translocation to the nucleus. The latter activates Ca2+-dependent transcription factors that control PBMC functions such as proliferation, differentiation, and cytokine production.To conclude, the co-citation analysis supports the crucial role of Ca3.3Bibliographic coupling is based on the idea that two publications sharing common references have similar content. This analysis enables the formation of thematic clusters in the literature corpus obtained with our query. Consequently, recent niche publications may appear in our analysis. This section aimed to determine what drove the calcium and inflammation research field on PBMCs over the last three decades. The analysis was done on 998 articles, and 8 clusters were identified Figure 6rd quartile and refers to the SOCE. This cluster can be further divided into subgroups related to lymphocytes and macrophages, respectively.The red cluster contains 57 out of 232 articles in the 32+ sensor and ORAI1 channel-forming protein were determined by Feske et al. The CRAC composition depends on cell type, localization, and activation state, but ORAI1 is the dominant channel member in several immune cells, mainly in neutrophils and mast cells (3) binding to IP3 receptors (IP3R) in the endoplasmic reticulum (ER) membrane following TCR or BCR activation. IP3 leads to IP3R opening, resulting in Ca2+ release from the ER into the cytosol. The resulting ER Ca2+ decrease induces a conformational change of STIM1, which binds to ORAI1 channels to open its pore in the plasma membrane. The importance of Ca2+ influx through plasma membrane channels in T cells was characterized by studying the Ca2+ conductance triggered by the TCR. An absence of this current was demonstrated in human T cells from patients with immunodeficiency diseases  components in the lymphocyte cluster. In 2005 – 2006, both STIM1 Cast cells –39. In pdiseases . Missensdiseases , 41.+) channels were identified in 1983 by Matteson and Deutschand in 1984 by DeCoursey et al. The latter publication shows their implication in functional processes such as mitogenesis  production and cell proliferation in T cells are partly mediated by K+ channels . In contrast, in vitro M1 polarization induced by IFNɣ is associated with the recruitment of the transient receptor potential cation channel 1 (TRPC1) to enhance Ca2+ entry, leading to high expression of inflammatory genes , involved in the physiological response in some immune cells, i.e., monocytes and macrophages, through intracellular Casurvival .2+ signaling in the immune function of macrophages and lymphocytes. It therefore represents the largest cluster in our query, gathering around 23% of the articles in the bibliographic coupling map.In summary, the red cluster highlights the central role of Card quartile, from 2003 to 2016. It is well known that Ca2+ signaling enhances mitochondrial adenosine triphosphate (ATP) production in activated T cells   –52. The  (NF-κB) –59. Therrd quartile. A part of it is located close to the red cluster and covers articles related to Ca2+ signaling and transcription factors (rd quartile and focuses on chemokine receptors (CCR) and their ligand. Chemokine receptors are G-protein-coupled-receptors serpentine receptors. Chemokines, a particular type of small cytokines, are known to contribute to the trafficking of leukocytes to the inflammation site through a signaling cascade. Depending on the environment, chemokines activate neutrophils to attract and activate monocytes, basophils, eosinophils, or lymphocytes , also known as monocyte chemoattractant protein 1 (MCP-1), in this pathology. This cluster contains 2/15 publications in the 3ormation . At the ormation . Of note2+ signaling is widely studied in physiological conditions and several pathologies involving an immune response. Since intracellular Ca2+ signaling partly controls cytokine production, it was expected to see such a coupling between the Ca2+ signaling clusters and the inflammatory ones. Therefore, the resulting map aligns with our current field knowledge.Altogether, this bibliographic coupling showed that Ca3.42+ signaling is crucial in controlling immune cell functions. We next wanted to determine the evolution of flow cytometry-based studies to evaluate the Ca2+ kinetics in PBMCs over the last three decades. To perform this analysis, another query was defined to focus on flow cytometry, as specified in the “Materials and Methods” section. Ranging from 1993 to 2022, we obtained 184 articles. These articles were split into two flow cytometry usage categories: (1) for the phenotyping of immune cell profiles and (2) to analyze Ca2+ kinetics in PBMCs. We reported and focused our review on the 21 publications falling in the latter category . As for the techniques employed, both non-ratiometric and ratiometric probes are used, mainly to assess cytosolic Ca2+ levels, therefore putting aside several actors of Ca2+ signaling in other cell compartments. From this particular query, only one paper performed intracellular stimulations on a reticular player: Kushnir et al. , using caffeine as an agonist. They specifically looked at the cytosolic Ca2+ level with Fluo4 in human B lymphocytes targeted with an anti-CD19 antibody in PBMCs. The Ca2+ response amplitudes obtained by a 50 mM caffeine stimulation were recorded in normal or congestive heart failure lymphocytes and revealed an ER Ca2+ leakage as a signature of the pathology.Overall, this query highlights that the study of Car et al.  focused 42+ signaling revealed 3865 articles published between 1984 and 2022, which supported the design of a bibliometric analysis. We reported that conventional flow cytometry is extensively used in clinical routine as a tool for diagnosis and monitoring of inflammatory diseases. A significant advantage of this method lies in the compensation process to correct fluorescent spillovers, increasing the number of parameters studied and enabling target analysis of specific cell populations. Clinical biomarkers of inflammatory pathologies were identified with this quantitative multi-parametric analysis. More recently, spectral flow cytometry collecting the full light spectrum has enabled the distinction of unique fluorophores with overlapping emission spectra (Our literature review query on inflammation and Ca spectra , 97. Alo spectra , 95.2+ signaling, we highlighted that Ca2+ players involved in human PBMC models were studied and described in physiological settings. Indeed, Ca2+ ions are ubiquitous intracellular second messengers with a crucial role in the immune cell function: from the cell-specific membrane receptor activation to the SOCE, all contributing to the transcription of immune response genes (2+ fluxes in immune cell subtypes, even when underrepresented. So far, we have reported only 21 articles using flow cytometry to study Ca2+ signaling focused on cytosolic Ca2+ level variations bypassing the contribution of organelles, notably the ER as the main Ca2+ store and mitochondria whose function depends, in part, on Ca2+ signaling. Interestingly, a Ca2+ coupling exists between ER and mitochondria to control mitochondrial functions. In this regard, Assis et al. suggested that the width of ER-mitochondria coupling was associated with the macrophage activation status (2+ transfer from the ER to mitochondria requires further investigation. Additionally, a recent study demonstrated a reduced mitochondrial Ca2+ uptake via the mitochondrial Ca2+ uniporter during aging in macrophages as a potential contributor to inflammation in humans (2+ homeostasis. Osipchuk et al. reported that pharmacological inhibition of RyR leads to intracellular Ca2+ alteration and alters immune cell functions in vitro and in vivo (2+ leakage in B cells as a biomarker of this pathology. Regarding the SERCA pump, its role is yet to be described in PBMCs. Therefore, the contribution of the different organelles, notably ER, mitochondria, and their coupling, in regulating the immune cell function remains to be deciphered. Albeit combining two Ca2+ chemical probes to study two cellular compartments simultaneously remains unexplored. However, with the increasing development of new ratiometric sensors targeting several cell compartments, multiparametric analyses of Ca2+ fluxes by flow cytometry may become a promising alternative for human immune cell research in the coming decades.Regarding Case genes Figure 7n status . Howevern humans . The Rya in vivo . In PBMC in vivo  reportedCB: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Resources, Software, Visualization, Writing – original draft, Writing – review & editing. LC: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Writing – review & editing. FM: Conceptualization, Formal Analysis, Investigation, Writing – review & editing. TB: Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. SD: Conceptualization, Supervision, Validation, Writing – review & editing. MP: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing. CCDS: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing – review & editing.


We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca The activity of SERCA1a  or SERCA2a (cardiac isoform) is essential for muscle relaxation (diastole), restoring SR Ca2+ following its release via Ca2+ channels  for muscle contraction (systole). Decreased SERCA activity and excessive RyR leak results in failure to maintain the high gradient of [Ca2+] between the cytoplasm (sub-μM) and the SR (mM) during diastole and are associated with heart failure (HF) in humans and animals1. Decreased SERCA activity is related to multiple factors, including reduced SERCA gene expression, increased post-translational modifications, and altered interaction with regulatory proteins1. Overall, decreased SERCA activity and increased Ca2+-leak can lead to a pathophysiological state of the cardiac myocyte2 , skeletal myofiber (Brody’s disease and myotonic dystrophy)3, or non-muscle cells 4. Altered SERCA interactions with regulatory proteins (regulins), e.g., phospholamban (PLB), have been linked to HF5. Of the seven known regulins6, the dwarf open reading frame (DWORF) peptide is the only one known to activate SERCA, both by direct activation8 and by competing with PLB binding10, preventing HF in a mouse model of dilated cardiomyopathy11.Sarco/endoplasmic reticulum calcium ATPase (SERCA), integral to the sarcoplasmic reticulum  or endoplasmic reticulum  membrane in most mammalian cells, uses Ca2+ cycling. Discovery of small molecules that target specific transporters, and their interaction with modulatory proteins, is needed to exert improved control of Ca2+ homeostasis for positive therapeutic outcomes. Here, we seek primarily SERCA2a activators to alleviate HF and related arrhythmias, a goal supported by numerous reports13, although some controversy remains14. Compounds that uncouple ATPase and transport activities of SERCA are of interest as enhancers of metabolism or thermogenesis, to reduce obesity or hypothermia16. SERCA inhibitors are proposed for treatment of cancer or malaria18.Current therapeutic measures for HF include beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin-receptor blockers (ARB). However, these do not directly target proteins responsible for dysfunctional Ca21, are coupled to Ca2+ transport. The interaction of small molecules with SERCA can induce structural changes, detectable by this biosensor, that correlate with function, making this a powerful tool for high throughput screening (HTS) discovery of SERCA-binding compounds22.SERCA2a is a large transmembrane protein, with the phosphorylation (P) and nucleotide-binding (N) domains forming the catalytic site, influenced by the actuator (A) domain Fig. A. Large intermolecular FRET biosensor designs24. We have also validated intramolecular FRET biosensor constructs of SERCA25, to detect binding of compounds directly to SERCA. For this, we engineered a “two-color” SERCA , we previously validated this biosensor using the NCC (727 compounds)22 and LOPAC (1280 compounds)25 libraries. The next logical step, in the present study, is to use 2CS in HTS of a 50,000-compound DIVERSet library, a diverse collection of drug-like small molecules that has yielded effective hit compounds in other drug discovery projects29. HTS was enabled by the FLT-PR (fluorescence lifetime plate reader), which scans a 1536-well plate with unprecedented precision and speed, determining FLT with ~ 0.3% CV  in 2.5 min28, making possible a high-precision 50,000-compound screen in 2 days. To remove false positives, a spectral unmixing plate reader (SUPR) was used to provide complementary spectral measurement of compound-induced FRET changes26.In previous early-stage drug discovery campaigns, we focused on the SERCA regulator, PLB, via 26, the GFP/RFP 2CS biosensor has been thoroughly validated for screening the DIVERSet compound library (DIVERSet-CL) using HEK293 cells. To validate selected hit compounds and prioritize those with lead potential, we acquired concentration response curves (CRCs) using FRET and functional assays (Ca2+-ATPase activity and Ca2+-uptake). We hypothesized that the combination of improved fluorescence technology and screening a larger library of compounds would yield a larger and more diverse collection of hit compounds that improve cardiac SERCA function, thus increasing the potential for discovering lead compounds for new heart failure therapeutics.Although other biosensors are under development 25 was incubated with compounds or DMSO (control) for FLT acquisition in the FLT-PR. FLT measurements had a median coefficient of variation (CV) of 0.4% across all plates . FLT changes induced by potential hit compounds . 295 compounds remained.Compounds that significantly altered the structure of 2CS were determined from their change in lifetime (Δτ) vs DMSO controls (2CS plus DMSO), and Δτ was compared to the normal statistical fluctuation of the biosensor by computing the robust (r)ved Fig. B, step 231, (b) increasers offer greater reproducibility26, and (c) most previously identified SERCA modulators have been shown to be FLT increasers25. Therefore, we prioritized 160 FLT increasers  more FLT decreasers were found to fail these tests25, to rule out compounds that directly bind to the fluorescent proteins and alter FLT. ΔFLT (Δτ) from the FLT-PR and Δ(G/R)  samples22) from the SUPR were determined for each compound. As these are two complementary measures of FRET (FRET decreases both), a strong correlation was observed in 2CS for compounds that induced a structural change much greater than observed in the null-biosensor  with measurable FRET-EC50  vesicles enriched for SERCA2a23 , [Ca2+]MID , and [Ca2+]BAS . For the Ca2+-ATPase activities, the values at [Ca2+]BAS were < 10% of those at [Ca2+]MAX and < 25% of those at [Ca2+]MID, whereas for Ca2+-uptake rates, values at [Ca2+]BAS were negligible. Functional data acquired at [Ca2+]MAX and [Ca2+]MID were corrected by subtracting the appropriate basal rate at pCa 8.0 and the % effect due to the compound was fitted to the Hill’s function  and inhibitors (Compounds 10–18)  and functional efficacy . Under ideal conditions, a maximum coupling ratio (CR) of 2 Ca2+ transported per molecule of ATP hydrolyzed has been reported35. CR defines the efficiency of Ca2+ uptake by SERCA, which was determined from the ratio of the measured maximal Ca2+-uptake rate (VU) at [Ca2+]MAX, to the maximal Ca2+-ATPase activity (VA) at [Ca2+]MAX (VU/VA) (Table SERCA2a Activators). CR was also used to classify and prioritize compounds. Activators were compounds that increased Ca2+-ATPase activities and/or Ca2+-uptake (at one or both [Ca2+]) (Table 2+-ATPase activity and Ca2+-uptake to increase CR (Compound 7), (2) increases Ca2+-ATPase activity more than Ca2+-uptake to decrease CR , and (3) increases Ca2+-ATPase activity but inhibits (induces divergent effects on) Ca2+-uptake . We define “divergent” to indicate that the compound induces opposing effects at two different [Ca2+] (an increase at one [Ca2+] and a decrease at the other) in one assay.To assess the impact of hit compounds on SERCA2a function, we used an absorbance-based Caa23 Fig. B, step 5ion Fig.  to yield2+-ATPase activities and Ca2+-uptake rates , 2) moderate inhibition of Ca2+-ATPase activities and strong inhibition of Ca2+ -uptake (Compounds 15 and 17), 3) mild inhibition of Ca2+-ATPase activities and moderate-to-strong inhibition of Ca2+-uptake (Compounds 14 and 16), and 4) mild inhibition on Ca2+-ATPase activities and mild-to-moderate inhibition on Ca2+-uptake (Compounds 10 and 18) (Table SERCA2a Inhibitors).Inhibitors induced strong (≥ 68%), moderate 34 to 67%), and mild (≤ 33%) inhibition of SERCA2a and were defined in four subcategories: 1) strong inhibition of Ca%, and mi36 scores above 0.4 were binned as clusters, while those with scores below 0.4 were classified as singletons. The analysis yielded diverse scaffolds37 of hit compounds 1, 2, and 3) have a common 5-(aryloxymethyl)oxazole-3-carboxamide)38; those in cluster B (4 and 5) share a N-heteroaryl-N-alkylpiperazine. Cluster C (9 and 10) is defined by an amide linkage and cluster D  by a piperidine scaffold. Clusters E-L  contain a singleton with no common scaffold with any other hit compound in this study. All hit compounds have physicochemical properties39 that are conducive of favorable drug disposition in vivo, including low molecular weight (< 500), low cLogP , low non-H rotatable bonds that describe the molecular flexibility (< 10), low probability of hydrogen bond formation , and low total polar surface area (tPSA < 140 Å) (Supplementary Table Four clusters of compounds (A–D in Table 1–9) induced varying effects on SERCA2a function  decreased FRET of 2CS in live cells with FRET-EC50 = 0.3 μM, suggesting stabilization of an open conformation of SERCA2a. It accelerated Ca2+-ATPase activity at both [Ca2+] to induce ΔFMAX = 14% and ΔFMID = 7%  and ΔFMID (19%) , Compound 8 (singleton G)  , and were ~ 2 × greater than the FRET-EC50 (4.9 μM). C10 was 4.3 M ([Ca2+]MAX) and 7.8 μM ([Ca2+]MID), indicating significant ATPase activation at low dosage. C10 for uptake at [Ca2+]MAX was 25 μM and was not determined at [Ca2+]MID because Compound 8 did not induce changes higher than 10%.In the second category of activators (Compounds  G) Fig. A decreas2 (cluster A) induced similar Ca2+-ATPase activation  as Compound 7, with slightly smaller increases in Ca2+-uptake (Table 4 (cluster B) increased Ca2+-ATPase activities at both [Ca2+]MAX and [Ca2+]MID by ~ 10%, and increased Ca2+-uptake at both [Ca2+]MAX (0.5%) and [Ca2+]MID (11%). Compound 9 (cluster C) increased ΔFMAX (21%) and ΔFMID (10%) for Ca2+-ATPase activity, though with smaller increases for Ca2+-uptake (3–5%). Thus, Compounds 4 and 9 decreased CR similarly . Thus these four compounds join Compound 7 as promising future lead compounds , 5 (cluster B), and 6 (singleton E) increased Ca2+-ATPase activities at both [Ca2+]MAX and [Ca2+]MID, but induced divergent (inhibitory) effects on Ca2+-uptake at [Ca2+]MAX, decreasing CR (0.39—0.48). In cardiomyocytes, this decrease in CR would likely result in a decrease in SR Ca2+-uptake, while increasing ATP hydrolysis. Compound 6  , which can disrupt Ca2+ homeostasis41. C10 was similar at both [Ca2+] (9.7 μM and 8.7 μM), but was significantly different for Ca2+-uptake , decreasing CR to 0.48.In the third category, Compounds 5 increased Ca2+-ATPase activity moderately at [Ca2+]MAX (25%) and [Ca2+]MID (35%). Ca2+-uptake was inhibited slightly at [Ca2+]MAX (2%), but activated at [Ca2+]MID (20%) , but they inhibited Ca2+-uptake at [Ca2+]MAX (10% and 20%) (Table 1 and 5 induced similar decreases in the CR (to 0.49 and 0.47), while Compound 3 induced a slightly smaller CR of 0.39 (Table 42.Compound %) Table . Compoun2, 7, 8, and 9 induced Ca2+-dependent activation of the Ca2+-ATPase activity over the range of [compound] studied. Compounds 1, 4, and 6 showed similar activation of Ca2+-ATPase activity at the two [Ca2+] measured, while Compounds 3 and 5 showed higher activation at [Ca2+]MID compared to [Ca2+]MAX.Compounds 18. Compounds 10–18 decreased Ca2+-ATPase activities and Ca2+-transport at [Ca2+]MAX and [Ca2+]MID to varying extents; strong (S), moderate (M), or mild and are in four subcategories , most of the inhibitors (Compounds 10–18) showed weaker affinity, with FRET-EC50 values in the range of 5–32 μM, but the maximum inhibitory effects (efficacies) of the inhibitors tended to be greater  showed similar inhibition of both SERCA2a functions, except that, compared with the Ca2+-ATPase activity, Ca2+-uptake inhibition at [Ca2+]MID required slightly higher [compound], as shown by the right-shift of the red curve  than Compound 12  and 17 (singleton K) induced moderate inhibition of both activities, decreasing ΔFMAX and ΔFMID by ~ 50% for Ca2+-ATPase activity and slightly more for Ca2+-uptake (70–95%) (Table 14 (singleton H) and 16 (singleton J) induced mild inhibition of Ca2+-ATPase activity, but a moderate-to-strong inhibition of the Ca2+-uptake  and 18 (singleton L) induced mild inhibition of Ca2+-ATPase and mild-to-moderate inhibition of Ca2+-uptake  Compounds rve Fig. D. Compouake Fig. D, E to l Compound2+] . This decrease in FRET implies that the actuator (A) and nucleotide-binding domains (N) of SERCA2a moved apart, supporting an open configuration at low [Ca2+] in HEK cells, possibly priming SERCA in a more open state to bind Ca2+. Previous studies with a different SERCA2a biosensor indicated that the addition of Ca2+ induces a decrease in FRET21. Our functional assays at high and mid [Ca2+] show activating, uncoupling, and inhibiting effects that may correlate with structural changes. Further future elucidation of the compounds’ effects on SERCA2a conformational states will require detailed analysis of FLT-detected FRET and transient kinetics data. We identified three categories of activators that (1) increase both Ca2+-ATPase activity and Ca2+-transport to increase CR   for activators  than for inhibitors  , low nM [Ca2+] in live HEK cells43 vs. μM [Ca2+] in the pCSR in vitro assays), which measure different properties (structure vs function). Functional CRC assays showed that inhibitors tend to induce larger changes (indicating higher efficacy) than activators, in both Ca2+-ATPase activity and Ca2+-uptake. Most inhibitors induced a larger change in Ca2+-uptake than in Ca2+-ATPase activity, decreasing CR.Most FRET-EC2+-ATPase activity than in Ca2+-uptake. A notable exception is Compound 7, which increases Ca2+-transport even more than it increases Ca2+-ATPase activity, increasing CR. This compound will be a high priority as a lead compound for future efforts in medicinal chemistry and assays of physiological function. Compounds 2, 4, 8, and 9 will have only slightly lower priority.Most of the activators reduced CR, inducing larger changes in Ca46. Only Compound 7 (singleton F) induced higher activation in Ca2+ transport than in the Ca2+-ATPase activity. Compounds 2 (cluster A), 4 (cluster B), 8 (singleton G), and 9 (cluster C) induced similar effects of moderate activation of Ca2+-ATPase activity, with smaller activation of Ca2+-transport. Compounds in activator clusters A (Compounds 1 and 3) and B (Compound 5) along with Compound 6 from singleton E, showed similar functional effects: moderate ATPase activation with mild inhibition of Ca2+-transport at [Ca2+]MAX. Inhibitor compounds in clusters D , C (10), and H–L (14–18) induced a range of effects at both [Ca2+], which will be useful in designing derivatives for structure activity relationship (SAR) analysis.Ten compounds were binned into four clusters (A–D); eight were singletons (E–L) Table . Many co27. There was 81% overlap in the fluorescent compounds detected (and thus rejected) in these two HTS studies, indicating that our FRET-HTS methodologies are effective and versatile29. In our previous study of the DIVERSet-CL, using the SERCA2a Ca2+-ATPase activity as the primary HTS assay (ATPase-HTS), we discovered 19 activators30. While no identical activators were found in that ATPase-HTS study30 and in the current FRET-HTS study, there were several compounds with similar scaffolds that showed similar functional results. These compounds share the oxadiazol scaffold and activated the Ca2+-ATPase activity but inhibited Ca2+-uptake. Another common scaffold is the amide group; six compounds30 identified with an amide induced a smaller increase in the Ca2+-transport than in the Ca2+-ATPase activity, reducing CR similar to Compound 9 in this study. It is not surprising that the two studies did not identify the same compounds, because (a) the FRET-HTS assay was performed with human cardiac 2CS in live HEK cells in low [Ca2+], while the ATPase-HTS assay was done in purified SR from rabbit skeletal muscle (SERCA1a) under high [Ca2+]30, (b) the FRET assay is much more precise than the functional assay, (c) the relationship between SERCA structure and function is complex, and (d) the binding sites on SERCA2a for these compounds are unknown. As discussed above, a ligand-binding site may recognize several different scaffolds46. It is also possible that a compound binds to PLB or competes with PLB for binding to SERCA, thus increasing SERCA2a activity, as was shown for the activator, istaroxime48. These observations highlight the value of complementary HTS assays for the same target.There was negligible overlap in hit compounds identified in our previous FRET-HTS of the DIVERSet-CL targeting tumor necrosis factor receptor 12+-transport by SERCA2a is needed when cardiac relaxation is impaired, as in diastolic dysfunction1 or diabetic cardiomyopathy49. SERCA2a activation is a promising strategy, in combination with current drugs such as β-blockers and ACE inhibitors50. Activation of SERCA also has therapeutic potential for Alzheimer’s disease51 or Duchenne muscular dystrophy (DMD)52. Until recently, very few compounds were known to stimulate SERCA2a: CDN1163 (stimulates Ca2+ transport)53, CP-154526 (increases the apparent Ca2+ affinity of SERC2a)54, Ro 41–0960 54, and istaroxime (stimulates SERCA2a activity)55. However, our recent ATPase-HTS assay identified ~ 19 new activators of SERCA30, and we identified nine in the present study. A SERCA activator from our previous work (CDN1163) shows promise as a therapeutic agent for Alzheimer’s disease51 and for DMD52. Of all these SERCA2a activators, only istaroxime has been in phase IIb clinical trials for treatment of heart failure56. However, because of its unsuitability for human usage56, istaroxime must be modified48.Activation of Ca1, 3, 5, and 6 induced small effects on the Ca2+-ATPase activity (~ 10–25% increase) and induced a negative effect on the Ca2+-transport , where SLN reduces Ca2+-transport without affecting the Ca2+-ATPase activity (SERCA1a uncoupling), thus reducing CR15. Uncoupling of SERCA1a leads to higher usage of ATP, which enhances non-shivering thermogenesis (NST)15. Another contributor to NST is Ca2+ leak from SR through resting RyR channels, stimulating SERCA to re-sequester Ca2+ into SR, thus using more ATP and generating heat58, which has been suggested as a potential therapeutic strategy for reducing obesity57.Compounds ort Fig. , thus de17. Similarly, our discovery of new SERCA inhibitors with a range of potencies and efficacies is likely to be advantageous for non-cardiac applications18.Six decades of research for SERCA inhibitors as oncology therapeutics have yielded hundreds of SERCA inhibitors with varying potencies and efficaciesa potential lead compound (Compound 7) that activates Ca2+-uptake more than the Ca2+-ATPase activity, increasing the CR, so this will be a high priority for future efforts in medicinal chemistry and assays of physiological function, along with four other promising SERCA2a activators. The innovative technology included two novel plate-readers – the FLT instrument used in the primary screen, and a spectral instrument – that were used to remove compounds with interfering fluorescence signals, allowing us to focus on valid SERCA activators and inhibitors. It is possible that some of the eliminated fluorescent compounds have potential as SERCA2a effectors, which could be evaluated in future work using our ATPase activity assay30. In future studies, we will evaluate these hit compounds in more functional detail, including the full range of [Ca2+], SERCA isoforms, Na+/Ca2+ exchanger, RyR, and L-type Ca2+-channels. Medicinal chemistry will be done to elucidate SAR and to design analogs with greater potency and specificity59, justifying studies in intact muscles and animals. Finally, we have shown that our primary screening technology can perform precise HTS on several thousand compounds per hour, making this approach capable of application on an industrial scale, screening millions of compounds.Here we successfully used the 2CS biosensor to identify novel small-molecule effectors of SERCA with diverse chemical scaffolds, resulting in an array of activator and inhibitor hit compounds. Most importantly, based on the amplitude of the functional effects on SERCA2a, we discovered 25. Briefly, tagRFP was genetically fused to the N-terminus of SERCA (A-domain) and eGFP was inserted as an intrasequence tag before residue 509 in the nucleotide-binding domain (N-domain)61. Donor-only and acceptor-only (1CS) biosensors were created in a similar manner as the 2CS biosensor but with the construct containing either only eGFP or only tag-RFP, respectively. The fluorescent proteins fused to SERCA in 2CS and 1CS do not significantly affect SERCA activity, in membranes purified from HEK cells25. A null-biosensor construct consisting of eGFP and tagRFP connected by a 32-residue unstructured flexible linker peptide (G32R) was created as described previously25. All constructs were cloned into expression vectors containing the genes for antibiotic resistance to G418, puromycin, or blasticidin.A two-color intramolecular human SERCA2a (2CS) biosensor, based on human cardiac SERCA2a fused to green fluorescent protein (eGFP) and red fluorescent protein (tagRFP) was developed to detect structural changes that are related to the functional changes of SERCA25. Briefly, cells were transiently transfected with 2CS, 1CS, or G32R null-biosensor plasmids using Lipofecatime 3000 or 293fectin (Thermo Fisher Scientific). Flow cytometry was used to select and enrich for the population of cells expressing respective biosensors. Stable HEK293 cell lines were maintained in phenol red-free DMEM media  supplemented with 2 mM GlutaMAX , 10% fetal bovine serum (FBS) , 1 IU/mL penicillin/streptomycin , and 250 μg/mL G418 (Fisher Scientific). Stable HEK293-6E cell lines were maintained in F17 media (Sigma Aldrich) supplemented with Kolliphor p188 , 200 nM/mL GlutaMAX, and either 1 μg/mL puromycin  or 2 μg/mL blasticidin (Goldbio) as a selection antibiotic. All cell lines were grown at 37 °C with 5% CO2.Stable cell lines were generated using either human embryonic kidney (HEK) HEK293  or HEK293-6E  cellsA 50,000 DIVERSet-CL was purchased from ChemBridge Corporation  at a 10 mM stock concentration for each compound. All compounds met the high quality standard of 100% identification by NMR and/or LC–MS and have a minimum purity of 85% and their identity verified using LC–MS/ELSD as confirmed by the ChemBridge Corporation. For the FLT HTS initial screens, the compound library was reformatted into 384 well Echo compatible plates using the Biomek FX  and 5 nL of either compound (columns 3–22 and 27–46) or DMSO  was dispensed into forty 1536 well black polystyrene assay plates  using an Echo 550 liquid dispenser (Beckman Coulter) to yield a final assay screening concentration of 10 μM. The low autofluorescence and low interwell cross-talk of these plates made them advantageous for FLT measurements. Plates were heat sealed with a PlateLoc Thermal Microplate Sealer  and stored at − 20 °C prior to use. The same methods were applied for subsequent FLT retesting of the hit compounds identified in the FLT screen  with repurchased compounds were made in a similar manner using with the Echo 550 (Beckman Coulter) using either 384 well transparent plates (Greiner Bio-One) or black-walled plates with transparent bottoms (Greiner Bio-One), respectively.FRET CRC assay plates (0.78–100 μM compound range) with at least ten different compound concentrations were made by adding the appropriate volume of compound or DMSO into black 384 well plates (Greiner Bio-One) using a Mosquito HV . Subsequent Ca g for 5 min. Cells were filtered using a 70 µm cell strainer and diluted to 1–2 × 106 cells/mL. Cell concentration and viability were assessed using the Cell countess (Invitrogen) and trypan blue assay. During assays, cells were constantly and gently stirred using a magnetic stir bar at room temperature, keeping the cells in suspension and evenly distributed to avoid clumping. HEK 293-cells expressing 2CS were dispensed at 5 μL or 50 μL per well into assay plates  pre-plated with compounds  using a Multdrop Combi liquid dispenser  and sealed until needed. Because the kinetics of membrane permeability, diffusion, and/or binding of the compound to live cells may be compound-dependent, we tested two incubation times, 20 min and 120 min, for the FRET CRC. FRET EC50 values determined from both incubations were similar, but the 120 min incubation yielded a more reproducible and sigmoidal curve. Plates containing eight-point concentration curves of three tool compounds 22 were used as positive controls for biosensor function and performance prior to running the full-scale FRET-HTS assay.On each day of screening, cells were harvested, washed three times with PBS, and centrifuged at 30023.The primary FLT-FRET-HTS assay was performed over two days with a custom HTS fluorescence lifetime plate reader (FLT-PR) with donor emission detected at 517 nm  was fit by a convolution of the measured instrument response function (IRF) (Eq. 62 in the absence (τD) and presence (τDA) of the acceptor as described in Eq. (A detailed description of the high-throughput fluorescence lifetime plate reader (FLT-PR) and spectral unmixing plate reader (SUPR), manufactured by Fluorescence Innovations Inc and provided by Photonic Pharma, LLC was described previouslyIRF) Eq.  and a sid in Eq. :1a\documE) was determined as the fractional decrease of donor FLT in the absence and in the presence of acceptor as in Eq.  was fit by least-squares minimization of a linear combination of component spectra for donor (G for green), acceptor (R for red), cellular autofluorescence (C) and water Raman (W), as described previously22. The change in ratio of the mole fractions of the G and R component spectra between compound and DMSO control (Δ(G/R)) provides a direct indication of a change in FRET due to biosensor structural changes, independent of the lifetime measurements. Together, these complementary metrics provide an effective method for eliminating false positives arising in either method.FLT-PR data was used as the primary metric for flagging potential hit compounds. After fitting waveforms with a single exponential decay to quantify donor lifetime, the change in fluorescence lifetime due to compound (Δτ) was computed by performing a moving median subtraction in the order the plate was scanned, with a window size of 24 wells, rather than subtracting DMSO controls. The reasons for this are twofold: 1) plate gradients are often observed due to heating of the digitizer during acquisition and 2) performing Δτ computations with DMSO controls can sometimes result in artifacts as a half of the DMSO wells are on the edge of plates, which occasionally exhibit artifacts due to processes needed for the preparation of the compound library being tested. As most compounds are likely to be non-hits, and therefore DMSO like, computation of a moving median is an effective alternative to solving both gradient issues and edge-effect distortion of the primary metric for hit selection, Δτ.63 – yielded a value of 0.6225. A value of 0.5 ≤ Z' < 1 indicates excellent assay quality, ready for large-scale HTS63 as we recently showed for an ATP-based HTS assay30. Data from the control (tool) compounds was not needed for assessment of assay quality in the full-scale HTS in this study, instead we used the coefficient of variance (CV), computed using wells containing only DMSO, to assess the quality of the plates, in order to separate within-plate and between-day variability, as in our previous ATP-based HTS study30.Previous validation in 1536-well plates indicated that the Z' parameter – a measure of HTS assay quality that factors in the signal window and the variance of positive and negative controls 30. Given the increased precision , where the median (M) and median absolute deviation (MAD) are used in place of the mean and standard deviation  of the donor-only spectrum (I(a)) for each well to that of the plate-wide average DMSO spectrum (I(b)) in the same wavelength band as described in Eq. 26. Compounds that reproducibly exceeded an SI rZ-score of 5 (corresponding to an SI of 2 × 10–4) were deemed likely fluorescent compounds and were removed from consideration.To remove interfering fluorescent compounds, the similarity index (SI)ion 500–50 nm of t25. The hit threshold was also set using an rZ-score of ± 3. While the FLT-PR data and SUPR data showed strong correlation, the FLT-PR data exhibited some clear outliers, presumably due to compounds directly modifying the donor lifetime. To eliminate these likely interfering compounds, correlation was enforced by eliminating compounds that exceed an rZ-score of ± 3 from the median value of the ratio of Δτ over the Δ(G/R) metric.Spectral (SUPR) data was processed similarly to FLT-PR data, with the Δ(G/R) metric being computed by applying the same moving median filter on the initial measurement of the ratio of donor (G) to acceptor (R) mole fractions (G/R)20. The SR vesicles were flash-frozen and stored at − 80 °C until needed. The SERCA concentration in the ER preparations purified from HEK cell homogenate is at least 10 times less than in purified porcine cardiac SR20, but there was sufficient expression of the fluorescent SERCA2a biosensor in HEK cells to detect FRET with high precision by FLT26.Cardiac SR vesicles were isolated from fresh porcine left ventricular tissue using differential centrifugation of the homogenized tissue as previously described20. An enzyme-coupled, NADH-linked ATPase assay was used to measure SERCA ATPase activity in 384-well microplates. Each well contained 50 mM MOPS (pH 7.0), 100 mM KCl, 1 mM EGTA, 0.2 mM NADH, 1 mM phosphoenol pyruvate, 10 IU/mL of pyruvate kinase, 10 IU/mL of lactate dehydrogenase, 7 µM of the calcium ionophore A23187 (Sigma), and CaCl2 was added to set free [Ca2+] to three different concentrations23. Ca2+-ATPase activity was measured at three [Ca2+]: [Ca2+]MAX , [Ca2+]MID , and [Ca2+]BAS . 10 μg/mL of SR vesicle, calcium, compound (0.048 to 50 μM), and assay mix were incubated for 20 min at room temperature before measurement of functional assays with each of the 18 hit compounds, because a shorter incubation time than the FRET live-cell assays achieved optimal responses. The assay was started upon the addition of MgATP, at a final concentration of 5 mM , and absorbance was read at 340 nm in a SpectraMax Plus384 microplate spectrophotometer .Functional assays were performed using porcine cardiac SR (pCSR) vesicles2+-transport assays were performed with similar porcine SR samples as in the Ca2+-ATPase assays described above. The compound effect on the Ca2+-transport activity of SERCA2a was determined using an oxalate-supported assay in which the change in fluorescence in a Ca2+-sensitive dye, Fluor-4, was determined as previously described23. A buffered solution containing 50 mM MOPS (pH 7.0), 100 mM KCl, 30 mg/mL sucrose, 1 mM EGTA, 10 mM potassium oxalate, 2 M Fluo-4, 30 µg/mL porcine cardiac SR vesicles, CaCl2 calculated to reach the free [Ca2+] , and compound (0.048 to 50 μM) was dispensed into 384-well black walled, transparent bottomed plates (Greiner Bio-One) containing the tested small molecule and incubated at 22℃ for 20 min while covered and protected from light. To start the reaction, MgATP was added to a final concentration of 5 mM, and the decrease in 485-nm excited fluorescence of Fluo-4 was monitored at 520 nm for 15 min using a FLIPR Tetra .CaE)  in the presence of acceptor  due to FRET. E was plotted as “FRET Effect (E/EDMSO)” vs [compound], fitted to the Hill’s function for determination of FRET-EC5031. This normalization of E corrects for variation of controls done on different days.FRET efficiency (E) Eq.  was dete2+ uptake), was measured at varying pCa and varying compound concentration. F measured at [Ca2+]MAX  or [Ca2+]MID  was corrected by subtracting the basal rate at pCa 8.0) and the % effect due to compound was reported. Concentration response curves (CRC) were fitted using the Hill function to determine VMAX (the activity at saturating [compound]), and EC50, the compound concentration at 50% effect54. When the CRC did not achieve saturation, the maximal change (Δ) in activity was determined, to yield ΔFMAX and ΔFMID at the [Ca2+]MAX and [Ca2+]MID conditions, respectively , and EC50 are reported in Table SERCA2a activity, F  using binning, multidimensional scaling (MDS), and compound similarity methods where the Tanimoto coefficient36 and maximum common substructure36 values were used to determine clustering  using the data analysis program Microsoft Excel . Data are presented as mean ± SEM calculated from a minimum of three separate experiments (n = 3).Supplementary Information.


Respiratory infectious diseases (RIDs) remain a pressing public health concern, posing a significant threat to the well-being and lives of individuals. This study delves into the incidence of seven primary RIDs during the period 2017–2021, aiming to gain deeper insights into their epidemiological characteristics for the purpose of enhancing control and prevention strategies.Data pertaining to seven notifiable RIDs, namely, seasonal influenza, pulmonary tuberculosis (PTB), mumps, scarlet fever, pertussis, rubella and measles, in the mainland of China between 2017 and 2021 were obtained from the National Notifiable Disease Reporting System (NNDRS). Joinpoint regression software was utilized to analyze temporal trends, while SaTScan software with a Poisson probability model was used to assess seasonal and spatial patterns.P = 0.009; APC = −40.87%, P = 0.02), while the other five RIDs peaked in 2019. Concerning seasonal and spatial distribution, the seven RIDs displayed distinct characteristics, with variations observed for the same RIDs across different regions. The proportion of laboratory-confirmed cases fluctuated among the seven RIDs from 2017 to 2021, with measles and rubella exhibiting higher proportions and mumps and scarlet fever showing lower proportions.A total of 11,963,886 cases of the seven RIDs were reported during 2017–2021, and yielding a five-year average incidence rate of 170.73 per 100,000 individuals. Among these RIDs, seasonal influenza exhibited the highest average incidence rate , followed by PTB , mumps , scarlet fever , pertussis , rubella , and measles . Males experienced higher incidence rates across all seven RIDs. PTB incidence was notably elevated among farmers and individuals aged over 65, whereas the other RIDs primarily affected children and students under 15 years of age. The incidences of PTB and measles exhibited a declining trend from 2017 to 2021 (APC = −7.53%, The incidence of PTB and measles demonstrated a decrease in the mainland of China between 2017 and 2021, while the remaining five RIDs reached a peak in 2019. Overall, RIDs continue to pose a significant public health challenge. Urgent action is required to bolster capacity-building efforts and enhance control and prevention strategies for RIDs, taking into account regional disparities and epidemiological nuances. With the rapid advancement of high-tech solutions, the development and effective implementation of a digital/intelligent RIDs control and prevention system are imperative to facilitate precise surveillance, early warnings, and swift responses.The online version contains supplementary material available at 10.1186/s40249-023-01147-3. Given the distinct transmission routes and modes of infection associated with respiratory infectious diseases (RIDs), many emerging and re-emerging RIDs have the potential to spread rapidly among susceptible populations worldwide. These RIDs persist as a significant public health challenge, imposing a global disease burden and jeopardizing individuals’ health and well-being , 2. In tWhile several studies have explored the epidemiological features of various RIDs across different time frames and geographical locations in China –14, the This study draws upon surveillance data for seven RIDs in the mainland of China spanning the years 2017 to 2021, sourced from the NNDRS. Among these RIDs, PTB, pertussis, measles, and scarlet fever are classified as category B infectious diseases, while seasonal influenza, mumps, and rubella fall under category C infectious diseases. The NNDRS is an internet-based real-time disease-reporting system that encompasses various healthcare facilities  across the mainland of China, boasting coverage of 55,077 health facilities in 31 provincial-level administrative divisions (PLADs) , 16. Thehttp://www.stats.gov.cn/english/Statisticaldata/AnnualData, accessed on April 20, 2023) [http://bzdt.ch.mnr.gov.cn/, accessed on April 20, 2023) under the Ministry of Natural Resources of the People's Republic of China.Demographic data by age and sex for 31 PLADs and the country were culled from the National Bureau of Statistics of China  . The staIncidence rates for both national and provincial levels were calculated on a monthly and annual basis, along with stratifications by sex and age groups. Visual representations in the form of bar graphs and scatter line plots were employed to depict trends in cases and incidence rates. Stacked plots and heat maps elucidated trends in occupation proportions and laboratory-confirmed cases, respectively. Seasonal attributes were visualized via radar charts, generated using SaTScan  outputs. Figures were crafted using R software with the ggplot2 package  and OriginPro , while spatial characteristics were mapped using ArcGIS software .P < 0.05) denotes an increasing trend in incidence rates, whereas an APC < 0 (P < 0.05) signifies a decreasing trend. Conversely, trends lacking significant changes are denoted by APC values falling outside these ranges [ y is incidence rate, x is year, β1 is regression coefficient.Temporal trends were subjected to analysis using Joinpoint regression software  . The defe ranges . The APCLLR) was deemed the most likely cluster, while others were ranked as secondary clusters in a specific sequence [RR) denoted the ratio of estimated risks within and outside the cluster, serving as an indicator of the elevated infection risk faced by individuals residing within the cluster compared to those outside it [The examination of seasonal and spatial characteristics was conducted using the SaTScan software. This software employed a Poisson probability model to identify clusters of RIDs in terms of seasonality, with a temporal window encompassing 50%. The software detected spatial clusters of RIDs in different regions for the years 2017 and 2021, employing the Poisson probability model and a spatial window covering 50% of the study areas . In ordesequence . The contside it .In the context of the seasonal analysis, the 31 PLADs in the mainland of China were stratified into two distinct regions: southern China  and northern China . This classification was based on meteorological attributes .A total of 11,963,886 cases of seven RIDs were reported in the mainland of China between 2017 and 2021. Of these, 7,941,892 (66.38%) were clinically diagnosed cases, while 4,021,994 (33.62%) were laboratory-confirmed cases. The five-year average incidence rate for all seven RIDs combined was 170.73 cases per 100,000 population. The highest incidence rate was recorded in 2019, reaching 343.18 cases per 100,000  , while clinically diagnosed cases decreased , though neither trend proved statistically significant , with clinically diagnosed rates also decreasing significantly  , and similar patterns emerged for laboratory-confirmed rates  and clinically diagnosed rates     Table .Table 2TRR = 3.68, P < 0.001) in 2017 and spanning 10 PLADs  in 2021 . In 2021, four clusters spread across 15 PLADs, with the most likely cluster encompassing 10 PLADs  (Table P = 0.009), mirroring the national pattern  in 2017, while two clusters covered 14 PLADs in 2021 with the most likely cluster encompassing 13 PLADs  (Table P = 0.004), Guizhou , Chongqing , Shaanxi , and Ningxia   in 2017 and six PLADs  in 2021 . In 2021, two clusters spanned seven PLADs, with the most likely cluster covering six PLADs  (Table P = 0.02) . In 2021, four clusters covered six PLADs, with the most likely cluster in Qinghai   in 2017 and five PLADs  in 2021 (Table P < 0.001), consistent with the national trend  and measles  decreased, while the remaining RIDs showed no significant trends from 2017 to 2021 (Table The five-year average incidence rate of laboratory-confirmed cases for all seven RIDs combined was 35.64/100,000, peaking at 96.70/100,000 in 2019 (Fig. The proportion of laboratory-confirmed cases among all seven RIDs increased from 26.33% in 2017 to 48.21% in 2021, with a peak of 48.96% in 2020 (Fig. This study primarily aimed to uncover the demographic, temporal, seasonal, and spatial distribution patterns of seven RIDs using recent national surveillance data. Additionally, we conducted a more comprehensive analysis of RIDs diagnosis categories, gender distribution, and occupation distribution, aspects that were not covered in our previous study . The resThis study underscores that the incidences of seasonal influenza, PTB, mumps, and scarlet fever remain substantial, while pertussis, rubella, and measles have reached lower levels. Findings from our study align with previous research on various aspects of RIDs characteristics in different areas and time periods –16, 20. A staggering 12 million reported cases of seven RIDs were documented in the mainland of China between 2017 and 2021. Among these, the incidences of all seven RIDs were notably higher in males than females, particularly for PTB, mumps, and scarlet fever, findings consistent with several reports , 20, 23.Significantly, our study highlights a decrease in the incidences of PTB and measles across the Chinese mainland, a trend supported by previous reports , 20, 27.The evolving healthcare landscape, marked by enhanced diagnostic capabilities and surveillance systems, particularly in diagnostic proficiency, has contributed to improved laboratory diagnostics in the mainland of China , 29, 30.In summary, infectious diseases, particularly RIDs, remain critical public health concerns. Effective control and prevention strategies are of paramount importance. Leveraging recent national surveillance data from the NNDRS, spanning 31 PLADs and featuring detailed information, this study has provided insights into the epidemiological characteristics of clinically diagnosed and laboratory-confirmed RIDs cases. Notably, while PTB and measles incidence rates have significantly declined, other RIDs have exhibited stable or fluctuating trends over the 2017–2021 period. These findings offer crucial guidance for shaping tailored strategies for RIDs control and prevention. However, the five-year duration of available surveillance data and potential variations in reporting across regions and levels present certain limitations.The incidence rates of seasonal influenza, PTB, mumps, and scarlet fever remain high, whereas pertussis, rubella, and measles have reached lower levels. The reported incidence rates of PTB and measles have experienced significant declines between 2017 and 2021. Peaks in reported incidence rates for seasonal influenza, mumps, scarlet fever, pertussis, and rubella were observed in 2019. Each of the seven RIDs demonstrates distinct epidemiological characteristics. While significant progress has been made in RIDs control and prevention, challenges persist. Urgent measures are needed to strengthen surveillance efforts and develop effective digital/intelligent systems for precise RIDs surveillance, early detection of emerging or re-emerging events, and prompt responses.Additional file 1: Figure S1. The spatial distributions for incidence rates of the seven RIDs in 2017 and 2021.


Individuals diagnosed with Fanconi anemia (FA), an uncommon disorder characterized by chromosomal instability affecting the FA signaling pathway, exhibit heightened vulnerability to the onset of myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML).Herein, we employed diverse bioinformatics and statistical analyses to investigate the potential associations between the expression/mutation patterns of FA pathway genes and MDS/AML.The study included 4295 samples, comprising 3235 AML and 1024 MDS from our and nine other online cohorts. We investigated the distinct proportion of race, age, French-American-British, and gender factors. Compared to the FA wild-type group, we observed a decrease in the expression of FNACD2, FANCI, and RAD51C in the FA mutation group. The FA mutation group exhibited a more favorable clinical overall survival prognosis. We developed a random forest classifier and a decision tree based on FA gene expression for cytogenetic risk assessment. Furthermore, we created an FA-related Nomogram to predict survival rates in AML patients.This investigation facilitates a deeper understanding of the functional links between FA and MDS/AML.The online version contains supplementary material available at 10.1186/s12920-023-01730-5. Fanconi anemia (FA) is a rare autosomal recessive hematological disorder commonly observed in children as a congenital bone marrow failure syndrome. It is closely associated with the FA signaling pathway –5. The FMDS, a clonal disorder originating from hematopoietic stem cells, has a high propensity for transformation into AML –13. AML In this study, we conducted a comprehensive investigation by combining the cohort in our center with the other nine cohorts. Utilizing available gene expression, mutation, and corresponding clinical traits, we analyzed various aspects of MDS/AML patients, including the FA mutation spectrum, expression profile, prognosis evaluation, cytogenetic risk classifier, and nomogram prediction model. This was achieved through various approaches, such as batch correction, principal component analysis (PCA), correlation analysis, nomograms, and the random forest/decision tree.n = 13), TCGA-LAML (n = 137), TARGET-AML (n = 291), BeatAML (n = 650), MDS_mskcc_2020 (n = 858), Papaemmanuil_MDS_2013 (n = 573), Papaemmanuil_NEJM_2016 (n = 1319), LAML-CN (n = 77), LAML-KR (n = 205), and WASHU (n = 136). To display the bar graph of each cohort, we used the “ggbarplot” R package. Additionally, we obtained a hollow pie chart to represent the proportion of MDS and AML using the “ggplot2” and “ggforce” R packages. The SELF_FA cohort specifically included mutation data from 13 FA patients who developed MDS or AML. The mutation data of the other cohorts was obtained from the cBioPortal database [https://ocg.cancer.gov/). The expression and clinical characteristics within the BeatAML cohort were obtained from the supplementary materials of the publication [Our study included 10 cohorts with a total of 4295 samples. The cohorts were named as follows: SELF_FA  among AML or MDS patients. We presented the findings using the “ggbarplot” function from the “ggpubr” R package.“barplot” function.The specific mutation sites of FA pathway genes were illustrated in the protein structure of patients with AML or MDS. The mutation number and percentage of FA pathway genes in the patients of AML and MDS were visualized using a “ggplot2” R package. Additionally, we utilized the “pheatmap” R package to display the relationship between the mutation, expression, and related clinical traits of the FA pathway gene. FLT3, FA, CEBPA, IDH1, NPM1, and specific FA pathway gene mutation rates in AML and MDS patients were further analyzed and presented as a radar diagram by an “fmsb” R package. We also compared the differences in race, age, FAB, gender, type, and cytogenetic risk factors between the FA wild-type and mutation groups. The “plotrix” R package was used to visualize the data. Furthermore, the Wilcox test was performed to analyze the difference in age index, and the “ggbarplot” function was utilized for the visualization. Finally, we analyzed the mutation percentages of IDH1, NPM1, CEBPA, and FLT3 between the FA wild-type and mutation groups, and these results were visualized using the The expression matrices of TCGA-LAML, TARGET-AML, and BeatAML datasets were processed using the R language. Batch correction was performed using the “sva” R package. PCA was conducted using the “prcomp ” function, and the effect before and after the correction was visualized using the “ggplot2” R package. To examine the expression pattern of FA pathway genes in different FAB groups, the “ggballoonplot” function was used.Based on the corrected matrix, the difference in the expression of each FA gene between the wild-type and overall mutant-type of FA pathway genes was analyzed using a Wilcox test and “ggplot2” R package. Also, a Wilcox test was performed to determine the correlation between mutation and expression of specific FA pathway genes, including XRCC2, BRCA1, FANCE, FAAP100, FANCC, and MAD2L2. Visualization was performed using the “ggviolin” function.We performed a Spearman correlation analysis using the “cor.Test” function to explore the links of the FA pathway gene expression with TMB. We visualized the correlation between TMB and expression of each FA gene as a radar diagram using an “fmsb” R package. We displayed the correlation among genes using a “Corrplot” R package as a heat map. Additionally, we utilized the “iGraph” R package to create a network visualization in the form of a tree layout. Finally, we generated scatter plots using the “ggplot2” R package to analyze the correlation between TMB and the hub gene expression within the network, such as BRIP1, FANCE, ERCC4, and MAD2L.We utilized the “survival” and “survminer” packages to evaluate the overall survival (OS) prognosis of patients with MDS + AML, MDS, and AML. We focused on the wild-type and overall mutant FA pathway genes, specifically FANCA. The subgroup survival analyses took into consideration factors such as cytogenetic risk, TMB, FLT3 mutation, and NPM1 mutation. To conduct the OS prognosis analysis, we used the expression matrix of the FA gene and employed the “surv_cutpoint” function to select the optimal cutoff. In addition, we performed receiver operating characteristic curve (ROC) analyses to assess the OS prognosis of TARGET-AML at 1-, 3-, and 5-year survival time points. The area under the ROC curve (AUC) value was calculated.A cph  modeling analysis was conducted on the training set using a “regplot” R package. Subsequently, a Nomogram was generated. The calibration curves were obtained using the calibrate  function of “ggstatsplot”. The “cph”, “validate”, “predict” functions of “rms” were used to yield the C_index along with a 95% confidence interval (CI).Cytogenetic risk was used as the outcome variable in our study. We utilized the “randomforest” R package to conduct the random forest analysis, which provided us with mean decrease Gini and mean decrease accuracy values. We employed the “ggdotchart” function to visualize the results. Further, we performed ROC analysis using the “pROC” R package. For our decision tree analysis, the samples were randomly divided into training and testing sets . We used the “rpart” R package for this analysis and visualized the decision tree using the “rpart Plot “R package.Figure Figure p = 8.4e-15). Analysis of FAB classification revealed that the individuals in the FA Wt group has the highest proportion of M4 subtypes (20.35%), while cases in the FA Mut group were more likely to be M2 carriers (23.44%). In comparison to the FA Wt group, there was a slight rise in the percentage of individuals identified as “male (gender)” . Further, we examined the correlation between specific FA gene expression and mutation. Notably, when XRCC2 was mutated, its expression level decreased , although this correlation was not observed in other FA genes.We first conducted batch calibration of gene expression matrices within BeatAML, TARGET-AML, and TCGA-LAML cohorts. Subsequently, a PCA analysis was conducted to assess the removal of batch differences among these cohorts Fig. a. Furthep = 0.028). To further explore this relationship, we depicted the correlation between TMB value and FA gene expression using a radar diagram .Next, we examine the relationship between FA gene expression/mutation and TMB. The TMB profile of AML and MDS patients who participated in this study is presented in Fig. ram Fig. c. Notablp = 0.031, R = − 0.122), although it was not statistically significant in the other groups.The expression correlation between different FA genes was analyzed using a heat map in Fig. p = 0.002), BRCA2 (p = 0.013), FANCD2 (p = 0.003), FANCE (p = 0.015), FANCI (p = 0.029), BRIP1 (p = 0.004), FANCL (p = 0.005), FANCM (p = 5.46e-04), PALB2 (p = 0.004), RAD51C (p = 0.002), SLX4 (p = 0.002), ERCC4 (p = 0.002), RAD51 (p = 0.004), BRCA1 (p = 0.003), UBE2T (p = 0.014), XRCC2 (p = 0.007), and MAD2L2 (p = 6.67e-04) genes. Conversely, we observed that low expression of FANCG (p = 0.011), RFWD3 (p = 0.043), and FAAP100 (p = 0.01) genes was associated with a poor prognosis.Here, we investigated the association between FA gene expression, mutation, and the clinical prognosis of MDS and AML patients. Our findings, as shown in Fig. Sp = 0.003), MDS (p = 0.023), and AML (p = 0.014) patients. Specifically, it was observed that MDS + AML patients with mutant FANCA also exhibited a better clinical OS prognosis . Although there was no statistical difference, MDS and AML patients with mutant FANCA also showed a tendency towards a better prognosis of clinical OS . However, the intermediate and poor risk groups did not differ significantly , MDS , and AML  patients with high TMB were associated with a poor prognosis of clinical OS. When we combined the FA mutation and TMB factors, the patients with high TMB but without FA mutation showed a poor OS prognosis . However, no statistical difference was detected in the subgroup of FA mutation . Additionally, we observed that MDS + AML  and AML patients  with the FLT3 mutation showed a poor prognosis, compared to the FLT3 wild-type group. Furthermore, we obtained a similar result in the subgroup analysis of AML patients without FA mutation . NPM1 mutations were slightly related to OS risk in patients with AML , but no significant correlation in other analyses  and AML (n = 3235) using various research strategies.There is a link between Fanconi anemia and a variety of tumor types, including AML , 24. TheIn the FAB classification, seven subtypes of AML (M0-M7) have been identified, each with distinct pathogenesis and treatment response , 27. AmoIn addition to FANCA gene mutation, we also detected the mutations in FANCE (R371W), FANCL , FANCM , SLX4 , and FANCD2 (R1236H) in the cases of our center. As evaluated by the KM survival curves, high expression of FANCE was related to a poor OS prognosis, but no statistical link emerged between FANCE mutation and OS. Furthermore, the mutation rate of these genes was not high among the 4259 subjects included in this study, and there were only a few patients with the same specific FA gene mutation site. The most frequent mutation detected was the N991D mutation of the BRCA2 gene, found in four samples. Therefore, we categorized the cases into wild-type and mutant FA groups for further analysis. Due to the low frequency of mutations in the FA gene, we did not filter the mutation sites. We objectively presented the specific mutation sites of the FA gene in the protein structure without classifying the variants as benign or malignant. A preliminary analysis was conducted to evaluate the clinical significance of the mutation sites by utilizing publicly accessible ClinVar databases. Most of the identified variants were categorized as Benign/Likely benign or of uncertain significance. For example, the N991D variant of the BRCA2 gene was identified as benign, while the I917V variant of FANCM was classified as benign/likely benign. Nevertheless, the clinical significance of the R163H variant of the FANCA gene remains undetermined.Interestingly, the FA mutation group exhibited a more favorable clinical overall survival prognosis in patients with MDS/AML, MDS, or AML. This observation could be attributed to the increased sensitivity of tumor cells carrying FA mutations to chemotherapy drugs. Furthermore, a higher TMB was closely associated with the response to immunotherapy in cancer cases, including AML , 31. NotPediatric patients with FA and AML have been reported to benefit from Azacitidine . When suIt is essential to determine the treatment plan for AML patients based on cytogenetic risk . Our finThe limited prevalence of individuals affected by rare diseases and rare mutations contributes to the paucity of available data. Our study has compiled an extensive dataset encompassing more than 4000 patients diagnosed with AML and MDS, explicitly focusing on FA mutations and, notably, the expression patterns of FA genes. Nevertheless, it is crucial to acknowledge that many patients lack information regarding their FA status and prognostic indicators. As a result, the inadequate sample size for specific subgroup analyses and the omission of additional prognostic assessments hindered a comprehensive evaluation of the association between FA expression, mutation, and prognosis of AML-MDS. To achieve a more impartial assessment, expanding the sample size to encompass a wider spectrum of clinical prognosis and survival outcomes is crucial.In summary, this study analyzes data collected from 4259 AML/MDS patients across 10 cohorts. We investigated the mutation spectrum and expression profile of FA signaling pathway genes, and examined the correlation between the FA gene expression/mutations and the survival prognosis of AML/MDS patients. Additionally, a Nomogram has been developed to predict the overall survival rates of AML patients. Moreover, an efficient FA-related random forest/decision tree classifier has been established to assess the cytogenetic risk of AML.Additional file 1:Fig. S1. Mutation feature of FA pathway genes with relatively low mutation frequency in cases of MDS/AML. (a) RAD51; (b) UBE2T; (c) RAD51C; (d) RFWD3; (e) FAAP100; (f) FANCE; (g) FANCC; (h) BRIP1; (i) FANCL. Fig. S2. Heat map data. We employed the “pheatmap” R package to visually represent the correlation between the mutation, expression, and relevant clinical traits of FA pathway genes. Fig. S3. Correlation between FA pathway gene expression and FAB. The expression pattern of FA pathway genes in different FAB groups was visualized using the “ggballoonplot” function. Fig. S4. KM survival curve analysis of FA gene expression. Based on the expression matrix of the FA gene, the R packages of “survival” and “survminer” were used for the overall survival prognosis analysis. The “surv_cutpoint” function selected the optimal cutoff. Fig. S5. Prognosis evaluation of FA mutation and cytogenetic risk. Based on FA mutation and cytogenetic risk factors, we performed the overall survival prognosis analysis of AML patients using the R packages of “survival” and “survminer”. (a) overall AML patients; (b) favorable risk subgroup; (c) intermediate-risk subgroup; (d) poor-risk subgroup. Fig. S6. Prognosis evaluation of FA mutation and TMB. Based on FA mutation and TMB factors, we performed the overall survival prognosis analysis of AML patients, using the R packages of “survival” and “survminer”. (a) MDS + AML; (b) MDS; (c) AML; (d) subgroup analyses of AML patients with or without FA mutation. Fig. S7. Prognosis evaluation of FLT3/FA mutation. Based on FLT3 and FA mutation factors, we performed the overall survival prognosis analysis of AML patients, using the R packages of “survival” and “survminer”. (a) MDS + AML; (b) AML; (c) subgroup analyses of AML patients with or without FA mutation. Fig. S8. Prognosis evaluation of NPM1/FA mutation. Based on the factors of NPM1 mutation and FA mutation, we performed the overall survival prognosis analysis of AML patients, using the R packages of “survival” and “survminer”. (a) AML; (b) MDS + AML; (c) subgroup analyses of AML patients with or without FA mutation.


The previous studies have revealed that abnormal RNA-binding protein Musashi-2 (MSI2) expression is associated with cancer progression through post-transcriptional mechanisms, however mechanistic details of this regulation in acute myeloid leukemia (AML) still remain unclear. Our study aimed to explore the relationship between microRNA-143 (miR-143) and MSI2 and to clarify their clinical significance, biological function and mechanism.Abnormal expression of miR-143 and MSI2 were evaluated in bone marrow samples from AML patients by quantitative real time-PCR. Effects of miR-143 on regulating MSI2 expression were investigated using luciferase reporter assay. Functional roles of MSI2 and miR-143 on AML cell proliferation and migration were determined by CCK-8 assay, colony formation, and transwell assays in vitro and in mouse subcutaneous xenograft and orthotopic transplantation models in vivo. RNA immunoprecipitation, RNA stability measurement and Western blotting were performed to assess the effects of MSI2 on AML.We found that MSI2 was significantly overexpressed in AML and exerted its role of promoting AML cell growth by targeting DLL1 and thereby activating Notch signaling pathway. Moreover, we found that MSI2 bound to Snail1 transcript and inhibited its degradation, which in turn upregulated the expression of matrix metalloproteinases. We also found that MSI2 targeting miR-143 is downregulated in AML. In the AML xenograft mouse model, overexpression of MSI2 recapitulated its leukemia-promoting effects, and overexpression of miR-143 partially attenuated tumor growth and prevented metastasis. Notably, low expression of miR-143, and high expression of MSI2 were associated with poor prognosis in AML patients.Our data demonstrate that MSI2 exerts its malignant properties via DLL1/Notch1 cascade and the Snail1/MMPs axes in AML, and upregulation of miR-143 may be a potential therapeutic approach for AML.The online version contains supplementary material available at 10.1186/s12967-023-04106-6. Acute myeloid leukemia (AML) is a heterogenous hematological malignancy characterized by inhibited differentiation and uncontrolled proliferation of hematopoietic stem/progenitor cells (HSPC). It is the most common leukemia in adults with the 5 year relative survival rate up to 29.5% . While tMusashi (Msi) gene family encodes a group of RNA-binding proteins (RBPs) that post‐transcriptionally regulates mRNA processing by binding to recognition motifs located in the 3’ untranslated regions (UTRs) of target mRNAs [Msi2 mRNA is associated with poor prognosis in AML, as patients with as low as 1% of total bone marrow cells expressing elevated MSI2 levels still have a short survival [et mRNAs . In mammet mRNAs –8, as thet mRNAs –13. Accusurvival , 15. TheExtramedullary AML sometimes involves the central nervous system and other solid organs, which is commonly associated with the former French-American-British (FAB) subtypes of AML-M5 . AccordiThe miRNAs are a class of endogenous small non-coding RNAs that regulate their cognate mRNA transcripts at the translation level, in consequence playing important roles in cellular signaling cascades . MiR-143Here, our mechanistic studies revealed that MSI2 activated the Notch signaling pathway and metastasis-related pathway through post-transcriptional upregulation of DLL1 and Snail1, thereby promoting AML cell proliferation and migration in vitro and accelerating AML progression in vivo. Further experiments validated that miR-143 could directly bind to MSI2 and enforced expression of miR-143 via intratumoral delivery had been shown to reduce the growth of AML xenografts in vivo.Human AML cell lines HL-60, HEL, THP-1 and KG-1α were acquired from ATCC. Primary AML cells were obtained from bone marrow (BM) aspirates of 58 newly diagnosed AML patients and the leukemia blasts were enriched by CD34 Diamond Microbeads . BM aspirates from 14 healthy donors served as negative control. All of these participants were from the First Affiliated Hospital of Wenzhou Medical University between January 1, 2019 and December 31, 2021  and then identified by quantitative polymerase chain reaction (qPCR) analysis. The sequences of siRNAs and miRNAs were shown in Additional file ™ RT Master Mix  . Then, the cDNA was amplified using TB Green®Premix Ex Taq II (Takara) on a 7500 Real-Time PCR System . MiR-143 was determined using Hairpin-it™ microRNA and U6 snRNA Normalization RT-PCR Quantitation Kit (GenePharma). The mRNA levels of protein-coding genes were normalized to GAPDH endogenous control and calculated according to the Pfaffl method [AML cells were transfected with siRNAs or miRNA mimic  using Lipofectaminel method . Primers3/well in 96-well plates for 0, 24, 48, 72, 96 h. Cell viability was detected by Cell Counting Kit-8  assay and the absorbance at 450 nm was read on a Synergy H1 microplate reader . After transfection as described above for 24 h, cells were plated into complete methylcellulose-based medium containing hIL-3, hGM-CSF and hSCF  to assess their clonogenic capacity. Localized clusters of ≥ 50 cells were counted as colonies after 7–14 d. Then, the wells were washed twice with PBS and cells were counted manually.Cells transfected with siRNAs or miRNA mimic were seeded at a density of 5 × 105 cells/well) were suspended in 200 µl serum-free medium and seeded in the top chambers with or without matrigel coated, and 600 µl complete medium containing 20% FBS was added to the bottom chambers as a chemoattractant. After 24 h, cells penetrating into the bottom chamber were captured using a IX53 fluorescence microscope . In matrigel invasion assays, cells in the bottom chambers were fixed, stained with crystal violet and photographed.Transfected AML cells  dual staining assay  on a Navios flow cytometer . Data were analyzed using FlowJo v10 .®, CST), NANOG , OCT4 , SOX2 , SNAIL1 , MMP2 , MMP9 (Clone: (D6O3H) XP®, CST), β-actin  and GAPDH , at 1/1,000 dilution for incubation overnight at 4 °C, respectively. The chemiluminescence was detected using Fluor Chem E  and the quantification of the blots was analyzed with Image J software .At 72 h post-transfection, AML cells were harvested and lysed immediately using RIPA Lysis Buffer  supplemented with protease inhibitor phenylmethanesulfonylfluoride fluoride . Then the protein was determined using antibodies against MSI2 , DLL1 , HES1 , Beverly, MA, USA), cleaved NOTCH1 (Clone: (D1E11) XP7 Lenti-MSI2 vector-transfecting HEL (MSI2-OE. HEL) cells were collected and treated with lysis buffer  containing 1% protease inhibitor and 1% RNase inhibitor. 10% lysis supernatant served as Input, and 45% lysis supernatant was incubated in immunoprecipitation reactions with 5 μg MSI2 antibody bound to magnetic beads (named Anti-MSI2), and 45% lysis supernatant was incubated with 5 μg IgG antibody  bound to magnetic beads as isotype control (named IgG). RNA was extracted and detected by qRT-PCR as described previously [1 × 10eviously .AML cells were treated with actinomycin D  at a final concentration of 5 mg/mL for 0.5, 1, 1.5, 2 or 3 h and harvested for total RNA extraction. Then, the mRNA expression levels of DLL1 and Snail1 were detected using qRT-PCR to analyze their mRNA half-life as described previously .PrkdcscidIl2rgtm1Sug/JicCrl) and male 4–6 weeks old BALB/c-Nu mice were purchased from Charles River  and bred under SPF conditions in the Laboratory Animal Center of the First Affiliated Hospital of Wenzhou Medical University. NOG mice were injected intravenously with 5 × 106 MSI2-OE. HEL cells or NC. HEL cells to establish orthotopic AML xenograft model . As the subcutaneous tumor xenograft model is easy to dynamically observe the growth status of tumor, the subcutaneous AML xenograft model was built. For BALB/c-Nu mice, cyclophosphamide  at 80 mg/kg was administered intraperitoneally (i.p.) before tumor inoculation. Then a total of 5 × 106 MSI2-OE. HEL cells per mouse were subcutaneously (s.c.) injected into BALB/c-Nu mice with Matrigel matrix  at equal volume, while another group of mice were injected s.c. with 5 × 106 NC. HEL cells. When the tumor size reached about 150–200 mm3, mice were grouped randomly (N = 7 mice per group) and treated with 3 nmol micrOFFTM agomir-143 or agomir-NC .Data were analyzed as mean ± SD from at least three independent replicates with the level of significance defined as It has been reported that miR-143, as a tumor suppressor, has low expression in a variety of malignant tumors and is closely related to the occurrence, development and prognosis of malignant tumors –33. A he+ primary AML cells from patients  cells  from GEO Datasets (GSE22775) were identified, of which 250 genes were upregulated and 371 genes were downregulated when AML cell lines were treated with MSI2 shRNA  and glycine amidinotransferase (GATM) mRNA were predicted to have a binding site with miR-143-3p. VASH1 mediated miR-143-induced cell dissemination and angiogenesis [MSI2 mRNA was validated by luciferase reporter assay, providing evidence that miR-143 reversed the role of MSI2 in AML development. Pramanik et al. demonstrated that restitution of tumor suppressor microRNAs using a systemic nanovector could inhibit pancreatic cancer growth in mice [Recently, approaches to inhibiting MSI2 proteins based on RNA interference have achieved promising outcomes in preclinical research , 8. Zhenogenesis , while Gogenesis . Our preogenesis . And as  in mice . ConsistOur research has revealed a novel mechanism Fig.  by whichAdditional file 1: Table S2. Sequences of siRNA/miRNA. Table S3. Sequences of primers used for quantitative real-time PCR. Figure S1. Biological effects of miR-143 in AML. Figure S2. Overexpression of MiR-143 hardly unaffected the cell cycle, but suppressed cell migration in AML. Figure S3. Overexpression of MiR-143 inhibited cell growth and induced cell apoptosis in AML. Figure S4. Biological effects of MSI2 in AML. Figure S5. Overexpression of MSI2 promoted growth and inhibited apoptosis in AML. Figure S6. Migration effect of MSI2 in AML. Figure S7. Flow cytometric analysis for human antigen expression in mouse tissues. Figure S8. IHC analysis for human antigen expression in mouse tissues. Figure S9. MSI2 activity was negatively modulated by miR-143 in AML cells. Figure S10. Expression of CD45 in spleen was detected by IHC. Figure S11. Flow cytometric analysis for human antigen expression. Figure S12. DLL1 and Snail1 gene expression in clinic.Additional file 2:Table S1. Baseline characteristics of participants.


It is now widely recognized that Ca2+ represents an important and universal second messenger responsible for the regulation of various cellular processes such as proliferation, differentiation, migration and death . MoreoveSala et al. demonstrated that the modulation of the Ca2+ levels mediated by the ether a-gò-gò related gene 1 (ERG1) influences selection and differentiation pathways of lymphoid clones. In particular, the authors underlined the importance of ERG1 activity to sustain an adequate electrochemical gradient necessary to achieve the Ca2+ influx during B and T cell receptor activation. Dysregulation of ERG1 leads to alterations in Ca2+ signals that allow the wrong selection of proliferating tumorigenic lymphoid clones. In line with these results, it has been demonstrated the aberrant expression of ERG1 is found in leukemia and it is associated with chemoresistance and worse prognosis  reduces tumor growth through CD8+ stimulation  . High comulation . CAMKK2 mulation . Huang eHan et al. identified for head and neck squamous cell carcinoma (HNSCC) a 12-ion-channel-gene signature useful to predict the prognosis. Among these channels, ANO1 is a calcium-activated chloride channel whose overexpression promotes distant metastasis of HNSCC whereas TRPC1 is a cation channel that mediates Ca2+ influx and found to be associated with tumorigenesis. The 12-ion-channel-gene signature also correlates with the immune infiltration: in the low-risk group the levels of B lymphocytes and CD8+ T cells are high while the high-risk group is characterized by the presence of resting-state macrophages seem to be associated with a negative prognosis.A lot of attention has been focused on ion channels in cancer recently, especially those that modulate Ca2+ flux. In fact, their aberrant expression and dysfunction, in both cancer and anti-tumor immune cells, affect immune escape mechanisms and, consequentially, tumor progression. About this, Despite many calcium-mediated pathways involved in tumor progression and antitumor immunity being investigated, integrating the findings obtained so far with those derived from new and sophisticated proteomic analyses will be the new challenge. Omics-based strategies, fundamental to biomedical and translational research, present a chance to discover new pharmacological targets in cancer immunotherapy  by identCA: Writing – original draft. MM: Writing – review & editing.