The underlying mechanisms, elucidated by studies at the molecular, biochemical, cellular and intact animal levels, indicate that transversion of Ser96 to Ala results in abolishment of an HRC phosphorylation site by Fam20C kinase and dysregulation of SR Ca2+ cycling. This is mediated through aberrant SR Ca2+ release by the ryanodine receptor (RyR2) quaternary complex, due to the impaired HRC/triadin interaction, and depressed SR Ca2+ uptake by the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2) pump, due to the impaired HRC/SERCA2 interaction. Pharmacological intervention with KN-93, an inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), in the HRC Ser96Ala mouse model, reduced the occurrence of malignant cardiac arrhythmias. Herein, we summarize the current evidence on the pivotal role of HRC in the regulation of cardiac rhythmicity and the importance of HRC Ser96Ala as a genetic modifier for arrhythmias in the setting of heart failure.Sudden unexpected cardiac death (SCD) accounts for up to half of all-cause mortality of heart failure patients. Standardized cardiology tools such as electrocardiography, cardiac imaging, electrophysiological and serum biomarkers cannot accurately predict which patients are at risk of life-threatening arrhythmic episodes. Recently, a common variant of the histidine-rich calcium binding protein (HRC), the Ser96Ala, was identified as a potent biomarker of malignant arrhythmia triggering in these patients. HRC has been shown to be involved in the regulation of cardiac sarcoplasmic reticulum (SR) Ca This is based on accumulating evidence on the crucial role of SR Ca2+ homeostasis in cardiac contraction, the direct association of human mutations in SR Ca2+ release components with arrhythmias, as well as alterations in the expression levels and activity of key Ca2+ handling proteins in human heart failure  a 13-residue stretch of polyglutamic acid; and (4) a cluster of 14 closely spaced cysteine residues, with the repeating pattern of Cys-X-X-Cys suggestive of a heavy metal binding domain, at the carboxyl terminus , indicating the high capacity of HRC for Ca2+ binding  patients  implantation) and 96 healthy controls, the HRC genetic variant Ser96Ala was found to have a statistically significant association with malignant ventricular arrhythmias and SCD in the patients . Specifi2+ handling, employed rat cardiomyocytes overexpressing HRC, and demonstrated that increased HRC levels lead to a 30% increase in SR Ca2+, accompanied by a 40% decrease in SR Ca2+ release, ultimately resulting in depressed cardiomyocyte shortening and re-lengthening. The depressed contraction of the HRC-infected cardiomyocytes remained even after maximal isoproterenol stimulation. At the molecular level, HRC overexpresssion was accompanied by increase of the ryanodine receptor (RyR2) quaternary complex components junctin and triadin . Genetically modified mouse models, as well as their explanted hearts and isolated cardiomyocytes, have long been used to delineate the effects of various genes and mutations thereof on cardiac arrhythmogenesis. Although these models have a number of limitations, they remain one of the major tools available for in depth cellular and physiological studies at the o levels .2+ uptake (by 35%) and attenuated cardiomyocyte Ca2+ transient decay (by nearly 40%). Possibly as a compensatory mechanism, there was a marked increase of the Na+-Ca2+ exchanger (NCX) and triadin protein levels in myocytes isolated from these mice. At the cellular level, however, neither NCX mediated Ca2+ extrusion nor RyR2 SR Ca2+ release was elevated. In fact, the depressed SR Ca2+ sequestration was associated with an attenuated rate of Ca2+ extrusion. Interestingly, as the mice aged, they presented with impaired cardiomyocyte Ca2+ cycling, leading to abnormal cardiac remodeling and congestive heart failure -mediated HRC knock-down in mice with or without transverse aortic constriction (TAC)-induced heart failure, led to decreased fractional shortening and increased cardiac fibrosis compared with control. At the molecular level these findings were associated with increased phosphorylation of RyR2, CaMKII, p38 MAPK, and phospholamban (PLN). Thus, down-regulation of HRC led to significant deterioration of cardiac function following TAC-induced heart failure  mice were generated. Although morphologically and histologically they were normal, compared to wild-type, at the cellular level, the HRC-KO mouse cardiomyocytes exhibited significantly enhanced contractility, Caformance .2+ transients, the prolongation of Ca2+ decay, and the caffeine-induced SR Ca2+ release, indicating a higher SR Ca2+ load. hHRC Ala96 had a stronger suppressive effect on the maximal SR Ca2+ uptake rate compared to hHRC Ser96. Furthermore, hHRC Ala96 increased the frequency of spontaneous Ca2+ sparks, while the hHRC Ser96 reduced them compared to GFP expressing controls. Importantly, expression of hHRC Ala96 in cardiomyocytes from a rat model of post-myocardial infarction heart failure induced dramatic disturbances of rhythmic Ca2+ transients. These findings reproduce the arrhythmogenic propensity of the human HRC Ser96Ala variant carriers when their cardiac function is compromised in the setting of heart failure . Indeed, the open probability of RyR2 was significantly increased in hHRC Ala96 cardiomyocytes. Under stress conditions of 1 μmol/L isoproterenol and 5 Hz stimulation, isolated cardiomyocytes from hHRC Ala96 hearts presented with 6-fold more aftercontractions and 3-fold more delayed afterdepolarizations when compared to hHRC Ser96 cardiomyocytes. RyR2 was found to be hyperphoshorylated by CaMKII at Ser2814. Importantly, treatment of the hHRC Ala96 derived cardiomyocytes with KN93, a specific inhibitor of CaMKII, reversed the increase of Ca2+ sparks and waves , was shown to be significantly decreased in 3–4-month-old Ser81Ala mice, when compared to the wild-types, whilst the Ser81Ala mice showed a slight decrease in intraventricular septum and left ventricular posterior wall thickness. Further age-related decreases in contractile function were similar between the two strains. Taken together, these data indicated that the increased mortality observed in HRC Ser81Ala mice was unlikely due to acute heart failure, and other factors should be considered  when compared to the wild-type, with more than 50% of Ser81Ala mice going into ventricular tachycardia, compared to none of the wild-type animals. We extended these studies by carrying out experiments in isolated myocytes, where an increase in aftercontractions, calcium after-transients and delayed afterdepolarizations were observed in Ser81Ala derived cells, further supporting the hypothesis that the increase in mortality is due to arrhythmogenic events  under baseline conditions in Ser81Ala myocytes, whilst no differences were observed at the protein kinase A (PKA) site of RyR2, or in PLN phosphorylation. However, there were no alterations in the levels of the RyR2 protein, whilst an increase was seen in junctin and NCX, and a decrease in triadin. Additionally, increases in action potential (AP) duration were noted in the Ser81Ala myocytes, which went along with an increase in inward (Ca2+) current. Taking all of these data together, we hypothesized that CaMKII played a key role in the arrhythmogenic phenotype in the Ser81Ala mouse. At both the cellular and in vivo levels, inhibition of CaMKII with KN-93 was indeed able to reduce the number of arrhythmogenic events.Interestingly in heart failure, where arrhythmias may also ensue, it has been shown that SR Cancreased . In this2+ leak and longer APs, and this is, at least in part due to CaMKII activity. This alteration of CaMKII activity could be driven by the alterations in the local Ca2+ concentration, although the exact mechanisms of CaMKII activation in this model remains to be elucidated. Taking all of these data into account, CaMKII inhibition could be a potential therapeutic target in patients with similar polymorphisms/mutations.Despite the fact that further studies should be carried out in this model, including the potential dyadic structural remodeling in myocytes, we can summarize that the HRC Ser81Ala mutation causes arrhythmia via increased spontaneous Ca2+ compartmentalization within the cardiomyocyte. During each cycle of cardiomyocyte activation Ca2+ fluxes rapidly back and forth between the cytosol and the sarcoplasmic reticulum. The SR Ca2+ release occurs through the opening of the gigantic RyR2 multiprotein complex under the regulation of triadin and junctin . Defects in HRC function due to genetic mutations lead to aberrant SR Ca2+ cycling and enhanced propensity toward arrhythmia triggering.Indeed, acute overexpression of the HRC Ser96Ala human variant in cell culture systems significantly increased the binding of HRC Ser96Ala to SERCA2, an effect that was associated with enhanced inhibition of SERCA2 activity and thus reduced maximal SR Caake rate . Importa release . Similarhythmias . Collectin vitro, these phosphorylations may not occur in vivo since HRC is a luminal protein and is not accessible to these cytoplasmic kinases  in wild-type hearts and can be dephosphorylated in vitro by alkaline phosphatase . Collectively, these data suggest that Fam20C is, at least in part, responsible for HRC phosphorylation, and that the variant Ser96Ala changes the affinity of HRC interactions with triadin and SERCA2  that phosphorylates S-x-E/pS motifs on proteins was found to be responsible for the majority of the extracellular phosphoproteome . Locatedin vivo . These fd SERCA2 . Furtherin vitro and in vivo approaches at the molecular, cellular, tissue, organ, and whole organism levels have demonstrated its importance in cardiomyocyte physiology and pathophysiology. HRC mediates a cross-talk between SR Ca2+ uptake and release, regulating both systems and acting as a Ca2+ sensor. Importantly, the function of HRC is regulated by its phosphorylation at Ser96 and human carriers with the Ser96Ala variant are predisposed to cardiac arrhythmias under stress conditions. Thus this variant may serve as a biomarker for the identification of cardiovascular disease patients at risk for SCD.Studies on HRC function over the past decade, using All authors contributed in manuscript preparation and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The aim of this work was to research in Arabidopsis whether [Ca2+]cyt is restored to initial levels when B is resupplied and elucidate whether apoplastic Ca2+ is the major source for B-deficiency-induced rise in [Ca2+]cyt. The use of chemical compounds affecting Ca2+ homeostasis showed that the rise in root [Ca2+]cyt induced by B deficiency was predominantly owed to Ca2+ influx from the apoplast through plasma membrane Ca2+ channels in an IP3-independent manner. Furthermore, B resupply restored the root [Ca2+]cyt. Interestingly, expression levels of genes encoding Ca2+ transporters  were upregulated by ethylene glycol tetraacetic acid (EGTA) and abscisic acid (ABA). The results pointed out that ACA10, and especially CAX3, would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.Boron (B) is a micronutrient for plant development, and its deficiency alters many physiological processes. However, the current knowledge on how plants are able to sense the B-starvation signal is still very limited. Recently, it has been reported that B deprivation induces an increase in cytosolic calcium concentration ([Ca Multiple stimuli induce specific spatio-temporal changes in cytosolic Ca2+ levels ([Ca2+]cyt), termed as “Ca2+ signatures” cyt as well as the expression of Ca2+-related genes such as CNGC19 (Ca2+ channel), several ACAs (Ca2+-ATPases), CAX3 (Ca2+/H+ antiporter), various CMLs , and CDPKs (Ca2+-dependent protein kinases) in Arabidopsis roots [2+]cyt in the Malus domestica pollen tube tip cyt, and elucidate whether the rise in [Ca2+]cyt triggered by B deprivation is due to a Ca2+ influx from the extracellular medium or from intracellular Ca2+ reservoirs. For these purposes, in vivo fluorescence measurements of [Ca2+]cyt in Arabidopsis seedlings subjected to B starvation and, subsequently, resupply experiments were performed. In addition, [Ca2+]cyt was determined in the presence of several chemical agents known to affect calcium homeostasis.An issue of increasing interest is how plants sense B availability. Cachannels . In addiis roots . Furthertube tip . Althoug2+ influx when compared to control cells [2+]cyt and expression of Ca2+-related genes described in Arabidopsis plants upon 6 and 24 h of B deficiency [2+]cyt to initial levels prior to B starvation stimulus, Arabidopsis seedlings expressing YC3.6 were subjected to B deprivation for 24 h and, subsequently, were grown with 2 µM B for 1, 3, 6, or 24 h. At indicated times, fluorescence measurements were performed in Arabidopsis roots. Interestingly, a gradual decrease in fluorescence signal and, hence, in [Ca2+]cyt was observed when plants were resupplied with 2 µM B  could be differently affected by B deficiency, total root B contents were determined. There was a remarkable decrease in the root B concentration in Col-0 wild type and seedlings expressing YC3.6 after 24 h of B deprivation  treatment induced activation of Cais roots ,34,35. A compare C,D,I,J. ongation . In addiongation . Moreove and ABA D,J, as it growth ,38.Arabidopsis root [Ca2+]cyt when ABA and EGTA were simultaneously added was also observed using the aequorin-emitted luminescence method [2+]cyt as well as that triggered by B deficiency were mainly a consequence of Ca2+ influx from the apoplast. Remarkably, in both B treatments, simultaneous application of ABA and EGTA highly reduced E,F,K,L fe method . Togethe2+ channels in this B-deficiency response, ruthenium red (RR), a specific compound reported to inhibit Ca2+ release from vacuole to cytosol, was used. RR is a membrane-permeable Ca2+ channel blocker that inhibits vacuolar cyclic ADP-ribose (cADPR)-dependent Ca2+ channels [2+]cyt under B deficiency was not decreased significantly when seedlings were treated with RR -regulated Ca2+ channels could be involved in the calcium response triggered by B starvation, U73122, an aminosteroid inhibitor of phospholipase C that reduces IP3 production and thereby inhibits the activity of these channels, was used [2+]cyt triggered by B deprivation  and triggered an increase in [Ca2+]cyt , ACA10 (plasma membrane PIIB-type Ca2+-ATPase), and CAX3 (vacuolar cation/proton exchanger) genes were overexpressed in Arabidopsis seedlings subjected to 24 h of B deficiency . ABA-induced overexpression of CAX3 gene was especially associated with a remarkable rise in [Ca2+]cyt observed in B-deficient seedlings treated with ABA -based Ca2+ sensor  were surface-sterilized in a 5% (v/v) hypochlorite solution for 15 min and then washed three times in ethanol and three times in sterile H2O. Sterile seeds were sown in square (12 cm x 12 cm) Petri dishes containing a culture medium (CM) supplemented with 2 µM H3BO3 cyt measurements were performed using an A. thaliana line expressing Yellow Cameleon 3.6 (YC3.6) cyt after 6 and 24 h of this nutrient stress, which is due mainly to Ca2+ influx across the plasma membrane from the apoplast, even though it cannot be ruled out that Ca2+ also comes from the vacuole through the tonoplast CNGC19 channel. When B-sufficient conditions are re-established, [Ca2+]cyt is gradually restored. CAX3 would play a major role in the restoration of Ca2+ homeostasis after 24 h of B deficiency.In summary, it can be concluded that B deficiency elicits increased [Ca


Escherichia coli strains, while in preweaning and growing rabbits, a coinfection of two or three pathogens is the most prevalent situation. Clostridium spiroforme and E. coli are the main bacterial agents detected in preweaning rabbits, but enterotoxigenic Bacteroides fragilis has just appeared as a new possible emergent pathogen. Coinfections between bacteria (C. spiroforme and E. coli), parasites (Eimeria spp.), and viruses (rotavirus) are much more frequent than simple infections in growing rabbits; for this reason, complete laboratory studies are required to establish on-farm disease control measures.Digestive disorders are the main cause of economic damage in rabbit farms and, usually, antibiotic treatment is the first choice to control them. Nevertheless, a broad range of infectious agents can be involved in such disorders, as we have observed in our diagnosis work as a veterinary diagnostic laboratory. In this study, a global and updated overview of the frequency of detection of those etiological agents is provided. We have seen differences depending on the age of the affected rabbits, with young rabbits (<15 days old) being the most affected by enteropathogenic Escherichia coli (EPEC), Clostridium spiroforme, C. perfringens, rotavirus A, Bacteroides fragilis, and Eimeria spp. Also, 40 EPEC and 38 non EPEC isolates were investigated for the presence of other colonization factors  by qPCR. EPEC is the most prevalent agent in young rabbits, and although different virulence profiles have been found among EPEC isolates, the liftA+, ral+, and paa+ profile is the most prevalent. C. spiroforme and EPEC are the more frequently detected pathogens in preweaning rabbits, but B. fragilis appears to be a new possible emergent pathogen. In growing rabbits, diverse co-infections between C. spiroforme, Eimeria spp., EPEC, and rotavirus are much more frequent than infections due to only one of them. Other pathogens detected in very few cases are Salmonella spp. and Enterococcus hirae.Digestive disorders are the main cause of economic damage to rabbit farms. This article provides a global and updated overview of the diverse etiological agents causing them, since 757 clinical cases were analyzed during 2018 and 2019—Ninety-five from young rabbits (<15 days old), 117 from preweaning rabbits (15–35 days old), and 545 from growing rabbits. Etiological diagnosis was carried out by bacteriological culture and a set of real time polymerase chain reaction (qPCR) tests for the detection of enteropathogenic  Oryctolagus cuniculus) producer of the European Union [Spain is the largest rabbit  strains are one of the main etiological agents causing these disorders. These EPEC strains can be any serotype or biotype and, moreover, they do not produce known enterotoxins or Shiga toxins; their virulence is associated with intestinal epithelial cell damage by effacing the microvilli and attaching intimately to the cell membrane, producing “attaching and effacing” lesion and diarrhea [eae) among other virulence-associated genes related to the type III secretion system [afr2, ral, lifA, and paa genes, have also been found in rabbit EPEC isolates, but their roles remain unclear since there have been EPEC strain isolates lacking these rabbit-specific fimbrial adhesins, suggesting the presence of further potential adhesins that are not yet known [Several pathogens have been described as causative agents of enteric disease in rabbits. Enteropathogenic diarrhea ,6,7,8. Sn system . In addiet known .Clostridium spiroforme. This Clostridium produces a binary toxin, similar to the iota toxin from Clostridium perfringens, which is considered as the principal virulence factor that causes enterotoxemia in rabbits [Eimeria spp. Have been described, and the severity of the process has been related to the Eimeria species implicated and the infection degree [In addition to EPEC, some other bacterial species have been defined as pathogenic to rabbits; one of the most important is  rabbits . It can  rabbits . Another rabbits . Besidesn degree .C. perfringens [Enterococcus hirae [Bacteroides fragilis has been pointed out as an enteric pathogen that causes severe disorders around the weaning period [Other etiological agents that have been accepted as potential pathogens, whose presence has been related to several risk factors  and can induce enteric problems, are group A rotavirus , C. perffringens , and Entus hirae . More reg period . TherefoThe objective of the present study was to describe the etiological agents identified in samples of the digestive processes of young rabbits (<15 days old), in pre-weaning rabbits (15–35 days old), and in growing-finishing rabbits, sent by veterinarians to our laboratory during 2018 and 2019.A total number of 757 clinical cases (samples received from an affected farm at an outbreak) coming from 363 different commercial rabbitries were analyzed in this work. We received samples in the laboratory from January 2018 to June 2019 coming from elsewhere in the Iberian Peninsula . Received samples were consistent with digestive organs or caecal swabs collected by practitioners from clinically affected growing rabbits. In each clinical case, samples from 3 to 5 animals were analyzed. Of the 757 clinical cases, 95 were due to gastrointestinal disorders in young rabbits (<15 days old), 117 cases were from 15–35 day-old rabbits before weaning, and 545 affected growing rabbits.C. spiroforme, C. perfringens, and Group A rotavirus were carried out in 100% of clinical cases (n = 757), whereas Eimeria spp. and enterotoxigenic B. fragilis were only studied by qPCR in 542 and 349 out of 757 total clinical cases, respectively, as those two latter qPCR tests were included in September 2018 for Eimeria spp. and January 2019 for B. fragilis in our routine diagnosis service.Bacteriological cultures and a set of real-time Polymerase Chain Reaction (qPCR) assays for the detection of EPEC, The number of clinical cases in which pathogens were analyzed are presented in an age distributed format in n = 40) and non-EPEC (n = 38) E. coli isolates obtained by bacteriological culture in this study were selected for the investigation of other colonization factors such as adhesive factor/rabbit 2 (afr2), rabbit EPEC adherence locus , linfocite inhibitory factor (liftA), and the porcine attaching and effacing-associated (paa) gene. On the other hand 78 EPEC  and grown at 37 °C under aerobic conditions for 24–48 h. MALDI Biotyper  was used to identify bacterial isolates.For every single case, up to 5 samples were pooled for acid nucleic acid extraction to make only one determination per case and pathogen. The content of swabs used for bacteriological culture was afterwards removed in 700 µL of PBS solution through rotation movement and vortex usage. After that, total nucleic acids were extracted from 200 µL of that suspension using an automatic device  and the respective commercial kit , following the manufacturer’s instructions.E. coli colonies were extracted with a simple thermal shock. The colonies were put in 1 mL of distilled water and boiled for 10 min in a water bath. After that, the tube was centrifuged for 5 min at 15,600× g, and the eluted DNA was transferred to a new tube.The DNA from E. coli eae gene, EXOone oneMIX C. spiroforme, EXOone oneMIX C. perfringens Alpha Toxin, EXOone oneMIX Rotavirus A, EXOone oneMIX Bacteroides fragilis, and EXOone oneMIX Eimeria spp. . The gene targets of each commercial kit and their coded proteins are shown in The above-mentioned pathogens were detected simultaneously from the same DNA elution. Real-time PCR reactions were performed using commercial kits available on the market following the instructions of the manufacturer: EXOone oneMIX Escherichia coli isolates were investigated for the presence of the eae gene as well as other colonization factor genes, which are also related to the adherence of the bacteria: afr2, ral, lifA, and paa. For that purpose, real-time PCR assays were performed using their respective commercial kits available on the market following the instructions of the manufacturer: EXOone oneMIX E. coli afr2, EXOone oneMIX E. coli ral, EXOone oneMIX E. coli lifA, and EXOone oneMIX E. coli paa .® software , but the Venn diagrams were generated using the online webpage http://www.interactivenn.net/ [All graphics were generated using Graphpad Prism 5enn.net/ .C. perfringens, C. spiroforme, EPEC, rotavirus A, Eimeria spp., and enterotoxigenic B. fragilis) distributed by age groups are shown in Results obtained by qPCR for each of the studied pathogens ,  culture . Other bE. coli isolates studied in this work are listed in n = 38) were also negative for the other colonization factors investigated, whereas all of the EPEC isolates (n = 40) were also positive at least to one other colonization factor. Different virulence profiles have been found among EPEC isolates, but the more common one was that positive to liftA, ral, and paa (n = 28), while isolates positive for afr2 were scarce (n = 6).The virulence attributes of the 78 C. spiroforme is the pathogen that is most frequently detected in preweaning and growing rabbits , 48% in preweaning rabbits, and 58% in growing rabbits  [n = 40) studied in this work were also positive at least to one other colonization factor, and although different virulence profiles have been found, the one positive to liftA, ral, and paa (n = 28) was more frequently found, while isolates positives for afr2 were scarce (n = 6). These results are in contrast with those described by Dow et al. [afr2 positive EPEC pathogenic strains, while lifA was rarely found. This can reflect the complexity to also define the pathogenic capacity of a strain by genotypic traits.Although the frequency of detection of EPEC was lower than  rabbits . The hig rabbits  and ther rabbits . Colibacabbits <1 days olda, etc.) ,7,8,10. Eimeria spp., 70% of growing rabbits and 30% of preweaning rabbits were positive, mostly in coinfection with C. spiroforme, EPEC, or rotavirus A    and is arabbits < days oldClostridium species, Clostridium cuniculi [Epizootic Rabbit Enteropathy (ERE) is a severe gastrointestinal syndrome that was first described in French intensive rabbit farms in 1997, which mainly affects post-weaning rabbits, causing high mortality rates in the absence of medication, but its etiology is still not completely understood , and itscuniculi , althougConcerning wild rabbits, the main pathogens of the digestive system described in Spain are parasites . The preC. spiroforme and enterotoxigenic B. fragilis are usual co-infections with EPEC and C. spiroforme and, less frequently, with Eimeria spp. Concerning growing rabbits, we cannot find any preeminent pathogen over the rest, finding a large variety of coinfections among C. spiroforme, Eimeria spp., EPEC, and Rotavirus A and thus showing a much more complex scenario where a single cause for the disease cannot be attributed.Our study provides an overview of the etiological agents present in rabbit enteric diseases in the Iberian Peninsula during 2018–2019. From the high number of samples included in the study, we conclude that colibacillosis by EPEC is very important in young rabbits (<15 days old) as a single infectious disease; meanwhile, the older the rabbits are, the more complex the digestive diseases become. In preweaning animals EPEC remains as an important pathogen, but other pathogens grow in importance, for example,


Stem cells can be conceptualized as computational processors capable of sensing, processing, and converting environmental information (input) to yield a specific differentiation pathway (output). In this study, we employ a temperature-controlled polymer sheet actuator to interpret and transfer information, controlled by the material’s programming, to mesenchymal stem cells. The cell’s interpretation of mechanical, thermal, and biochemical signaling is shown to be dependent on the actuator’s activity, utilized to accelerate differentiation toward bone cells, further elucidating the role of microenvironmental parameters on mammalian cells. Our method provides a unique approach to processing two discrete stimuli into one biochemical signal, calcium ions, providing a basis for the logical control of the flow of biological signals and the design of cellular functions. 2+) influx, and focal adhesion assembly, could be visualized and quantified. This paper gives compelling evidence that the temperature sensing and mechanosensing of MSCs are interconnected via intracellular Ca2+. Up-regulated Ca2+ levels lead to a remarkable alteration of histone H3K9 acetylation and activation of osteogenic related genes. The interplay of physical, thermal, and biochemical signaling was utilized to accelerate the cell differentiation toward osteogenic lineage. The approach of programmable bioinstructivity provides a fundamental principle for functional biomaterials exhibiting multifaceted stimuli on differentiation programs. Technological impact is expected in the tissue engineering of periosteum for treating bone defects.Stem cells are capable of sensing and processing environmental inputs, converting this information to output a specific cell lineage through signaling cascades. Despite the combinatorial nature of mechanical, thermal, and biochemical signals, these stimuli have typically been decoupled and applied independently, requiring continuous regulation by controlling units. We employ a programmable polymer actuator sheet to autonomously synchronize thermal and mechanical signals applied to mesenchymal stem cells (MSCs). Using a grid on its underside, the shape change of polymer sheet, as well as cell morphology, calcium (Ca In bioinformatics, the mass collection and mass distribution of biological information has enabled the unparalleled collaborative examination of complex cellular systems, detecting previously hidden patterns in the behavior of a variety of organisms. Current cell-signaling methods, however, rely on bulky controlling devices that regulate the transmission of decoupled thermomechanical information, limiting our control of the cellular microenvironment, but because of their expense, also the standardization of protocols that comes with widespread adoption by the scientific community , 2. Prog2+) influx , a type of multipotent stem cell, are highly sensitive to physical characteristics of their microenvironment such as temperature, differentiated stiffness stress, topography, and extracellular matrix mechanics . A range6) influx , 17. HowTo explore these questions, this study used programmable shape-memory polymer actuator (SMPA) sheets, capable of reversibly and responsively changing between two different predefined shapes (actuation) –21. SMPA2+ signaling, leading to the enhancement of Ca2+ influx. YAP signaling is activated by mechanical stimulation but suppressed by Ca2+ influx. Acetylation at histone H3 lysine 9 (H3K9ac) on osteogenesis-related gene promoters is promoted by dual stimuli, enhancing osteogenesis and suppressing adipogenesis of hADSCs.Here, a temperature decrease from 37 to 10 °C leads to a simultaneous elongation of the sheet in the direction of deformation during programming, and compression in the perpendicular direction. The unstretched shape can then be recovered by increasing the temperature to 37 °C again B. This cSI Appendix, Fig. S4A), a high cell viability over 7 d (>80%) was sustained under cyclically changed temperature , suggesting the 37 °C temperature (22 min per cycle) allowed for cell recovery. For each cycle, elongation of p-SMPA sheets occurred in the direction of deformation during programming during cooling, with compression occurring in the perpendicular direction, and an overall increase of the material surface area . The average elongation of the p-SMPA at 10 °C in cell culture medium was measured as 13.6 ± 0.8% after 24 temperature cycles (day 1), while the np-SMPA displayed an elongation of less than 1% with no distinguishable difference in both macroscopic shape and grid dimensions .In this work, semicrystalline linear poly(ε-caprolactone) (PCL) polymers were used as starting materials for the preparation of the polymer networks forming the actuating sheets. The low and broad melting transition (25–50 °C), relatively slow degradation rate, and commercial viability make these materials attractive candidates for many biomedical applications. Its elastic deformation in the temperature range 10–37 °C fulfills the requirements of this study . The shaSI Appendix, Fig. S1B). The uniformity of the elongation of the grid squares at different locations of the p-SMPA sheet suggested homogeneous actuation during temperature change .To ensure that the shape change of the sheet was sufficiently fast relative to the migratory movement of the cells, a temperature program was used to cool from 37 to 10 °C in 8 min and heat from 10 to 37 °C in 8 min. This greatly increased the speed of the shape change of the sheet . By measuring the change in the distance of two points initially separated by 100 µm, the speed of elongation and compression was measured as 3.6 ± 2.6 µm/min (temperature decrease) and (5.6 ± 1.5) μm/min (temperature increase). The movement speed in the compression direction was measured 2.4 ± 2.2 μm/min (temperature decrease) and 2.7 ± 1.5 μm/min (temperature increase) . The migration velocity of hADSCs on SMPA sheets was ∼0.6 ± 0.3 µm/min, as quantified via time-lapse microscopy using a previously reported method . This is supported by work elsewhere . The changes in several key spatial cell parameters, such as the area covered and the length in the direction of elongation and compression, were similar to that of the p-SMPA sheet . Furthermore, the influence of both stimuli on cell proliferation was assessed. The cell proliferation was inhibited by cyclic temperature change but promoted by mechanical stimulus, as evidenced by the staining of cell proliferation marker Ki67 as well as the quantification of cell number .Given that the magnitude of the material shape change is dependent on direction, with a greater shape change in the direction of elongation, cyclic actuation of the sheets should influence the cell orientation. To verify this, cells were seeded at a lower density to ensure the sufficient freedom of movement. After 3 d (72 cycles of actuation), the cells on the p-SMPA sheet aligned preferentially along the direction of elongation, when compared to those on a np-SMPA sheet  exposed to cyclic temperature change displayed visible Ca2+ influx (Movies S5 and S6). In contrast, only a small fraction of cells showed visible Ca2+ influx when cultured at 37 °C (7.3 ± 2.6% on np-SMPA and 8.1 ± 4.8% on p-SMPA) and at 10 °C (5.9 ± 4% on np-SMPA and 4.9 ± 4.7% on p-SMPA) .As a signaling messenger, Ca of MSCs , 26. Herr stores . Given t p-SMPA) A, while 2+ levels suggested a rapid variation Ca2+ concentration with temperature change . The cyclic temperature change between 37 and 10 °C induced a greater Ca2+ influx and activation than with the temperature range 37 and 30 °C . This observation verified that certain thermosensitive channels were responsible for the temperature-induced Ca2+ influx. For example, the thermosensitive TRPM8 channel is activated below 20 °C, facilitating the intracellular entry of Ca2+ . Despite the actuation’s negligible influence on the percentage of cells which experienced Ca2+ influx, it contributed to the Ca2+ signaling by increasing the intracellular Ca2+ level. The cells exposed to dual stimuli presented a higher concentration of intracellular Ca2+ than those exposed to thermal stimulus . Compared to the control, a significantly higher intracellular Ca2+ level was observed in the group with only mechanical stimulus . This confirmed that the actuation of the sheet might activate some mechanosensitive channels, such as TRPM7 , indicating the preservation of spontaneous Ca2+ oscillations under the dual stimuli.The analysis of the dynamic intracellular Cae change D–F. Give of Ca2+ . The inhas TRPM7  and PIEZas TRPM7 . Previoullations . DynamicSI Appendix, Fig. S7A). Compared to the cells without stimuli and only thermal stimulus, the cells in the group of dual stimuli expressed increased integrin β1 as well as enhanced fibronectin fibrillogenesis , suggesting an effect of mechanical stimuli on matrix protein assembly and activation of integrin adhesion complexes.When incident on cells, an external mechanical signal can regulate integrin activation, focal adhesion composition, and cytoskeleton organization to transduce the mechanical signal intracellularly and activate a series of downstream pathways. Talin, a vital component of integrin adhesion complex which mediates integrin activation and connects integrin to cytoskeleton , was enrSI Appendix, Fig. S8). Inhibiting of actin polymerization with cytochalasin D (CytoD) to block the mechanotransduction pathway resulted in significantly decreased YAP and RUNX2 activity . However, the nuclear localization of YAP was suppressed by the cyclic temperature change , which confirmed the role of Ca2+ in regulating YAP activity. The SMPA sheet’s geometric and temperature changes could regulate RUNX2 activity through a signaling network consisting of multiple pathways , and osteocalcin (OCN), was significantly enhanced in the cells treated with dual stimuli . These results suggested that both Ca2+ and mechanical signals could modulate histone modification, with different role for different types of histone modification.To gain insight into the effect of the dual stimuli at the epigenetic level, histone modification was evaluated after 10 d. The H3K9 acetylation (H3K9ac) at promoters of osteogenesis-related genes, including  stimuli A. Inhibiromoters B. The ef2+ signaling. For example, HDAC1 expression was down-regulated by cyclic stretch  is an important regulator of histone acetylation. Phosphorylation of HDAC1 at Ser421 increases its deacetylation activity. Previous studies have demonstrated the response of HDAC1 expression, nuclear localization, and phosphorylation to external mechanical cues and Ca stretch . Mechani stretch . Intraceof HDAC1 , 40. Herof HDAC1 C and D. ALP mRNA and protein activity . The OCN level under dual stimuli was significantly higher than that under mechanical stretch only , suggesting a cumulative effect of temperature change and p-SMPA actuation on osteogenesis. The adipogenesis of hADSCs was shown to be inhibited by temperature change and p-SMPA actuation. The adipogenesis marker FABP4 was down-regulated in samples exposed to cyclic temperature change and was further decreased in the presence of both stimuli .To investigate the effect of dual stimuli on hADSCs’ fate decision, differentiation markers were quantified at mRNA and protein levels . Both teactivity A–C. The activity A–D, and  stimuli A and E. 2+. Ca2+ dynamics regulate the intercellular connection of mechano- and thermosensing components, serving as a cellular basis for the osteogenic differentiation of MSCs. Polymer actuation activates the mechanosensor YAP, and subsequently promotes RUNX2 nuclear localization. Meanwhile, the cyclic temperature change and interlinked polymer sheet actuation decreased nuclear localization of phosphorylated HDAC1 via Ca2+ signaling, and maintained a high level of histone H3K9 acetylation on osteogenesis-related gene promoters. Thermal and mechanical dual effects work cooperatively to promote osteogenic differentiation of hADSCs. This dual-stimuli system could serve as a culture device to modulate stem-cell function, demonstrating the cooperative use of multiple external signals to achieve biological outputs. The p-SMPA sheet shows great potential to meet the clinical requirements for the tissue engineering of periosteum to treat bone defects. MSCs could be cultured in vitro where both thermal and mechanical stimuli are used to induce a high level of osteogenesis. After transplantation, the shape-memory effect of a p-SMPA sheet could allow self-tightening when exposed to the relatively higher temperature of the body, facilitating the attachment of the cell-laden sheet onto bone and accelerating its restoration process. In the future, detailed knowledge of signaling mechanisms with both spatial and temporal resolution is required. It is important to use a systemic approach, logically dissect the thermal and mechanical stimuli, and establish a precise and faithful model system, which enables a more effective combination of different stimuli.In summary, a thermally controlled SMPA sheet was employed to control the differentiation process of MSCs. In this work, we demonstrate that multiple physical stimuli  provided by a programmed polymeric actuator sheet could be used as basic inputs to guide the construction of the internal signal processing architecture of living cells . The resSI Appendix.Detailed descriptions appear in The cyclic temperature change between 37 and 10 °C was realized by computer-controlled thermochambers (Instec). Thermal and mechanical dual stimuli were generated by subjecting p-SMPA to temperature cycles. In the case of np-SMPA sheet only a thermal stimulus is provided, and a mechanical stretching device was used to generate mechanical stimulation.Temperature and cell morphology were measured using a digital thermometer and laser scanning microscopy, respectively, where the 50 × 50-µm grid printed on the SMPA sheet was used to provide information about the actuation of the cell substrate during characterization.The raw data for Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


TNF‐α rs361525, rs1800750, rs1800629, IL‐10 rs1800896, rs1800872, IL‐6 rs1800795, TGF‐β1 rs1800470, IFN‐γ rs2430561 single nucleotide polymorphisms (SNPs) on AML risk, the multivariate effect of SNPs on overall survival (OS) in AML and the association between the investigated SNPs and prognostic factors in AML.Cytokines were correlated with survival and disease progression in acute myeloid leukemia (AML). We aimed to evaluate the multivariate effect of FLT3 , DNMT3A (R882), and NPM1 type A mutations.All SNPs were genotyped in 226 adult AML cases and 406 healthy individuals. AML patients were investigated for P < .001). The presence of the rs1800750 variant genotype (P = .005) or FLT3‐ITD mutation (P = .009) in a cytogenetic high‐risk group (P = .003) negatively influenced OS. A negative association was observed between Eastern Cooperative Oncologic Group Scale status > 2, lactate dehydrogenase (LDH) level, platelet (PLT) count <40 000 cells/mm3, and OS. Multivariate Cox regression analysis showed that the presence of the rs1800750 variant genotype was a risk factor for death (P = .007), and that blast percentage, LDH level (≥600 IU/L), and cytogenetic high‐risk were independent significant predictors for death in AML .Univariate analysis revealed that age above 65 years had a negative influence on survival  on AML risk, the multivariate effect of SNPs on overall survival (OS) in AML and the association between the investigated SNPs and prognostic factors in AML. A negative association was observed between Eastern Cooperative Oncologic Group Scale status >2, lactate dehydrogenase (LDH) level, platelet (PLT) count <40 000 cells/mm3, and OS. Ages above 65 years, PLT count, TNF‐α rs1800750 variant genotype, blast percentage, LDH level, and cytogenetic high‐risk may be used as independent risk factors to assess AML mortality. Therefore, it is necessary to evaluate more than one SNP in the same study to take into account the polygenic model of inherited cancer susceptibility.To the best of the authors' knowledge, there are no published studies describing the possible association between cytokine polymorphisms and biological parameters, overall survival (OS), and AML prognostic impact.TNF‐α 238G>A (rs361525); 376G>A (rs1800750); 308G>A (rs1800629)], IL‐10 −1082T>C (rs1800896); −592T>G (rs1800872), IL‐6 174C>G (rs1800795), TGF‐β1 869C>T (rs1800470), IFN‐γ +874T>A (rs2430561)] on AML risk; (b) the multivariate effect of the eight SNPs on OS in AML patients; (c) the association between studied polymorphisms and clinical prognostic factors in AML patients.The objectives of this study were to evaluate: (a) the multivariate effect of eight SNPs  count, AML type).The distribution of OS time was presented using medians with their 95% CI for the studied groups. In order to quantify the multivariate effects of all studied SNPs on OS, we used Cox regression, the full model tested consisting in eight SNPs , four somatic mutations  and an EPV (number events per predictor) equal to 7 (EPV = 7), we used the Minimizing approximated Information Criterion (MIC) as a new sparse estimation method to select a set of predictors for OS in AML patients.The sample size of patients used for the Cox regression analysis was 204 cases with 170 events. The MIC estimation method retained seven out of 24 candidate predictors for OS in AML patients. The effect size of selected predictors on OS in the reduced model was described by corrected HR determined from the shrinkage estimators of regression coefficients and adjusted HR determined from classical estimators.Cox proportional hazards regression analysis with parameters estimated via the MIC method was performed using the “coxphMIC” package in the R statistical computing environment.IL‐10 (rs1800896 and rs1800872) and TNF‐α  SNPs, we used the Haplotype AnalysisFor the haplotype analysis of 33.1Demographic data, as well as clinical and biological features of our AML patients, are presented in Table P ≥ .05). Seventy‐four AML patients  were ≥65 years of age.The mean age ± SD of the AML groups was 54.44 ± 16.78 years (range 19‐87 years old), whereas the mean age ± SD for the controls was 56.01 ± 15.567 years (range 20‐85 years old). The control group included 213 females (52.5%) and 193 males (47.5%), whereas the AML group was made up of 113 (50%) females and 113 (50%) males. Age and gender distributions were similar in both the AML and controls  and TNF‐α , genotypes demonstrated deviation from the Hardy‐Weinberg equilibrium (HWE) in either the AML cases or controls. TGF‐β1 rs1800470 and IL‐6 rs1800795 genotypes were not consistent with HWE (P < .05) in AML patients but were in HWE on controls. The TGF‐β1 rs1800470 heterozygous genotype was associated with AML development risk and IFN‐γ rs2430561 heterozygous genotype was a protective factor for AML , while the other studied SNPs were not associated with AML risk. Regarding allele distribution, no differences were observed between the investigated groups except for the variant A allele of TNF‐α rs1800750 (P = .002).The genotype distribution for all investigated P = .059, OR = 1.45, 95% CI: 0.99‐2.13).We analyzed whether there were any differences between the investigated groups in the presence of at least two variant genotypes and observed that the presence of four variant genotypes from the investigated SNPs were associated with AML risk with a tendency towards statistical significance (3.3IL‐10 rs1800872 variant genotype in patients <40 years vs patients ≥40 years of age (P = .03), with a higher frequency of the variant genotype in elderly patients. There was an association between age ≥65 years and IL‐10 rs1800896 (P = .019).The clinical features of AML patients according to the presence of wild‐type and variant genotypes for each cytokine polymorphism are presented in Table TGF‐β1 rs1800470 variant genotype .A higher frequency in females was observed for pe Table . We obseTNF‐α rs1800629 variant genotype was found in patients with LDH <600 IU/L .Furthermore, a higher frequency of the IL‐10 rs1800896 (P = .072).No associations were observed between blast percentage and investigated SNPs, but there was a tendency towards statistical significance between blasts <70% and IL‐6 rs1800795 variant genotype (P = .03) and a higher frequency of variant genotypes between the AML de novo cases, with a trend towards statistical significance (P = .071) in the case of TNF‐α rs1800629 SNP.Regarding AML subtypes, we found an association between the IL‐10 rs1800896 variant genotype (P = .007) and a trend towards statistical significance (P = .083) in case of TNF‐α rs1800629 for high cytogenetic risk.Regarding the relationship between cytogenetic risk groups and cytokine gene polymorphisms, there was a significant association in the case of IFN‐γ rs2430561 (P = .043), TNF‐α rs1800750 (P = .005) while TNF‐α rs1800629 variant genotype was associated with ECOG grade ≥2 (P = .044).We observed an association between each ECOG performance status grade and the variant genotypes of TNF‐α rs1800629 variant genotype was associated with FLT3‐ITD (P = .049), FLT3 (ITD+D835) (P = .048), and DNMT3A somatic mutations (P = .008), whereas the FLT3‐D835 or NPM1 type A mutations showed no association (P > .05).Regarding the association between somatic mutations and investigated cytokines SNPs, the presence of FLT3‐ITD mutation (P = .081) and six variant genotypes with the NPM1 mutation (P = .081) showed a trend towards statistical significance.We also tested the association between somatic mutations and combinations of 3, 4, 5, or 6 variant genotypes in the studied SNPs, but only the presence of five variant genotypes with the P > .05) on all AML cases (n = 226).There was not found significant associations between studied SNPs and type of treatment (3.4P < .001). Patients under 65 had a better survival than patients over 65 , whereas gender and AML subtype showed no difference (P = .523). On the other hand, there was a significant difference in survival curves distributions (P < .001) between low, intermediate, and high cytogenetic risk groups .There was an association between AML patients' age and survival  vs 6.0 (4.4‐7.6)). Survival time showed a significant difference (P < .001) in AML patients treated with high‐dose (HD) chemotherapy , low‐dose (LD) chemotherapy , and HD chemotherapy + hematopoietic stem cell transplantation (HSTC) .We observed a better outcome in AML patients with an ECOG performance status ≤1 compared to those with a status ≥2 .Patients with WBC >50 000/mm3 at diagnosis had an increased rate of survival compared to those with lower PLT levels . When we analyzed the univariate impact of LDH level on OS, we observed that LDH levels above 600 IU/L were significantly associated with a shorter OS ].Kaplan‐Meier analysis showed that AML patients with PLT >40 000/mmFLT3‐ITD mutation had a significantly shorter OS (P = .004) than those with FLT3‐ITD negative status . The Kaplan‐Meier curves depicted in Figure FLT3‐ITD positive and negative status. Moreover, in AML patients with FLT3 mutation, the presence of the D835 mutation did not affect OS (P = .689). When we analyzed FLT3 (ITD+D835) positive status, we observed a trend towards statistical significance for this association (P = .078) with survival. No association was found between NPM1 or DNMT3A status, hemoglobin, hematocrit levels, and bone marrow blast percentage at diagnosis and OS (P > .05).Patients with the TNF‐α , IL‐10 (rs1800896 and rs1800872), IL‐6 (rs1800795), TGF‐β1 (rs1800470), and IFN‐γ (rs2430561) variant genotypes were not associated with patients' OS (P > .05), and neither were the combined variant genotypes (presence of >3 variant genotypes). Kaplan‐Meier curves were plotted to show the crude survival rates for the eight SNPs. . Patients lacking cytogenetic risk group data were not included in this analysis, therefore only 204 AML cases were investigated, with death occurring in 170 of these patients.The results of the Cox proportional hazards regression regarding the effects of the studied cytokine SNPs on mortality in AML patients are presented in Table P < .001, HR = 2.15, 95% CI: 1.55‐2.99). Presence of the TNF‐α rs1800750 variant genotype , or the FLT3‐ITD mutation , and inclusion in the cytogenetic high risk group  negatively influenced OS. In the case of AML patients with a cytogenetic risk established (n = 204) the treatment type  was significantly associated with OS  = 25.7, P < .001), the risk of death was higher in those treated with HD than in patients with LD . In addition, the effects of cytokines on OS were not modified after controlling for the treatment effect, of all polymorphisms only TNF‐α rs1800750 SNP having a significant effect on OS after controlling treatment effect .The univariate effect of investigated parameters on OS indicated that an age above 65 years had a significant negative influence on survival . Based on our Cox proportional hazards regression findings, we considered that age above 65 years was an independent predictor for mortality in AML cases . Following multivariate analysis, we observed a trend towards statistical significance for the association between TNF‐α rs1800629 variant genotype, PLT count, and mortality .Multivariate Cox regression analysis based on the MIC estimation method retained only seven predictors from the 24 candidate predictors for OS in AML patients . Furthermore, we observed that blast percentage, LDH level, and cytogenetic high‐risk were independent significant predictors for death in AML .The 4Our study provides data regarding the impact of cytokine gene polymorphisms in AML patients and AML mortality predictors in a Romanian case‐control study.TGF‐β1 rs1800470 and IFN‐γ rs2430561 variant genotypes were associated with AML susceptibility for the additive and dominant models. Furthermore, no evidence of association was observed between the investigated cytokine SNPs and AML in the allelic model, except for TNF‐α rs1800750 A allele (P = .002).We found that the TGF‐β1 rs1800470, our findings were similar to those of a study performed in Brazil which demonstrated that the TGF‐β1 rs1800470 TT homozygous genotype was associated with an increased risk of developing hematological malignancies , with a 4‐fold increase in the risk of developing hematological cancers.TGF‐β1 rs1800470 variant genotype.Regarding IFN‐γ +874T>A (rs2430561), no association was detected between the variant genotype or variant allele and AML risk in Turkey.IFN‐γ +874T>A polymorphism and leukemia risk for all comparison models. In the case of subgroup analysis by leukemia type, a significantly increased risk for CML was observed in the dominant model  and a decreased risk for CLL was found in the allelic, co‐dominant, and dominant models when separately using the fixed‐effect model .Regarding TNF‐α rs1800629, IL‐10 rs1800872, and IL‐10 rs1800896 SNPs were not risk factors for AML development in our population, with similar findings being reported by Pehlivan et al.TNF‐α rs1800629 polymorphism and leukemia risk.TNF‐α rs1800629 in CLL patients compared to controls (P = .00003 and P = .00007).The TNF‐α rs361525 did not confer susceptibility to AML in the additive and dominant models. No studies are currently available regarding the role of rs361525 in AML susceptibility, however, a closely‐related hematological malignancy case‐control study  showed that the rs361525 and rs1800629 variant genotypes were significantly higher in MPN cases (P = .04 and P = .02), providing a risk factor for developing MPN of 2.21 and 1.82, respectively.P = .02).P = .002). In the allelic model, we noticed that TNF‐α rs1800750 A allele was associated with AML susceptibility (P = .002).In addition, our findings indicated that IL‐6 rs1800795 genotypes or alleles among AML patients and controls, our data being similar to the results reported by Nursal et al.Furthermore, we observed no differences regarding the frequency of IL‐10 SNPs in the predisposition to leukemia are contradictory. The IL‐10 −592C>A promoter SNP was investigated in 115 AML patients and 137 controls from China, where a significant difference regarding the −592AA genotype percentage (P = .014) and −592A allele frequency (P = .004) was observed. The −592AA genotype prevalence risk was 2.492 times higher than in CC genotype carriers .IL‐10 rs1800872 variant allele was associated with a slightly increased risk of AML (adjusted OR = 1.30 95% CI: 1.01‐1.72).IL‐10 (rs1800896 and rs1800872) gene may have a significant association with AML etiopathogenesis.Research results regarding the role of IL‐10 rs1800896 allele or genotype frequency and CML risk, a recent study indicated that IL‐10 could be a useful survival biomarker in CML.Considering that no association could be detected between the IL‐10 rs1800872 and rs1800896 and AML predisposition. These findings are in line with certain studies reported previously In the present study, no relationship was found between IL‐10 rs1800896, our findings were in line with those of Fei et al who found no significant differences between AML patients and controls when comparing rs1800896 allele and genotype frequencies.IL‐10 rs1800896 variant genotype and IL‐10 plasma levels and concluded that rs1800896 was not associated with acute lymphoblastic leukemia (ALL) susceptibility nor with relapse risk.Regarding IL‐10 and TNF‐α haplotypes and AML risk. The most frequent haplotypes of the IL‐10 rs1800896 and rs1800872 SNPs were CTGG (23.5% patients vs 25.1% controls) and CTGT (24.3% patients vs 20.7% controls). Logistic regression did not confirm any significant association between CTGT haplotype and AML risk , with similar results being found for the CTGG haplotype .We also investigated the potential association between the TNF-α haplotypes  were h5 AGGGGG haplotype (30.1% patients vs 30.3% controls) and GGGGGG (60.6% patients vs 63.5% controls), but neither was correlated with AML risk . Moreover, we analyzed the impact of the IL‐10 and TNF‐α haplotypes on OS in our AML cohort. We did not find any differences in OS regarding the CTGG and CTGT haplotypes or any other IL‐10 haplotype , nor between AGGGGG and GGGGGG haplotypes and the other TNF‐α haplotypes .We observed that the most frequent FLT3‐ITD mutation negatively influenced overall AML survival. The investigated SNPs had no effect on OS in AML, either when they were tested individually or in the case of combined variant genotypes . The fact that no associations were found between AML patients' OS and the presence of TNF‐α , IL‐10 (rs1800896 and rs1800872), IL‐6 (rs1800795), TGF‐β1 (rs1800470), or IFN‐γ (rs2430561) variant genotypes suggests that these SNPs may not represent independent survival biomarkers in AML.The current study showed a significantly lower survival rate in elderly AML patients compared to patients under 65 years of age. A shorter OS was observed in AML cases with cytogenetic high risk, and in those with decreased WBC counts. High LDH (>600 IU/L) levels and TNF‐α rs1800750 variant genotype, FLT3‐ITD mutation, cytogenetic high risk, ECOG performance status ≥2, LDH level ≥600 IU/L, and PLT count lower than 40 000 cells/mm3 had an effect on OS in AML.Moreover, in the univariate Cox regression analysis, we found that age above 65 years, TNF‐α rs1800750 SNP, blast percentage, LDH level, and cytogenetic high‐risk were found to be independent significant predictors for OS in AML. We observed a trend towards statistical significance regarding the association between the TNF‐α rs1800629 variant genotype and OS. In contradiction, Kim et al reported a lower OS (estimated 20.1 months) in case of the GA variant genotype compared to GG homozygous genotype (estimated 54.6 months) for the IL‐10 rs1800896 SNP in AML patients.IL‐10 rs1800871 and rs1800872 variant genotypes did not have an effect on OS.IL‐10 rs1800871 have been previously reported.In multivariate Cox PH regression analysis, only age above 65 years, TNF‐α rs1800629, IL‐10 rs1800872, rs1800871, and rs1800896, IFN‐γ rs2430561, and TGF‐β1 rs1800470 (codons 10 and 25) polymorphisms and OS in their Turkish CML patients,Pehlivan et al found no association between TGF‐β1, IFN‐γ, TNF‐α, IL‐6, and IL‐10 SNPs and AML risk was not evaluated because we only had a few cases with only one variant genotype and no cases with wild‐type homozygous genotype for all of the analyzed cytokine SNPs.The relationship between any two combined variant genotypes of the RUNX1 and CEBPA mutations, the lack of data about the plasma level of TNF‐α, IL‐10, IL‐6, TGF‐β1 and IFN‐γ in the patient samples. However, the robustness of this study is represented by the fact that it investigated a representative AML cohort in Romania, since, to our knowledge, there is currently no published data on the role of these polymorphisms in AML on Eastern European populations. This is the first study to investigate the association between cytokine SNPs and AML risk, and, as a result, our report presents novel observations not previously described in a complex heterogeneous disease characterized by genomic changes and the accumulation of blasts, most commonly in the bone marrow, resulting in bone marrow failure and leading to death.Our study contains a number of limitations, including the lack of data regarding cytokine expression and the lack of information regarding Furthermore, our study investigated, for the first time, the relationship between somatic mutations in AML and cytokine SNPs in a single multivariate model, in which the estimation method allowed the selection of variables, model fitting, and stable regression coefficients.In conclusion, based on our findings, we consider that TGF‐β1 rs1800470 and IFN‐γ rs2430561 variant genotypes were associated with AML susceptibility. Our study revealed that age above 65 years, PLT count (<40 000 cells/mm3), TNF‐α rs1800750 variant genotype, blast percentage (>70%), LDH level (≥600 IU/L), and cytogenetic high risk may be used as independent risk factors for assessing AML mortality.The authors report no conflict of interests.


It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP Membrane contact sites between the ER and mitochondria are known to convey calcium signals between these two organelles via IP3 receptors, but the molecular mechanisms are currently unclear. Here, the authors show that IP3 receptors play a structural tethering role in maintaining ER-mitochondrial contacts. ER-mitochondrial contacts are relevant for intracellular signaling through calcium, reactive oxygen species and Bcl-2 family proteins, biosynthetic pathways, membrane dynamics, and even for mitochondrial DNA replication (reviewed in ref. 5). The alteration of the physical contact between the ER and mitochondria and the malfunction of the interorganellar Ca2+ transfer was linked to various complex symptoms involved in human diseases, such as progressive cognitive and motor disorders, neurodegeneration, or cancer9. Thus, determining the structure of the interface between the ER and mitochondria, the molecules involved in the physical interaction and their regulation is expected to aid understanding of the pathogenesis of such diseases and identification of potential new drug targets. In the past decade more than 60 proteins have been localized at the contacts11, among them the IP3 receptors (IP3Rs), but their distinct roles in shaping the ER-mitochondrial contacts have never been directly addressed.In the past decade, interorganellar signaling and its pathophysiologic connections to various human diseases have received growing attention. One of the most intensely pursued paradigms has been the local communication between the endoplasmic reticulum (ER) and mitochondria at their close contacts, which are supported by direct physical links referred to as tethers2+ release from the ER, giving rise to a cytoplasmic [Ca2+] ([Ca2+]cyto) signal, which controls a variety of cytoplasmic targets and is also effectively delivered to the mitochondrial matrix to regulate oxidative metabolism and cell survival13. Because the global [Ca2+]cyto usually peaks at ≤ 1μM and the affinity of the mitochondrial Ca2+ uptake machinery is relatively low12, and because clamping the global [Ca2+]cyto with a slow Ca2+ buffer does not prevent calcium signal propagation to the mitochondria15, the IP3R-mitochondrial Ca2+ delivery has to involve a privileged local mechanism. Indeed, direct local [Ca2+] measurements showed the IP3R-mediated Ca2+ release can expose the adjacent mitochondrial surface to 10–30 times larger [Ca2+]cyto than the global [Ca2+]cyto increase17.IP3Rs are large-conductance cation channels that mediate Ca2+, ATP, and post-translational modifications , but each isoform shows different activation and deactivation patterns24. The expression pattern and subcellular distribution of the three IP3R isoforms show heterogeneity in different tissues and cell types27. Overall, the three IP3R isoforms seem to have different functions and regulation, which are expected to result in heterogeneity in their communication with mitochondria. Among the three different IP3R isoforms, IP3R1 and IP3R3 have been described to co-IP with the voltage dependent anion channels (VDAC1) in the mitochondrial outer membrane (OMM) or to facilitate Ca2+ signal transmission from the ER to the mitochondria30. However, no systematic comparison of the three IP3R isoforms in the same cell type has been conducted.There are three isoforms of IP3Rs, encoded by three distinct genes, all regulated by IP3, Ca31. This model is also broadly relevant since several functional consequences of IP3R-mitochondrial communication have also been described in DT40 cells33. In the current study, we investigate a possible role of IP3Rs in shaping the interface between the ER and mitochondria and assess the role of each IP3R isoform in delivering Ca2+ signals to the mitochondria in IP3R-deficient  DT40 cells stably and evenly rescued by biochemically tagged individual mammalian IP3R isoforms. To test the broad relevance of the findings made in DT40 cells, we also employ a very recently established IP3R-deficient (TKO) HeLa cell model34.The first vertebrate cellular model available to compare individual IP3R isoforms in the same cellular background was established in DT40 cells3-mediated Ca2+ signaling was completely absent  and IP3R-deficient (TKO) DT40 cells  and ER targeting sequences coupled with the two components of the FKBP-FRB heterodimerization system and YFP and CFP, respectively, were overexpressed in WT and TKO cells. Addition of rapamycin causes trans heterodimerization between interfacing FKBP and FRB domains to rapidly connect the ER-and OMM-targeted anchors. Induction of the bridge formation is initially confined to the areas where the ER and OMM were naturally close. Addition of rapamycin (100 nM) led to rapid redistribution of the majority of the CFP fluorescence to the mitochondria and an increase in the FRET between CFP and YFP . The two IP3R1 clones (R1 A and R1 B) had similar sensitivities . IP3R3 has the lowest sensitivity   of the cell lines were in the range of 50–80%. The IP3 sensitivity and pool sizes were then compared to what we recorded previously in double knockout (DKO cells) DT40 cells expressing each individual endogenous avian IP3R isoform19  and mammalian (rescue) IP3Rs with different affinities  and one clone each of IP3R2 and IP3R3. As a first approach, we examined the relative distribution of ER and mitochondria along the tor Fig. . We fountor Fig.  as compator Fig. , similartor Fig. . The comtor Fig. . The higtor Fig. , demonstWe employed three techniques to visualize directly the relationship between IP3Rs and mitochondria in stable-rescued DT40 cells. First, the localization of FLAG-tagged IP3Rs and OMM was visualized by single-molecule localization super-resolution microscopy Fig. . A clustNext, we sought to validate these findings using 3D stimulated emission depletion (STED), super-resolution microscopy. We were able to visualize FLAG-tagged IP3R2 and IP3R3 together with the OMM marker TOM20 and to localize areas where IP3R clusters overlapped with OMM signal  levels, simultaneously in intact cells. To trigger IP3-dependent ER Ca2+ release we stimulated the cells with trypsin, which activates the IP3-linked protease-activated receptors (PAR). Addition of trypsin was followed by cytoplasmic and closely coupled mitochondrial Ca2+ signals in each IP3R isoform expressing cell line  knockout (cR2 DKO) cells, and found higher maximal [Ca2+]mito levels in cR2 DKO cells following direct stimulation by IP3  uncaging light overlaps with the excitation wavelengths of fura2, [Ca2+]cyto was measured with a single wavelength probe, Fluo-8. When Ca2+ was present, the peak amplitudes of [Ca2+]cyto signals were similar for each IP3R isoforms . To understand this phenomenon, we measured baseline [Ca2+]cyto levels in R1 cells with fura2, which, as opposed to Fluo-8, allows the precise calibration of the measured Ca2+ concentration  has been used to globally expose mitochondria to elevated [Ca2+]cyto in intact cells. We found that in store-depleted cells, the presence of any IP3Rs facilitates SOCE, especially IP3R1  and a non-conductive pore mutant clone of IP3R1  that fails to mediate Ca2+ release35  expressing TKO clone, In both R2 GS3 and M3 clones IP3-dependent Ca2+ signaling was absent  in cardiomyocytes. Studies have reported that IP3R3 (1) is a marker of the ER-mitochondrial contacts, (2) is required for apoptotic mitochondrial Ca2+ signals30, (3) concentrates to the MAMs where it is stabilized by sigma-1 receptors40 or BAP16 and (4) similarly to IP3R1, co-IPs with VDAC141. However, in most cases IP3R3 has been put forward in contrast with IP3R1 but not IP3R2, or without validating the distribution/contribution of the other isoforms41.We have examined the properties of the three mammalian IP3Rs in the context of the structural organization and Ca45. Furthermore, DT40 cells expressing only one IP3R isoform are available , as well as TKO cells rescued with each mammalian IP3R isoforms, providing the possibility of side by side comparison of the individual IP3Rs in the same cellular environment46.The model system we chose was the chicken B cell (DT40) line that endogenously expresses all three IP3R isoforms and is available without any IP3Rs (TKO). This model has been widely used to evaluate the IP3R-dependence of various cellular functionsWe demonstrated that the presence of IP3Rs affects the physical associations between the ER and mitochondria using techniques that go beyond the resolution limit of conventional optical microscopy. In live cells, FRET measurement of the tight ER-mitochondrial associations showed dependence on IP3Rs Fig. . In fixe28, other ER-mitochondrial linker species might have supporting role in the interaction of IP3Rs with the mitochondrial outer membrane . Indeed, the role of plasma membrane-ER junction localized IP3R1 in mediating SOCE was reported47. Our results show that IP3R1 maintains sustained cytoplasmic calcium signal, which feeds more Ca2+ to the mitochondria as compared to IP3R2 and IP3R3. Complementing this result, in store-depleted cells every IP3R isoform facilitated the Ca2+ influx through the plasma membrane but IP3R1 was more effective in this regard than IP3R2 or IP3R3  and IP3R-deficient  DT40 cells were provided by Prof. Tomohiro Kurosaki  and were cultured in suspension, in RPMI 1640 medium with glutamine  supplemented with penicillin/streptomycin, 2 mM L-glutamine, 10% FBS , 1% chicken serum  and 0.4% 2-mercaptoethanol in 5% CO2 and 95% air at 40 °C. DT40 cells stably expressing any IP3R or M3 construct were produced by David I. Yule,  and were kept under the same conditions as above but in medium supplemented with 2 mg/ml G418 (Enzo Cat# ALX-380-013-G005).Wild-type (WT) and IP3R-deficient (TKO) HeLa cells were provided by Katsuhiko Mikoshiba . Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM), with 4.5 g/l glucose  supplemented with 10% FBS, 2 mM glutamate, 100 units/ml penicillin/streptomycin ,6 cells in 350 μl medium) were electroporated (BTX ECM 830) with the following constructs: mitoRCaMP, mito-mCherry, ER-GFP, ER linker, OMM linker (15 μg cDNA each) using cuvettes with 4 mm gap-width with, 230 V in two, 25 ms pulses with 3 s interval then rapidly diluted in 10 ml medium and kept at 40 °C for 24 h.DT40 cells , 0.5% sodium deoxycholate (w/v), and 10% glycerol with a mixture of protease inhibitors (Roche). After 30 min of incubation on ice lysates were cleared by centrifugation at 16,000 × g for 10 min at 4 °C then transferred into fresh tubes. Protein concentrations were quantified with DC protein assay kit (Bio-Rad). Proteins were resolved on 5–7.5% SDS-PAGE gels then transferred to nitrocellulose membranes . Rabbit polyclonal antibodies recognizing IP3R1 (CT1) (1:5000) raised against the C-terminal 19 amino acid (aa) residues of rat IP3R1 and IP3R2 (NT2) (1:200) raised against N-terminal 320–338 aa residues in mouse IP3R2 , mouse monoclonal antibody against 22–230 aa residues of human IP3R3 , Calnexin rabbit polyclonal antibody , and mouse monoclonal Flag antibody  were used for immunoblotting. Corresponding mouse and rabbit HRP-linked secondary antibodies were obtained from Cell Signaling . Proteins were detected with chemiluminescence using HRP substrate for CCD imaging (Azure Biosystems).All reagents used for SDS-PAGE were obtained from Bio-Rad. Harvested cells were washed once with ice-cold PBS then solubilized in cell lysis buffer containing 10 mM Tris-HCl, 10 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM Na48. Interfaces between ER and mitochondria were analyzed using a custom Image J plugin/macro written by DW.DT40 cells were fixed and embedded using the following protocol: Cells were fixed using 2% glutaraldehyde, then with 1% OsO4 and stained with 0.5% uranyl acetate, pelleted in 2% agarose , dehydrated in a dilution series of acetone/water, and embedded in Spurr’s resin (Electron Microscopy Sciences). The sections were examined with a JEOL JEM1010 TEM fitted with a side-mounted AMT XR-50 5Mpx CCD camera or an FEI Tecnai 12 TEM fitted with a bottom-mounted AMT XR-111 10.5 Mpx CCD camera. Whole-cell images were taken with ×6700–14,000 (JEOL) or ×4400–6500 (FEI) direct magnification. Mitochondria were imaged using ×40,000  or ×15,000  magnificationFor immuno-EM experiments the following protocol was used: Plated DT40 cells were fixed with 5% paraformaldehyde followed by quenching, permeabilization, blocking, and labeling with primary antibody , followed by secondary blocking and incubating with secondary antibody anti-mouse IgG Nanogold . Silver enhancement  was applied, followed by further fixation and contrasting with uranylacetate. Samples were dehydrated in grade of alcohol and embedded in Durcupan (Electron Microscopy Sciences). After polymerization, ultrathin sections (65–80 nm) were prepared using by ultramicrotome  and studied with FEI Tecnai 12 electron microscope. Images were taken at ×3200–15,000 magnification.3, 10 mM Na-HEPES, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2 mM CaCl2, and 10 mM glucose, pH 7.4) containing 2% BSA (Roche). For measurements of [Ca2+]cyto, cells were loaded with 2 µM fura2/AM (Invitrogen) or 2 µM fluo8/AM (TEFLabs) in the presence of 0.003% Pluronic F-127 and 100 μM sulfinpyrazone for 10 min at 37 °C. Caged IP3 (D-23-O-Isopropylidene-6-O-(2-nitro-4.5-dimethoxy)benzyl-myo-Inositol 145-trisphosphate-Hexakis(propionoxymethyl) Ester,  was loaded under the same conditions for 30 min Subsequently, the cells were washed with fresh ECM containing 0.25% BSA and transferred to the thermostated stage (37 °C) of the microscope.DT40 cells were plated on Cell-Tak  coated 25 mm coverslips then dye-loaded in a serum-free extracellular medium  a ProEM1024 EMCCD camera (Princeton Instruments) fitted to a Leica DMI6000B inverted microscope with Lambda DG4 wavelength-switch xenon light source (Sutter Instruments) and operated by a custom software , and (2) an ImagEM EM-CCD camera (Hamamatsu) fitted to an Olympus IX81 microscope with Lambda DG4 light source and Mosaic targeted illumination module (Andor) operated by the Metamorph software (Molecular Devices). Uncaging light was provided by a mercury arc lamp  or a 365 nm LED source (Sutter Lambda TLED+). Both imaging setups used the UV-optimized Olympus UAPO/340 ×40/1.35N.A. oil immersion objective.Two imaging systems were used for Ca2+]cyto and [Ca2+]mito was measured fura2 fluorescence was recorded at 340 and 380 nm excitation while fluo8 at 485/15 nm excitation and the mitochondrial matrix-targeted RCaMP was either excited at 545/20 nm (w/fura2) or at 577/20 nm (w/fluo8), using dual-band dichroic and emission filters . Images were collected every 0.2 s or 2 s. [Ca2+]cyto measurements with fura2 were calibrated in vitro with adding 1 mM CaCl2, followed by 10 mM EGTA/Tris, pH 8.5. To determine [Ca2+], the following formula was used: [Ca2+] = Kd × (Sf2/Sb2) × (R − Rmin)/(Rmax − R), where Kd is the Ca2+-dissociation constant (0.224 µM), R is the ratio of the fluorescence intensities at 340/380 nm excitation, Rmin and Rmax are the fluorescence ratios in Ca2+-free and Ca2+-saturated conditions respectively, Sf2/Sb2 is the ratio of fluorescence intensities measured at 380 nm excitation in Ca2+-free (f)/Ca2+-saturated  conditions49.For simultaneous measurements of [Ca2+]cyto and [Ca2+]mito were recorded in permeabilized cells with Fura2FA added to the bulk buffer after permeabilization and rhod2AM accumulated by the mitochondria. One micromolar CaCl2 was added to optimize ER Ca2+ loading. IP3 receptors were stimulated with adding IP3 in a final concentration of 7.5 μM.cyto, 1.5 μM fura2/FA was added. Fluorescence was monitored in a fluorometer  using 340 nm and 380 nm excitation and 500 nm emission for fura2. Calibration of the fura2 was carried out at the end of each measurement adding 1 mM CaCl2, followed by 10 mM EGTA/Tris, pH 8.5 and using the formula described aboved49.DT40 cells in suspension were pre-incubated in Ca36 then AlexaFluor 647-conjugatedanti-mouse or anti-rabbit secondary antibody . Immunofluorescence was imaged using a Zeiss LSM 880 confocal system equipped with Airyscan detector, using 488, 561, and 633-nm laser lines for green, red and far-red fluorescent probes, respectively. Images were taken through a ×63 oil objective . Co-distribution of ER and mitochondria was calculated as the log2 ratio of the cumulative abundance of mitochondria and that of the ER in the 0–1 µm Z sections of each cells.DT40 Cells were transfected with ER-lumen targeted GFP and OMM-targeted mCherry (OMP25-mCherry) expression vectors. Transfected cells were incubated at 40 °C for 24 h then attached to Cell-Tak treated coverslips and further incubated for 4 h. Cells were fixed in 3% paraformaldehyde/PBS and labelled with anti-FLAG  or anti-IP3R1 primary antibody (CT1)3D super-resolution microcopy was performed using a Vutara 352 nanoscope (Bruker). Briefly, two focal planes were projected onto the sensor of an ORCA Flash v.2 sCMOS camera (Hamamatsu) through an Olympus UPlanSApo ×60/1.2 N.A. water-immersion objective. The point spread functions for the wavelengths used were calibrated and aligned using 100 nm Tetraspeck microspheres (ThermoFisher Scientific). Laser illumination at 640 and 561 nm were used for stochastic imaging of AlexaFluor 647 and CF-568 (Biotium), respectively, and 405 nm light was used to recover portions on CF568 from their dark state. The two colors were recorded sequentially  and several thousand frames were acquired for each fluorophore. Cells were plated, fixed and IP3Rs were labelled as described above, and TOM20 was labelled with anti TOM20  primary then with CF568 Goat Anti-Rabbit IgG (dilution: 1:200 Biotium Cat# 20103-1) secondary antibodies. Imaging was performed in freshly prepared buffer for oxygen-depleting and reducing conditions: 20 mM cysteamine, 1% 2-mercaptoethanol (v/v), 169 AU/ml glucose oxidase and 1400 AU/ml catalase in 50 mM Tris-HCl, 10 mM NaCl and 10% (w/v) glucose (pH 8.0). Calibration, alignment, acquisition and localization were all done in the Vutara SRX software.3.Cluster-based minimum distance analysis was also performed in Vutara SRX. Empirically determined settings were used to generate IP3R clusters  and large clusters that represented the volumes of whole mitochondria , which were used for all measurements. Control data consisted of randomly generated clusters containing 20 particles each  at a density of 12.5 clusters/µm3D STED microscopy was performed using an Abberior Instruments Expert Line STED microscope equipped with an Olympus UPLSAPO ×100/1.4NA oil immersion objective. Briefly, Tom20 was labelled with anti-TOM20 primary  and Alexa Fluor 594 anti-rabbit IgG secondary  antibodies. Flag-tagged IP3Rs were labelled with anti-FLAG primary  and STAR RED, goat anti-mouse IgG  secondary antibodies. Sequential confocal and STED images were obtained following excitation of Alexa Fluor 594 and STAR RED by 594 and 640 nm lasers, respectively. Both fluorophores were depleted in three dimensions with a 775 nm pulsed STED laser. Z-stacks were obtained by collecting images at 150 nm intervals using the 3D STED mode. RESCue STED was employed to minimize the light dosage. In this mode when the photon count returned in the confocal images was below 10, the STED laser was not engaged for that pixel. Blend mode depth projection images were generated and fluorophore volumes and interfaces between these volumes were analyzed using IMARIS 9.2.1 (Bitplane).t-test or Mann–Whitney Rank Sum Test if normality test failed. Multiple samples were compared with one-way ANOVA with post-hoc Holm method  or post-hoc Dunn’s method . Difference between the expected frequencies and the observed were tested with Chi-squared test.For the statistical tests Microsoft Excel or Systat SigmaPlot softwares were used. Significance was tested with two-tailed tests. Normality was tested with Systat SigmaPlot. Datasets of two independent groups were pairwise compared with unpaired Student’s All graphs were created in SigmaPlot 12 and were combined with the images in PowerPoint.Every element of each graph and image was created by the Authors. No previously created elements were used.Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummarySource Data


BCL2 protein inhibitor venetoclax (ABT-199) has been authorized by Food and Drug Administration for relapsed/refractory chronic lymphoid leukemia with 17p deletion. Although venetoclax/ABT-199 also caused cell death in acute myeloid leukemia (AML), whether it could be applied to clinical treatment needs further studies. Here, we revealed clinical implication of BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using venetoclax.BCL2 expression was analyzed in adult AML patients from public datasets The Cancer Genome Atlas (TCGA) and confirmed by another independent cohort from our own data.BCL2 expression showed up-regulated in AML patients among TCGA data and confirmed by our own data. BCL2 overexpression was correlated with FAB-M0/M1, whereas BCL2 under-expression was related to FAB-M5. However, BCL2 expression has no effect on overall survival (OS) and leukemia-free survival (LFS) of AML patients (determined in BCL2low and BCL2high groups). Interestingly, in the BCL2low group, patients undergoing autologous or allogeneic hematopoietic stem cell transplantation  had significantly better OS and LFS compared with patients only received chemotherapy, whereas, no significant difference was found in OS and LFS between chemotherapy and auto/allo-HSCT patients in the BCL2high group. BCL2 expression was found positively correlated with HOX family gene, and negatively correlated with tumor suppressor microRNA such as miR-195, miR-497, and miR-193b.BCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and cytogenetically normal AML.The online version of this article (10.1186/s13000-019-0841-1) contains supplementary material, which is available to authorized users. Acute myeloid leukemia (AML) represents for a molecularly, biologically, clinically, and etiologically heterogeneous disorder with variable outcome . DespiteBCL2 gene is found in human B-cell lymphomas, which is first identified through cloning the breakpoint of a translocation of t [BCL2 protein plays crucial role in inhibiting the influx of adenine nucleotides through the outer mitochondrial membrane, resulting in reducing ATP hydrolysis and inhibiting cytochrome-C release [BCL2, ABT-199, presents antitumor activity while sparing platelets [BCL2 overexpression in de novo adult AML, and may provide theoretical basis for targeted therapy using BCL2 inhibitor venetoclax.Located on chromosome 18q21.33, t . BCL2 pr release . Based olatelets . In 2016latelets , whetherlatelets . Herein,BCL2 expression data from The Cancer Genome Atlas (TCGA)  were identified and included in this study [A first cohort of 173 adult AML patients with is study . A totalA second cohort of 154 AML patients and 35 healthy donors was also enrolled in the study. The main clinical and laboratory features of the AML patients were presented in Additional file Bone marrow (BM) aspirate specimens were collected from 35 controls, 154 AML patients at diagnosis time, 48 AML patients at complete remission (CR) time, and 23 AML patients at relapse time. BM mononuclear cells (BMMNCs) were separated using Lymphocyte Separation Medium . Total RNA was extracted form BMMNCs using Trizol reagent . Reverse transcription was performed to synthesize cDNA using random primers as our previous reports –17.BCL2 mRNA using AceQ qPCR SYBR Green Master Mix . The primers used for BCL2 expression were 5′-CCCTGGTGGACAACATCG-3′ (forward) and 5′-CAGGAGAAATCAAACAGAGGC-3′ (reverse). Housekeeping gene ABL1 was detected by RT-qPCR using 2 × SYBR Green PCR Mix  [BCL2 mRNA levels were calculated using 2-∆∆CT method.Real-time quantitative PCR (RT-qPCR) was performed to examine , China) –17. RelaBCL2 expression in AML from TCGA data and controls was performed by GEPIA (http://gepia.cancer-pku.cn/detail.php) [http://string-db.org). The microRNA which could target BCL2 was identified by TargetScan (http://www.targetscan.org/vert_72/), mirDIP (http://ophid.utoronto.ca/mirDIP/), miRWalk , and miRDB (http://mirdb.org/miRDB/). All basic statistical analyses were performed using the base functions in R version 3.4 (https://www.r-project.org).The comparison of ail.php) . DiffereBCL2 expression on leukemia-free survival (LFS) and overall survival (OS) was analyzed though Kaplan-Meier analysis using Log-rank test. Univariate and multivariate proportional hazard regression analysis was performed using Cox regression. The P value (two-tailed) equal or less than 0.05 in all statistical analyses was defined as statistically significant.SPSS 22.0 and GraphPad Prism 5.0 were used for statistical analyses and figures creation. Mann-Whitney’s U test was used for the comparison of continuous variables, whereas Pearson Chi-square analysis or Fisher exact test was applied for the comparison of categorical variables. The prognostic effect of BCL2 expression data from public TCGA datasets was used for differential expression analysis. By using the GEPIA (http://gepia.cancer-pku.cn/detail.php), we found BCL2 expression in AML patients was significantly increased compared with GTEx normal BM samples  based on median level of BCL2 transcript. The comparison of clinical/laboratory characteristics of the AML patients between two groups were summarized in Table BCL2high and BCL2low groups in sex, age, BM blasts, and the distributions of cytogenetics (P > 0.05). However, BCL2high cases had significantly lower white blood cells (WBC) and higher peripheral blood (PB) blasts compared with BCL2low cases (P = 0.041 and 0.033). Additionally, significant differences in the distributions of FAB classifications and cytogenetics were found between two groups (P = 0.000). BCL2 overexpression was markedly correlated with FAB-M0/M1 (P = 0.038 and 0.015), whereas BCL2 under-expression was associated with FAB-M5 (P = 0.001). Among gene mutations, no significant differences were found, besides BCL2high tended to be associated with WT1 mutations (P = 0.057).In order to explore the clinical implication of BCL2high patients showed similar OS (median 26.3 vs 15.8 months) and LFS (median 11.1 vs 9.3 months) time compared with BCL2low patients  and CN-AML  , whereas the other 100 cases only received chemotherapy. In both chemotherapy and auto/allo-HSCT groups, BCL2 could benefit from auto/allo-HSCT, survival in patients with auto/allo-HSCT were compared among both BCL2high and BCL2low groups. In the BCL2low group, the patients undergoing auto/allo-HSCT had significantly better OS and LFS compared with patients only received chemotherapy among both total AML (OS median 56.3 vs 8.0 months and LFS median 13.8 vs 5.9 months) and CN-AML (OS median 56.3 vs 8.2 months and LFS median 13.8 vs 8.2 months)  and CN-AML (OS median 24.6 vs 15.5 months and LFS median 12.0 vs 8.6 months)  may affect prognosis in AML [BCL2 expression was not a valuable single factor that affecting prognosis in AML.In this study, we found and verified that  studies –28. Prev4 to 87% . Althougtigators , 20, we  reports . Interesotherapy –22, we drognosis , 24. In s in AML –28. ThusBCL2 acted as a negative regulator on cellular apoptosis and is a druggable target [BCL2 overexpression [BCL2 proteins to cause apoptosis is considered as a potential therapeutic approach in hematological malignancies [BCL2 inhibitor including ABT-737 and ABT-263/navitoclax were encountered with disappointment in clinic because of dose-dependent thrombocytopenia [BCL2 [BCL2 under-expression could benefit from auto/allo-HSCT, whereas patients with BCL2 overexpression did not benefit from auto/allo-HSCT.Apoptosis plays crucial roles in the command of tissue homeostasis, and is important in the clearance of infected, unwanted, or otherwise damaged cells . Meanwhie target , 30–32. gnancies –36. Earlytopenia . In 2013ia [BCL2 . By clinia [BCL2 , suggestBCL2 in AML to further get better understanding of AML biology. We found that BCL2 dysregulation was significantly associated with HOX gene family, which was reported highly correlated with hematopoiesis and leukemogenesis [BCL2 expression was negatively correlated with several microRNAs such as miR-195, miR-497, miR-135a, miR-196a, miR-193b, miR-455, miR-375, and miR-205, which were found to be associated with AML pathogenesis or patients prognosis by previous studies [miR-195 and miR-497 was identified as predicted microRNAs that could direct target BCL2. Obviously, further studies are needed to confirm the direct connections of BCL2 with microRNAs by luciferase assay.Herein, we further determined the molecular signatures associated with ogenesis , 39. Mor studies –44. Of tBCL2 overexpression identified specific FAB subtypes of AML, but it did not affect prognosis. Patients with BCL2 overexpression did not benefit from auto/allo-HSCT among whole-cohort-AML and CN-AML.Additional file 1:Clinic-pathologic characteristics in AML from our cohort. (DOCX 19 kb)Additional file 2:BCL2high and BCL2low. (XLSX 50 kb)Different expressed genes of microRNA for Additional file 3:BCL2high and BCL2low. (XLSX 1682 kb)Different expressed genes of RNA for Additional file 4:BCL2. (TXT 37 kb)Venn results of microRNAs targeting