To investigate the molecular background of the aggressive character of extramedullary disease and its differential treatment response, we performed amplicon-based next generation sequencing. An exon 4 (c.497_498insGA) frameshift RUNX1 mutation was exclusively found in all of the patient’s EM sites, but not in the BM or in peripheral blood samples at time of EM reoccurrence. In addition, we detected an exon 13 (c.3306G>T) ASXL1 point mutation only in the retroperitoneal tumor tissue at the time of the fourth relapse. In contrast to the patient’s intermediate-risk BM AML at diagnosis according to ELN2017, EM sites showed molecular adverse-risk features implicating intensified strategies like cellular therapies. Notably, disease relapse could only be detected by imaging throughout the course of disease. This case demonstrates both the necessity of continuous molecular profiling of EM to reveal differential molecular composition of EM and BM-derived AML, supposedly leading to divergent susceptibility to established therapies, as well as recurrent 18FDG-PET-imaging for detecting residual disease and assessment of treatment response in case of EM AML.Although extramedullary manifestations (EMs) are frequent in patients with acute myeloid leukemia (AML), they are often not detected during clinical workup and neither imaging- nor molecularly based diagnostic strategies are established to reveal their existence. Still, the detection of EM is essential for therapeutic decision-making, as EM present with aggressive and resistant disease and since mutational profiling might render patients within a different risk category, requiring personalized therapeutic strategies. Here, we report the case of an AML patient presenting with AML bone marrow (BM) infiltration and molecularly distinct EM at time of diagnosis followed by multiple EM relapses while undergoing several intensive chemotherapies including allogeneic hematopoietic cell transplantations .  NF1-deletion (del17q11). No other genetic abnormalities were detected using routine molecular analysis methods [fragment length analysis and amplicon-based next generation sequencing (NGS)].A 25-year-old man presented with fatigue, recurrent episodes of fever and infectious symptoms, night sweats, and inappetence. In addition, dyspnea and chest pain during inspiration were reported. Furthermore, rash on both arms was described. Hematologic workup revealed leukocytosis of undifferentiated cells, which were characterized as myeloid progenitors by immunophenotyping. Bone marrow (BM) aspiration and biopsy confirmed the diagnosis of acute myeloid leukemia (AML) . Cytogen2 daunorubicin (day 3–5) and 100 mg/m2 cytarabine (day 1–7) (DA) was initiated. As the patient complained of chest pain and newly onset of fever episodes, computed tomography (CT) of the thorax was performed on day 6 of DA, depicting a large mediastinal tumor and ground-glass opacities. 18Fluorodesoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT) demonstrated mediastinal vital tissue [maximum standardized uptake value (SUVmax) 5.3; Induction chemotherapy with 60 mg/mpos) with persisting del17q11 detection. A second cycle of DA was initiated leading to cytomorphologic and cytogenetic complete remission (CR). Follow-up CT showed the mediastinal tumor unchanged in diameter. No further extramedullary manifestation (EM) sites were traced in clinical examination and by imaging.BM assessment at day 15 showed moderate response and positive cytogenetic measurable residual disease (MRDversus host disease (GvHD) prophylaxis consisted of methotrexate (MTX) and cyclosporin (CsA). On day +8, the patient reported sudden and severe chest pain again and imaging revealed the mediastinal tumor with unchanged diameters. The patient developed full donor chimerism until day +56 and remained in cytomorphologic and cytogenetic CR. Immunosuppression was tapered rapidly and discontinued 6 months after transplantation.The patient underwent allogeneic hematopoietic cell transplantation  from a matched sibling donor after receiving myeloablative conditioning with 120 mg/kg cyclophosphamide and 12 Gy fractionated total body irradiation (TBI), which was tolerated without unexpected toxicities. Graft NF1-deletion. In addition, 18FDG-PET/magnetic resonance imaging (MRI) was further suspicious for localized iliac EM (SUVmax 5.4), while the initial mediastinal tumor (SUVmax 3.5–5.2) and the duodenal tumor (SUVmax 3.7) also showed metabolic activity. Surprisingly, BM aspiration showed a persisting CR (2 BID high-dose cytarabine (day 1–3) and 5 mg/m2 mitoxantrone  was started, which induced a metabolic CR of the EM according to 18FDG-PET/MRI. At the time of referral to the transplantation ward for second alloHCT, the patient reported about a cutaneous tumor on the chest that rapidly grew within the last few days. A punch biopsy verified again leukemic infiltration in this lesion . MTX and CsA were applied as GvHD prophylaxis. Full donor chimerism was achieved again; no further sites of chloroma appeared. BM aspirate revealed persisting CR. The patient developed acute GvHD grade III of the upper and lower gastrointestinal tract and the skin, which required repetitive escalation of immunosuppression. However, a follow-up 18FDG-PET/MRI demonstrated relapse of the mediastinal mass 1 month after the second transplantation (SUVmax 6.6). Local radiation therapy of the mediastinal tumor with a cumulative dose of 40 Gy was conducted leading to a significant shrinking of the mediastinal tumor burden. Three months later, the patient showed new signs of progression, this time with multiple EMs detected below the left kidney causing urinary obstruction, paravertebrally, and in the coeliac trunk  was used; for formalin-fixed, paraffin-embedded (FFPE) EM material, the QIAamp® DNA Mini Kit  was used. In the case of FFPE material, tumor area was marked before on a hematoxylin–eosin (HE) stained slide, and only tumor areas were utilized for DNA extraction. DNA concentration was quantified using the QuantiFluor® dsDNA System (Promega) in case of BM samples and the Qubit™ dsDNA BR Assay  for DNA isolated from FFPE samples. In addition, DNA extracted from a salivary sample obtained at diagnosis with the ORAgene One kit  was included as germline control.Genomic DNA was extracted from pre-treatment and relapse BM aspirates and EM sites obtained at time of occurrence. For BM samples, the Maxwell®), which was designed particularly for AML. The panel covers mutation hotspots or whole genes of ASXL1, BRAF, CBL, CEBPA, DNMT3A, FLT3, GATA2, IDH1, IDH2, JAK2, KRAS, MPL, NPM1, NRAS, RUNX1, SETBP1, SRSF2, TET2, TP53, U2AF1, WT1, and ZRSR2. During library preparation, unique molecular barcodes and sample-specific indices were incorporated according to the manufacturer’s protocol. Indexed libraries were then quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and paired-end sequenced (2 × 200 bp) on an Illumina® MiSeq platform . HG19 was used as reference genome for bioinformatic analyses. The bioinformatics evaluation was performed using the Biomedical Workbench from CLC  using a customized analysis algorithm with the following filters: coverage ⩾200 and allele frequency ⩾1%. Synonymous sequence variants and polymorphisms were not reported. Sequence variants with a change in the amino acid sequence were specified according to HGVSc and HGVSp with details of the underlying reference sequence and allele frequency. NGS-based assessment of MRD for single nucleotide variants (SNVs) was performed using an error-controlled deep sequencing procedure, as described in detail recently. The study was reported according to the CARE guideline (Supplementary File). Patient details were de-identified.In total, 100 ng of DNA was used as input for library preparation. The regions of interest were amplified using a custom designed amplicon NGS panel  revealed by cytogenetic analysis, no AML-specific genetic abnormalities according to the ELN2017 classification were found in the BM at diagnosis, classifying the patient’s AML as intermediate-risk. An RUNX1 frameshift mutation in exon 4 (c.497_498insGA) with a variant allele frequency (VAF) of 31.1% could be detected in the mediastinal EM from initial diagnosis  mutation (limit of detection 0.1%). In contrast, the duodenal AML manifestation harbored the same exon 4 RUNX1 mutation (VAF 26%) as the initial mediastinal manifestation (RUNX1 mutation (VAF 18.9%) (RUNX1 mutation (VAF 40.6%)  after achieving CR and MRDneg after first alloHCT. Furthermore, pB and BM samples demonstrated full donor chimerism throughout the following course of disease, making it impossible to detect AML reoccurrence via routine laboratory tests.Except for an iagnosis , howevernuous CR  without estation . MutatioF 18.9%) . All thrF 40.6%) , we furtM tissue . Again, 47 The prospective PETAML trial demonstrated EM occurring frequently at time of diagnosis (22%) with the majority of these AML patients still harboring active EM in follow-up imaging after chemotherapy despite CR in BM. Still, EMs are often under-reported during routine clinical workup, as some manifestations are only detectable by in-depth imaging series revealing only the ‘tip of the iceberg’. Interestingly, EM can occur and relapse with or without concomitant BM infiltration, suggesting differential disease biology.9 There have been a few reports demonstrating differences in metabolic pathways and forms of immune escape phenomena between BM AML and EM AML.11 Also, the clonal composition of EM in contrast to BM-derived AML as well as unique features and differences in genetic and phenotypic characteristics causing EM disease biology have not been investigated comprehensively. There is emerging evidence on significant differences and substantial heterogeneity in clonal composition of EM versus BM AML, however, begging the question of a (simultaneous) development of distinct AML clones and sites with different genetic features, disease characteristics and kinetics, as well as vulnerable targets to elaborate that might translate into adjusted therapeutic requirements.1216Although the prognosis for patients with AML has improved significantly over the last decades, EMs still represent a clinical dilemma because of a variety of affected tissues, highly resistant disease characteristics, and frequent relapse even in the face of intensive treatments.Supplementary Table 1) implicating worse prognosis and the need for intensified strategies like cell-based approaches. We assume that these differences of BM AML and EM AML are one of the components for differential response and the aggressive character of many EMs. We further hypothesize that the differential features of EM AML compared with BM AML may possibly be a general underlying concept, especially in AML cases with refractory behavior, aggravated by the ability of EM sites to undergo clonal evolution and intensify immune escape. The escape of EM at ‘sanctuary sites’ is also reflected by the frequent progression of EM after alloHCT despite the occurrence of clinically significant acute and chronic GvHD, as seen in our patient.17 As the clinical course described herein suggests, EM may undergo clonal evolution throughout sequential treatment by gaining additional mutations conveying resistance against the chosen salvage regimens. Further studies should focus on the clonal heterogeneity of extramedullary disease, the differences in phenotypic properties and genetic composition compared with their BM counterparts, and potential changes during the course of disease.Here, we report a paradigmatic case of a young patient with two distinct AML clones at diagnosis, which differed both in genetic markers, disease localization, as well as response to leukemic treatment and disease kinetics. In contrast to routine hematologic and genetic workup of the BM at diagnosis classifying the patient’s AML as intermediate-risk according to the ELN2017 classification, EM sites showed genetic adverse-risk features , a finding that was also confirmed in our patient with both skin and mediastinal involvement at diagnosis. A lesson learnt from this case vignette is the added information that may be derived from repetitive biopsies with histological and molecular workup. Also, repeated Importantly, treatment of AML patients with EM is an interdisciplinary effort, both in case of organ involvement but also during acquisition of biopsy material and diagnostic workup. Further research is needed to address drivers and pathways of clonal evolution, metabolic components, as well as characteristic traits of differential EM sites. Results may refine our concepts of EM and improve miscellaneous therapeutic strategies and future targeted approaches beyond standard AML therapies.Click here for additional data file.Supplemental material, sj-docx-1-tah-10.1177_20406207221115005 for Spatial heterogeneity and differential treatment response of acute myeloid leukemia and relapsed/refractory extramedullary disease after allogeneic hematopoietic cell transplantation by Desiree Kunadt, Sylvia Herold, David Poitz, Lisa Wagenführ, Theresa Kretschmann, Katja Sockel, Leo Ruhnke, Stefan Brückner, Ulrich Sommer, Frieder Meier, Christoph Röllig, Malte von Bonin, Christian Thiede, Johannes Schetelig, Martin Bornhäuser and Friedrich Stölzel in Therapeutic Advances in Hematology


Structural and physical properties of PA are well studied but recently two highly conserved structural motifs consisting of three amino acids each (clusters I and II), which contribute to the hydrophobic core of the EF-hand domains, have been revealed. Despite several decades of studies, physiological functions of PA are still poorly known. Since no target proteins have been revealed for PA so far, it is believed that PA acts as a slow calcium buffer. Numerous experiments on various muscle systems have shown that PA accelerates the relaxation of fast skeletal muscles. It has been found that oxidation of PA by reactive oxygen species (ROS) is conformation-dependent and one more physiological function of PA in fast muscles could be a protection of these cells from ROS. PA is thought to regulate calcium-dependent metabolic and electric processes within the population of gamma-aminobutyric acid (GABA) neurons. Genetic elimination of PA results in changes in GABAergic synaptic transmission. Mammalian oncomodulin (OM), the β isoform of PA, is expressed mostly in cochlear outer hair cells and in vestibular hair cells. OM knockout mice lose their hearing after 3–4 months. It was suggested that, in sensory cells, OM maintains auditory function, most likely affecting outer hair cells’ motility mechanisms.Parvalbumin (PA) is a small, acidic, mostly cytosolic Ca It was found in lower and higher vertebrates, including humans  is a small, acidic, mostly cytosolic Caews, see ,2,3,4). 2+-mediated relaxation phase in fast muscles . It is iewed in ,6,7. BioDespite many years of research on PAs, which have provided a good understanding of their structure and physicochemical properties, the physiological functions of PA are still far from being clear. This short review covers some new findings on structural and physicochemical properties of PAs and focuses on their potential physiological significance.PA consists of 106–113 amino acid residues and its molecular mass is ~11–12 kDa. Isoelectric points of PAs are within the range of 3.9 to 6.6 see . PAs areThe PA family diverged into α and β sub-lineages ,9,10. β-2+-binding domain is composed of two α-helices linked by a Ca2+-binding loop creating a structural motif, which is currently known as ‘EF-hand’.PA molecule is a compact globule composed mostly of α-helices and a small amount of β-strands (reviewed in ) Figure. Kretsin2+-binding loop of the EF-hand usually contains the most commonly observed consensus sequence DxDxDG. The negatively charged Ca2+-liganding oxygen atoms donated by carboxyl and carbonyl groups are located at vertices of a distorted octahedron. The side chains of loop residues at positions 1, 3, 5, and 12 provide the +x, +y, +z, and −z oxygen ligands, respectively. Water molecule oxygen is located at the −x position, and a backbone carbonyl oxygen occupies the −y position. In some cases, the carboxylate at −z position coordinates calcium by both oxygens and the octahedral geometry of the Ca2+ binding site turns into a distorted pentagonal pyramid geometry.Ca2+, since its AB-loop is too short and the DxDxDG motif is disturbed. Meanwhile, the AB domain shields the hydrophobic core of the protein and the hydrophobic parts of the functional EF-hands from solvent and influences their calcium affinities [PA molecule contains two active EF-hands, CD and EF domains. The AB domain of PA cannot bind Cafinities ,14,15. TSome of the PAs contain cysteine residues , but they do not form S–S links under the non-oxidizing conditions.2+-binding loops [Comparative analysis of the spatial structures of proteins of the EF-hand superfamily, including PA, made it possible to discover two highly conserved structural motifs, which form a supporting scaffold for the Cang loops . Each cong loops . In contng loops .2+- and Mg2+-loaded forms, and total energy of Ca2+ binding in comparison with the changes caused by similar amino acid substitutions in the cluster II. In agreement with these experimental results, it was found that local intrinsic disorder propensities and the overall levels of predicted disorder in rat β-PA are strongly affected by mutations in cluster I, whereas mutations in cluster II generate less pronounced effects [2+ by a protein with two binding sites is as follows [The general  follows ,18:2+ to PA occurs in two ways when the CD and EF binding sites are filled by Ca2+ simultaneously. At the same time, experiments carried out on mutant human and rat parvalbimins demonstrate that the substitution of one of the Ca2+ chelating Glu by Gln in the CD site completely abolishes the high metal affinity of parvalbumins, whereas similar mutation in the EF site abolishes the high affinity metal binding to the EF site and only causes a decrease in the metal affinity of the CD site by an order of magnitude [2+/Mg2+ to parvalbumin is a sequential process (1) and the CD site is occupied firstIn the agnitude . This su2+ binding to PA . At the EF site . Pauls e EF site  found th2+ ions. In fact, equilibrium Ca2+-association constants of PAs measured at low ionic strength lie in the range from 108 to 1010 M−1  and dissociation  (2):Equilibrium metal association constant K2+ and Mg2+ dissociation from cod PA after rapid mixing of the metal bound protein with a strong metal chelator, EDTA [off values for Ca2+ and Mg2+ are rather close to each other in the temperature region from 10 to 30 °C. Two exponential terms in the kinetic curves were interpreted as Ca2+/Mg2+ dissociation from the two binding sites of PA. The values of dissociation rate constants obtained from these analyses at temperatures from 10 °C to 30 °C are within the range 0.03–0.8 s−1 and 0.2–5 s−1 for Ca2+-PA complex and 0.9–4.5 s−1 and 4–33 s−1 for Mg2+-PA complex. It means that the differences in the equilibrium binding constant values for Ca2+ and Mg2+ (Ka) are mostly due to the differences in the kon values. The kon values for Ca2+ calculated from these data are within the range from 106 to 109 M−1s−1 (temperature range 10 to 30 °C); i.e., they are close to the diffusion-controlled limit. The kon values for Mg2+ are 2 to 5 orders of magnitude lower. The drastically lower kon values for Mg2+ this can be explained by the fact that Mg2+ ion in aqueous solution is much more strongly hydrated compared to Ca2+ ion due to the fact that it has 3.5 times higher charge density.Fluorescent stopped flow method was used to study kinetics of Caor, EDTA . Analysi2 and 84 ± 11 Å2, respectively) [2+ binding proteins, the interaction with calcium also slightly changes the contact surface area in the cluster II (68 ± 12 Å2). In the other group of Ca2+ binding proteins, calcium binding results in a dramatic increase in the interactions between the three residues of cluster II (contact surface area 11 ± 8 Å2). PA belongs to the first group of proteins: the binding of Ca2+ does little to change the interactions in both conservative clusters [Analysis of the 3D structures of the EF-hand proteins with and without bound calcium showed that the binding of calcium does not dramatically change interactions within cluster I  . In someclusters . 2+ from PA results in a decrease in its α-helical content [2+-induced changes in α-PAs and β-PAs is different [2+-loaded state has approximately the same three-dimensional structure. Ca2+ binding to α-PA causes changes mainly in the structure of the loop regions. In contrast to this, Ca2+ binding to rat β-PA results in a conformational change in the AB domain; rotation of the C, D, and E helices; changes in interdomain contacts; and rearrangement of the hydrophobic core. However, all these changes do not affect the overall protein folding. In the Ca2+-loaded rat β-PA, Phe70 is buried in the protein core, but in the apo-protein is located near the protein surface. Similarly, the single Trp of cod or whiting β-PA is buried deep inside the Ca2+-loaded protein, but Ca2+ removal causes a movement of this Trp to the protein surface [Removal of bound metal ions from PA (formation of apo-form) makes its structure looser and makes many of its groups more accessible to the solvent (reviewed in ). The re content ,23,24. Iifferent ,26. Rat  surface . mT, of apo-PAs under low ionic strength is within the range from 30 °C to 50 °C [2+ removal from such PAs decreases their α-helical content, and increases mobility of the aromatic residue environment, monitored by near-UV circular dichroism method. These results show that pike apo-α-PA is in the classical molten globule state [® VSL2 showed that the N-terminal region of PA—including α-helix A, AB-loop, and N-terminal half of α-helix B—was predicted to be less ordered in PAs with disordered apo-state [Thermal stability of apo-PA is low: mid-transition temperature, to 50 °C ,29,30. Cto 50 °C . Interesto 50 °C . This isle state ,32,33. Apo-state . It has 2+- and Mg2+-loaded states of five PAs  was studied by scanning calorimetry method in a temperature range from 15 °C to 125 °C [2+-loaded state (rat PAs). The second group is characterized by two distinct heat-sorption peaks in their Ca2+-loaded state (cod PA and pike α-PA). The unfolding behavior of the proteins of the last group (pike β-PA) above 100 °C is complicated by an oligomerization process. An analysis of the thermal denaturation curves of the Ca2+-bound PAs of the first group showed that their unfolding is not described by the simple ‘all-or-none’ scheme, and the process proceeds with a formation of an intermediate state. The multistage character of the thermal unfolding is evident in the melting curves of the PAs of the second group (pike α-PA and cod PA). They are characterized by two distinct heat-sorption peaks separated by 20–30 °C [Thermal denaturation of Cao 125 °C . The pro20–30 °C .2+-bound state, Mg2+- and Na+-bound states of pike α-PA are characterized by a single heat sorption peak [2+ binding causes expectedly less pronounced stabilizing effect in comparison with that induced by Ca2+ binding . Na+ binding results in even less pronounced stabilization (mT is ca 33 °C in 300 mM NaCl). The thermal unfolding of both Mg2+- and Na+-loaded states of pike α-PA obeys the simple ‘all-or none’ mechanism. In contrast to Caion peak . Mg2+ bi2+ by Ca2+ ions in the PA binding sites seriously alters its thermodynamic properties: the simple ‘all-or-none’ transition converts into a more complex multistage transition. Moreover, depending on its interactions with metal ions, PA  can be an intrinsically disordered protein (apo-form) or an ordered mesophilic (Na+-bound state), thermophilic (Mg2+-bound state), or even hyperthermophilic (Ca2+-bound state) protein [These results show that the exchange of Mg protein .2+-loaded PAs seems to arise due to stabilization of an intermediate state, which is absent in PAs exhibiting single peak in their heat-sorption curves. It has been found [® analysis showed the EF subdomain is included in the most stable thermodynamic domain in pike α-PA, whereas the CD and AB subdomains are contained in the less stable thermodynamic domain.The additional peak in the heat-sorption curves of some Caen found  that the2+/Mg2+ exchange in the binding sites of PAs results in alternation in the metal ion coordination mode: the coordination number decreases from 7 in the Ca2+-bound state to 6 in the Mg2+-bound state [2+-bound state and as a monodentate ligand in the Mg2+-bond state, which can change the protein conformation and seriously affect thermodynamic properties of the protein [It is well known that Cand state ,36,37. T protein . 6 M−1 at 18 °C), while their Ca2+- and Mg2+-loaded states do not bind this peptide [7 M−1 at 44 °C). Isolated Ca2+-binding fragment 38–108 of pike PA pI 5.0 (CD-EF) retains the ability for Ca2+-inhibited equimolar binding of melittin. So far, no target proteins have been found for rat or mouse PA. At the same time, it was shown that under certain conditions, PA is able to interact with short peptides. It was found that pike PAs pI 4.2 and 5.0 bind amphiphilic bee venom peptide melittin (26 amino acid residues) . In apo- peptide . Heating2+-sensor protein of the EF-hand family, calmodulin, binds melittin with a very high binding constant 1010 M−1 in the Ca2+loaded state [2+, calmodulin binds melittin with a much lower affinity, with the binding constant 105 M−1 [In contrast, the well-known Caed state ,40. In tSince both intracellular and extracellular PA is always found in an environment with high concentrations of divalent calcium and magnesium cations, it almost never exists in the apo-state. For this reason, its interaction with the amphiphilic peptide, discovered in , is unli2+ and Mg2+ ions to PA modulates its interaction with the DPPC vesicles. The interaction of the liposomes with Ca2+-bound PA shifts its thermal transition towards higher temperatures by 2 °C to 3 °C, while the interaction of the liposomes with the metal-free and Mg2+-bound protein results in similar shift of the thermal transition but towards lower temperatures. Moreover, the interaction of the protein with the liposomes causes changes in the calorimetric heat sorption peak of model synthetic liposomes. The denaturation curves measured by tryptophan intrinsic fluorescence method for free and liposome-bound cod PA are strikingly different. The thermally induced red spectral shift for the liposome-bound protein, which reflects an unfolding of the protein, begins at 10 °C lower temperatures than that for free PA, but the shift magnitude is less pronounced in this case. Tryptophan residue in the thermally denatured liposome-bound cod PA remains inaccessible to water, while the thermal unfolding of the free protein causes a translocation of its single Trp to the protein surface.It has been found that cod and pike PAs interact with model synthetic  and natural (phosphatidylcholine and phospatidylethanolamine) vesicles (mean diameter 300–500 Å) . It has Drosophila melanogaster [Interestingly, PA immunoreactivity was found in (or near) membranous systems, such as mitochondria and (or) microtubule in male gonads of nogaster .2+ translocation within the sarcoplasm and therefore could be a relaxation factor in fast-twitch muscles [Of the possible biological functions of PA, perhaps the one that has been explored the most is that in muscle cells. It has long been known that PA concentration in muscles correlates with the speed of muscle contraction and relaxation. PA concentration in sarcoplasm of fast skeletal muscles reaches a millimolar level, while slow-twitch muscles are characterized by about two orders of magnitude lower levels of PA ,44,45. T muscles ,44.2+ concentration in sarcoplasm of a resting muscle is very low (<1 μM), while free Mg2+ concentration in the muscle is in the millimolar region. For this reason, PA in the sarcoplasm of a resting muscle seems to be loaded by Mg2+ ions. Ca2+ transiently released from the sarcoplasmic reticulum diffuses through the sarcoplasm and activates muscle contraction interacting with troponin complex located on the actin fibrils. PA is located in the sarcoplasm of muscle cells right on the way of the Ca2+ movement, but since the exchange of Mg2+ for Ca2+ in the binding sites of PA is governed by the relatively slow process of Mg2+ dissociation, Ca2+ ions can reach the Ca2+-specific sites of troponin C [2+/Mg2+-sites of PA will compete for Ca2+ and promote its removal from troponin C, which will start muscle relaxation. Since the dissociation rate of Mg2+ in PA is low, it is reasonable to think that PA will not have time to remove calcium during a single contraction–relaxation cycle (twitch), especially in fast muscles. Instead, the Mg2+-Ca2+ exchange in PA can only happen after many successive twitches (after tetanus). Below are the results of some experiments indicating the important role of PA in accelerating the relaxation of fast muscles.Free Caoponin C . Later oCross-reinnervation of slow- and fast-twitch muscles results in a decrease in PA content in the fast-twitch muscle, and to an increase in PA content in the slow muscle ,48. PA c2+ level after a 20-ms stimulation of the isolated exterior digitorum longus (33% lower rate constant of Ca2+ decay), and in a reduction in the time to reach peak twitch tension [Direct gene transfer experiments were carried out to check whether PA really could work as a relaxing factor. PA cDNA was transferred in vivo to normal and regenerating rat soleus muscles, which do not normally synthesize PA . Conside tension .2+, while the fast relaxation recovery after the increase in rest interval by a formation of Ca2+-free PA. In flexor digitorum brevis muscles, the effects of tetanus duration on wild type and PA-deficient muscles were qualitatively similar to those observed in extensor digitorum longus muscles. The authors concluded that PA accelerates calcium movement from myofibrils to sarcoplasmic reticulum, mostly after tetanus. It was found that the relaxation rate of extensor digitorum longus in PA-deficient mice at 20 °C is low and does not depend on tetanus duration (<3.2 s) . In cont2+ released during the call and this Ca2+ moves back into the sarcoplasmic reticulum during the long intercall interval [Porichthys notatus) is another fish which produces sounds with a frequency 80–100 Hz at a temperature of 12–15 °C with a 100% duty cycle without any intercall intervals. In this case, PA seems to be of little use as a relaxation factor since it would become saturated at an early stage of calling. Tikunov and Rome [The superfast toadfish swimbladder muscle, the fastest vertebrate muscle known , is charinterval . Midshipand Rome  found th2+ dissociates from troponin C and binds to PA, which causes cardiac myocyte relaxation. Now PA is tested as a pharmacological agent to treat heart dysfunctions. For example, it has been found that PA fully normalizes the relaxation rate in diseased cardiac myocytes taken from an animal model of human diastolic dysfunction [2+ coordination in the CD binding site) to treat diastolic heart failure. E101D PA is characterized by 114-fold decreased Ca2+ affinity and 28-fold increased Mg2+ affinity compared to the wild type PA. E101D PA increased contraction amplitude of myocytes compared to both untreated myocytes and myocytes with E101Q PA, with slight improvements in relaxation. Moreover, E101D PA increased spontaneous contractions after pacing stress.In addition to fast skeletal muscles, PA has also been found in cardiac muscle tissue of rat, mouse, chicken, rabbit, and pig ,57, and function . PA genefunction . Similarfunction : they fofunction  used mut2+ decay [Summarizing, we can conclude that a lot of experimental data indicate that in many cases, PA can accelerate the relaxation stage of fast muscles, but this effect is not too pronounced, as the PA gene knock out results in 33% lower rate constant of Ca2+ decay . 2●–). Later O2●– is converted to hydrogen peroxide (H2O2) (reviewed in [2O2 is one of the intracellular reactive oxygen species (ROS). It is most abundant (0.1 μM) and long living  ROS, which acts both as an inducer of oxidative damage and a signaling molecule [2●– and H2O2. It is of interest that PA immunoreactivity was found in (or near) mitochondria [Interestingly, fast muscles with high PA concentrations are characterized by the highly efficient oxygen consumption (oxygen uptake up to 57 mL/min ). The moiewed in ). H2O2 imolecule . The majchondria .2+-loaded protein, while AOC for Mg2+-bound PA is similar to that of apo-PA. The interactions of PA with ROS causes oxidation of its Phe residues. It has been found that total antioxidant capacity of PA under in vivo conditions may reach the level of reduced glutathione. For this reason, one can suggest that one more physiological function of PA in fast muscles could be a protection of these cells from reactive oxygen species. Moreover, PA could change intracellular redox equilibria and signaling in a Ca2+-dependent manner.We have explored the possibility that PA could serve as a free radical scavenger. ORAC , TEAC , and hydrogen peroxide AOC assays were used to study antioxidant capacities (AOC) of various forms of intact rat α-PA . We have2+ buffering, Ca2+ transport, regulation of activity of various enzymes, and a protection of neurons against calcium overload. It is assumed that neurons containing high concentrations of these Ca2+-binding proteins would be more resistant to degeneration due to their Ca2+ buffering capacity [Calcium binding proteins of the EF-hand superfamily, parvalbumin, calbindin, and calretinin, have been found in various classes of inhibitory interneurons as well as in some pyramidal neurons in the mammalian neocortex  neurons    at GABAergic synapses between interneurons and Purkinje cells, both in wild-type (PA+/+) mice and in PA knockout (PA−/−) mice. They found paired-pulse depression in PA+/+ mice, but paired-pulse facilitation in PA−/− mice. 1 mM Ca2+ buffer EGTA rescued paired-pulse depression in PA−/− mice. The authors concluded that PA can effectively modulate short-term synaptic plasticity.Caillard et al.  suggeste2+ buffer but affects such fast processes as transmitter release at hippocampal and cerebellar GABAergic synapses. Eggerman and Jonas tried to resolve this paradox [It has been found that genetic elimination of PA results in changes in GABAergic synaptic transmission, such as an enhancement of synaptic facilitation, a reduction in asynchronous transmitter release, and an increase in the power of gamma oscillations ,76,77. P paradox . They meBoth in neurons and muscle fibers, PA expression strongly depends on their activity. Decreased neuronal activity or oxidative stress in PA containing neurons causes a decrease in PA-immunoreactive neurons, PA messenger RNA, and PA levels ,80,81.2+ transients in neurons are quite similar . It was muscles  and Purk muscles  of PA−/−myotubes  and MDCKmyotubes .PA containing neurons are involved in some neuropsychiatric diseases, in particular in schizophrenia and bipolar disorder and in neurodevelopmental disorders including autism spectrum disorder (ASD) . The deg2+ levels induced by oxidation will result in loading of PA with Ca2+ ions, which, in its turn, will decrease its AOC [2+ may initiate specific signaling cascades leading to cell death [Oxidative stress in PA-GABAergic interneurons and their dysfunction associated with a decrease in the levels of both PA and reduced glutathione (GSH) are hallmarks of the development of schizophrenia ,89,90,91 its AOC . For thell death .Mammalian oncomodulin (OM) is the β isoform of PA, which has at least 53% sequence identity with α-PA ,94. Init2+ buffer in these cells.Essential features of mammalian OM are similar to those of most other vertebrate β-PAs, such as a length of 109 amino acid residues, isolectric point < 5.0, and a cysteine at position 19. In spite of this, it has been found that mammalian OM and lower vertebrate β-PAs belong to different phylogenetic lineages (reviewed in ). In ver2+-free and Ca2+-bound rat OM [2+ from OM results in structural alterations , which are more pronounced in comparison with those caused by Ca2+ removal from α-PAs. This is illustrated by 2+-bound and Ca2+-free forms of rat OM , thfinities  (reviewefinities ). Thus, r et al.  suggeste2+ buffers, PA, calbindin 1, and calbindin 2, resulted in only minor impacts on hearing [2+ buffers cannot replace OM function. Climery et al. [Analysis of recent experimental data showed that OM may play an ambiguous role that depends upon the cell type in which it is expressed . Experim hearing , which sy et al.  suggesteOM has been found in macrophages and neutrophils. Benowitz et al. have studied the role of OM in an inflammatory-mediated nerve regenerative model ,109,110.Initially, OM was found in several types of mouse, rat, and human tumors, which made it attractive as a potential cancer marker . Later o2+ [OM does not cause transformation of cultured cells , but neo2+ . Glutath2+  that sho2+  and Clay2+  did not 2+/Mg2+ dependent chemoattractants. They elicit the vomeronasal organ-mediated predatory behavior in Thamnophis marcianus. These results show that PAs’ locations are not strictly intracellular, but that they can also be found in exocrine secretions.Two proteins were isolated from frog cutaneous mucus proteome, which take part in prey recognition by snakes of the genus Thamnophis . These pHigh PA immunoreactivity was found in the Leydig cells during testicular development at the stages of intensive testosterone synthesis . PA leve+-Cl- cotransporter (NCC) in the early DCT. Genetically modified (PA−/−) mice showed increased diuresis and kaliuresis at baseline with higher aldosterone levels and lower lithium clearance. Endogenous NCC expression was found to be Ca2+-dependent and modulated by PA expression level. It has been concluded that, in this system, PA regulates the expression of NCC by modulating intracellular Ca2+ signaling in response to ATP in DCT cells [It has been found that PA is selectively expressed in the early distal convoluted tubule (DCT) of kidney . PA is cCT cells ,126.https://thebiogrid.org/576309; accessed on 23 April 2022). Furthermore, analysis human oncomodulin by STRING platform [Although no target proteins have been described for the rat PA, human oncomodulin was reported to interact with mitochondrial creatine kinases 1A and 1B, syndecan binding protein (syntenin), ubiquitin-conjugating enzyme E2A, and WAP four-disulfide core domain 1  using a set of commonly utilized disorder predictors—such as PONDR® VLXT , PONDR®  [® VLS2 , PONDR® 2 [® VL3 , PONDR® 3 [® FIT , IUPred23 [® FIT ,133. Res[® VLS2B , which w[® VLS2B , 73.4% r2+-bound form in the presence of 100 mM NaCl.This classification is based on the accepted practice of grouping proteins based on their levels of ADS and/or percent of predicted disordered residues (PPDR). Here, proteins are considered as highly ordered, moderately disordered, and highly disordered if they satisfy the following criteria: ADS < 0.15 (PPDR < 10%), 0.15 ≤ ADS < 0.5 (10% ≤ PPDR < 30%), and ADS ≥ 0.5 (PPDR ≥ 30%), respectively . One mig® VLXT), all residues in oncomodulin are characterized by the disorder scores exceeding 0.15, suggesting that the entire protein is expected to be either flexible or disordered. This important observation suggests that the unique 3D structures reported for PAs are stabilized by interaction of these proteins with metal ions.2P2 platform  [To shed more light on the potential role of intrinsic disorder in functionality of PAs, we used Dil 2022) , which iTaken together, data presented in this review indicate that PAs can serve as illustrative examples of the “protein structure-function continuum” model ,148,149 2+ decay. Since fast muscles with high PA concentrations are characterized by highly efficient oxygen uptake, it was suggested that one more physiological function of PA in fast muscles could be a protection of these cells from reactive oxygen species. It was found that the oxidation of the PA by reactive oxygen species is conformation-dependent: antioxidant capacity (AOC) value for apo-PA 4–11-fold exceeds that for the Ca2+-loaded protein, while Mg2+-bound PA has AOC similar to that of apo-PA.Despite several decades of study of PA, its physiological functions are still poorly known. This is surprising since the concentration of PA in some organs and tissues reaches a millimolar level. No target proteins have been revealed for PA so far. It is believed that PA acts as a slow calcium buffer. PA concentration in muscles correlates with the speed of their contraction and relaxation. Numerous experiments on various muscle systems have shown that PA accelerates the relaxation of fast skeletal muscles, not during a single contraction–relaxation cycle, but after tetanus. The effect is not very pronounced and PA gene knock out results in about 30% lower rate constant of CaThe role of PA in neurons is even less understood. In the rat somatosensory cortex, PA is contained only in gamma-aminobutyric acid (GABA) neurons . PA is thought to regulate calcium-dependent metabolic and electric processes within such neurons. Genetic elimination of PA results in changes in GABAergic synaptic transmission, including an enhancement of synaptic facilitation, a reduction in asynchronous transmitter release, and an increase in the power of gamma oscillations.Mammalian oncomodulin (OM), the β isoform of PA, is expressed mostly in cochlear outer hair cells and in vestibular hair cells. It has been found that OM knockout mice start to lose their hearing at 1–2 months and are essentially deaf after 3–4 months. For this reason, it was suggested that, in sensory cells, OM maintains auditory function, most likely affecting outer hair cells motility mechanisms. In immune cells, OM seems to be secreted in response to inflammation and its function in this case is facilitation of axon regeneration.All of the above allows us to conclude that the elucidation of the physiological role of parvalbumins in various tissues and organs is undoubtedly worth continuing.


NPM1-mutated acute myeloid leukemia (AML) represents about one-third of all adult AML [NPM1 mutations, the most common genetic lesion in AML, abrogate the ability of the protein to localize in the nucleolus and create a new nuclear export signal (NES) at the C-terminus, leading to enhanced nuclear export of mutant NPM1 and its aberrant accumulation in the cytoplasm of leukemic cells [dult AML , is recodult AML . NPM1 muic cells , 4.NPM1-mutated AML, since relocalization of the NPM1 mutant by XPO1 inhibitors causes early downregulation of HOX genes that is followed by cell differentiation and growth arrest [We demonstrated that the interaction between mutant NPM1 and the nuclear exporter Exportin-1 (XPO1) causes the aberrant cytoplasmic delocalization of mutant NPM1 and is responsible for the high expression of HOX genes in h arrest , 5. The NPM1-mutated AML is the absent/low expression of CD34 [NPM1-mutated AML both preclinically [NPM1-mutated AML patients, a small subset of early CD34+ hematopoietic precursors carrying NPM1 mutations/cytoplasmic NPM1 is usually present [NPM1-mutated AML may derive from CD34-negative hematopoietic stem cells, raises the question of a possible relationship between absent/low expression of CD34 and cytoplasmic dislocation of mutant NPM1 [NPM1-mutated AML has been mostly associated with an adverse outcome [Another characteristic feature of  of CD34 , 7 that  of CD34 , strong  of CD34  and pres of CD34 , is remiinically  and in p present , suggestant NPM1 . Clinica outcome .NPM1-AML patients at diagnosis and relapse, using a highly specific monoclonal antibody against mutant NPM1. The results of these studies are presented below.To address this issue, we performed functional studies to assess whether the nuclear relocalization of the mutant NPM1 could result in the re-expression of CD34. Moreover, we searched for CD34+/NPM1 cytoplasmic precursors in the bone marrow (BM) biopsies of NPM1-mutated AML. To this end, we assessed the effect of nuclear relocalization of mutant NPM1 on CD34 expression, using the selective XPO1 inhibitor selinexor, which blocks the nuclear export of XPO1 cargo proteins, including NPM1. We treated the NPM1-mutated OCI-AML3 cell line that does not express CD34 and two NPM1-mutated patient-derived xenograft (PDX) cells (PDX2 and PDX3) [NPM1-mutated AML cells and showing that levels did not change following XPO1 inhibition (not shown).We first investigated whether cytoplasmic delocalization of mutant NPM1 could explain the absent/low expression of CD34 in nd PDX3)  that parNPM1-mutated CRISPR-engineered cell lines (OCI-AML3 and IMS-M2) in which endogenous mutant NPM1 is fused to the FKBPF36V degron tag and GFP  were double stained for CD34 and mutant NPM1. Fourteen out of the 15 cases showed the presence of no or rare CD34/mutant NPM1 double-positive cells  showed a higher percentage (up to 20%) of leukemic cells double stained for CD34 and cytoplasmic mutant NPM1 (Fig. NPM1-mutated AML (Supplementary Fig. BM paraffin sections from 15 lls Fig. . One casPM1 Fig. . MoreoveCollectively, the above results strongly suggest that, unlike HOX genes , CD34 exNPM1-mutated cells originate from an immature CD34+ hematopoietic cell [NPM1-mutated AML, particularly in association with FLT3-ITD [In conclusion, based on our functional and immunohistochemical studies, we hypothesize that most tic cell , but thetic cell , 14, maiFLT3-ITD , 15.Supplementary Information


Several factors contribute to increasing the peri-anaesthetic risk in rabbits; above all, the airway management is challenging due to their peculiar anatomic characteristics. In human anaesthesia, the Spritztube (ST), an extraglottic airway device, has recently been developed for airway management. In the present study, an anatomical evaluation of rabbit cadavers was first carried out with the aim of designing a specific ST for rabbits. In the second phase of the study, the STs designed were evaluated on privately owned rabbits undergoing general anaesthesia to test their feasibility and safety regarding airway management in this species. The results demonstrated that the correct positioning of the ST can be completed in 43 ± 21.4 s with an average of 2.1 ± 1 attempts. Respiratory distress during ST positioning was the only complication observed in one rabbit. In conclusion, STs specifically designed for rabbits can be used as a supraglottic airway device for the maintenance of isoflurane anaesthesia in spontaneously breathing animals.The Spritztube (ST) is an extraglottic airway device developed for humans. The aim of the study was to design an ST for rabbits and to evaluate its feasibility. The study was divided into two phases. Phase I: anatomical study on 12 rabbit cadavers to design 2 STs  for rabbits. Phase II: fourteen privately owned rabbits were anaesthetised, and intubation was attempted using a ST. Tube size, the method for confirming the correct positioning, the number of attempts, the time needed for the correct positioning of the ST and complications were recorded. The ST placement was feasible in all rabbits. The positioning of the ST was completed in 2.1 ± 1 attempts in 43 ± 21.4 s. A correct placement was confirmed by the visualisation of the proximal cuff at visual inspection of the oral cavity (14/14), by the detection of the airflow (9/14 rabbits) and by the visualisation of a capnographic wave (14/14 rabbits). Only one rabbit developed respiratory distress after the ST placement. The results of the present study allowed designing a ST specific for rabbits which was used a supraglottic airway device for the maintenance of isoflurane anaesthesia in spontaneously breathing rabbits. Rabbits are at high risk of developing peri-anaesthetic complications, one of which is the overall incidence of anaesthetic-related death in this species of up to 1.38%, with this being higher than that reported for other small companion animals [Several factors contribute to increasing the peri-anaesthetic risk in rabbits; above all, the airway management is challenging due to their peculiar anatomic characteristics. In fact, rabbits are characterised by a temporomandibular joint with a limited range of motion, a long and narrow pharynx with a large tongue and big incisors which hinder the visualisation of the larynx . In addi®) by adapting the human supraglottic airway device i-gel to obtain easy self-positioning over the laryngeal inlet of rabbits [With the aim of overcoming these difficulties, extraglottic airway devices have been investigated for rabbit airway management as an alternative to orotracheal tubes ,5,6. Sev rabbits ,11.The Spritztube (ST) is an extraglottic airway device, which has recently been developed for airway management in human patients and was first described by De Rosa and colleagues . The ST The aim of the study was to design an ST suitable for rabbits. The study was therefore divided into two phases. Phase I was conceived as an anatomical study on rabbit cadavers in order to obtain measurements of the upper airway for developing a prototype of the ST for this species. In phase II, an in vivo study was carried out to evaluate the ease of insertion and the safety of the ST in rabbits undergoing general anaesthesia.® - Soudal N.V., Turnhout, Belgium), diluted with 5 mL of acetone to prevent its over-expansion and with the addition of a few drops of yellow nitro dye . When the foam began to come out of the mouth, the injection was interrupted, the cannula was removed, and the lips were completely tightened. After finishing the casting procedure, the specimens were stored at 4 °C for 3 h to allow the foam to harden. A stepwise dissection of the lateral regions of the head and the neck was carried out starting from the skin. This allowed exposure of the pharynx, larynx, trachea, oesophagus and the mouth cavity, after the removal of the ramus and the caudal part of the body of the left mandible.Twelve fresh privately owned rabbit cadavers , with a mean body weight of 3.1 ± 2.1 kg, were included in the study. All the animals died from reasons unrelated to the upper respiratory and digestive tracts and were intended for cremation. Each rabbit was placed in the left lateral recumbency with the head and the neck at an angle as close as possible to the physiological position. The nostrils were blocked with gauze, while the lips were almost completely clamped with surgical forceps after inserting a plastic cannula in the mouth. According to the procedure described elsewhere , the casOnce the cast was exposed, the following measurements were taken and recorded: the distance between the incisors and the base of the tongue (IT) and the distance between the base of the tongue and the beginning of the oesophagus (TO) using a measuring tape. In addition, the oesophageal (OC) and tracheal (TC) calibres were evaluated .Fourteen privately owned rabbits  were included in the study. Only rabbits undergoing scheduled surgical procedures with general anaesthesia were included in the study. The rabbits were considered clinically healthy based on physical examination. The owner consent was obtained before the anaesthesia.The rabbits were premedicated intramuscularly with alfaxalone , xylazine  and ketamine  mixed in the same syringe for injection and administered between the semimembranosus and semitendinosus muscles. Concurrently, the rabbits were preoxygenated with 100% oxygen administered using a face mask at 3 L/min, until intubation was attempted. After the onset of sedation, an intravenous catheter was placed in the marginal auricular vein after the trichotomy and aseptic preparation of the area. In addition, before induction, the following measurements were taken using a measuring tape, maintaining the rabbits in the lateral recumbency: the distance between the naris and the external projection of the thoracic inlet (NT) in cm and the distance between the naris and the external projection of the cranial border of the larynx (NL). General anaesthesia was induced with 5% isoflurane in 100% oxygen delivered using a facemask 10 to 15 minutes after premedication. The placement of the ST was attempted, when the palpebral reflex, the ear-pinch reflexes and the mandibular jaw tone were lost. The two cuffs of the ST were checked for leaks before insertion.The ST was introduced blindly with the rabbit placed in the lateral recumbency and the head hyperextended. The operator positioned his/her non-dominant hand behind the mandibular angle. The tube was then advanced through the incisors towards the lateral wall of the pharynx with the dominant hand, until the operator felt the tip of the device reaching the oesophageal lumen below the index finger of the non-dominant hand. At that point, the distal oesophageal cuff was inflated, and the operator verified that the inflated oesophageal cuff was positioned caudally beyond the mandibular angle using percutaneous palpation. The central guide was then removed. The proximal cuff was inflated, and the ST was gently drawn back until the cuff stopped at the base of the tongue. Both the distal and the proximal cuffs were inflated using a syringe until resistance was felt upon insufflation, and the total amount of mL inflated in each cuff were recorded. The end of the ST was attached to a side-stream capnogram. A correct ST placement was confirmed by applying one of the following criteria: direct visualisation of the proximal cuff by opening the oral cavity, evaluation of the airflow movement or a normal capnographic wave. The tube size, the number of attempts necessary for the correct ST placement and the overall time needed for achieving the correct positioning were recorded. In addition, the death space of the ST, defined as the distance between the incisors and the machine end of the ST, was measured using a measuring tape. As soon as the correct placement of the ST was confirmed, the animals were attached to a Jackson-Rees modified Ayre's T-piece breathing system, and isoflurane was delivered in 100% oxygen. The isoflurane was initially delivered at 1.5%, and then during the procedure, the vaporiser was adjusted according with the anaesthetic depth.The safety of the technique was evaluated based on the following criteria: the presence or absence of gross lesions or bleeding from the oral cavity, the presence or absence of cyanosis of the tongue and/or the presence or absence of respiratory distress. In the case of complications during the ST placement, any attempt at inserting the ST ceased, and a face mask was used for maintenance of general anaesthesia. For additional confirmation of the correct placement of the ST, X-rays scans of the head and the cervical tract and computed tomography (CT)  were performed in one animal each. 2 (FE’CO2) using a calibrated side stream gas analyser; and isoflurane %. In addition, non-invasive blood pressure (NIBP) was evaluated using a Doppler device (MiniDop) by placing the probe on the metacarpal artery. At the completion of the first evaluation, the rabbits were positioned in the recumbency required for the surgery and the surgical procedure commenced.Soon after intubation and every five minutes up to the end of the procedure, the following variables were evaluated: heart rate (HR) from an electrocardiogram; peripheral haemoglobin oxygen saturation (SpO2) using a pulse oximeter applied on the tongue; body temperature (T°) using a rectal probe; respiratory rate (RR), fraction of expired COAt the end of the surgical procedure, isoflurane was discontinued, and atipamezole was administered IM at 0.1 mg/kg. When the rabbits regained the palpebral reflex, the ST was removed and checked for blood or damage. The rabbits were closely monitored until fully awake. Data distribution was tested using the Shapiro−Wilk test. Normally distributed data were reported as mean and standard deviation (SD); non-normally distributed data were reported as median and range. The categorical data were reported as proportions. Analyses were carried out using MedCalc 6.3 .The mean IT and TO lengths were 5.3 ± 0.8 cm and 2.2 ± 0.8 cm, respectively. The mean EC length was 0.7 ± 0.1 cm, and the mean CT length was 0.9 ± 0.3 cm.Based on these results, two ST were chosen to be used in the clinical part of the study: an 8 Ch and a 10 Ch device . n = 7), Mini lop (n = 5) and Champagne Argent (n = 1). All the rabbits underwent ovariectomy or orchiectomy except for one, which underwent tooth trimming. The mean NT length was 9.7 ± 0.9 cm, and the mean NL length was 6.1 ± 1.4 cm.Thirteen rabbits were involved in the entire period of the study. One rabbit was removed from the study, because it developed respiratory distress after the first attempt at the ST placement. The ST was therefore removed, and the respiratory distress disappeared. The repositioning of the device was not feasible due to an insufficient anaesthetic depth, and therefore, general anaesthesia was maintained using a face mask. Of the remaining animals, the mean age was 9.4 ± 4.7 months, and their mean body weight was 1.6 ± 0.5 kg. The rabbits included in the study belonged to the following breeds: Dwarf rabbits , by the detection of the airflow in 9/14 rabbits and by the visualisation of a correct capnographic wave (14/14 rabbits). The oesophageal cuff was inflated with a median of 5 mL (range: 2–6 mL), and the proximal cuff was inflated with a median of 5 mL (range: 2–6 mL).With the aim of confirming the correct positioning, an X-ray scan was taken in one rabbit, and a CT scan was performed in another rabbit. In detail, at the X-ray evaluation, the oesophageal cuff was detectable in the oesophagus as a circular radiolucent structure close to the thoracic inlet . The proAll the cardiorespiratory variables monitored remained within normal limits. During anaesthesia or at recovery, bleeding or gross lesions were not observed. In all the rabbits, recovery was uneventful, and they were discharged on the same day as soon as they were fully awake.In the present study, the technique for the ST placement was described for the first time. The results indicated that the ST is easy to place with minimal risk of trauma. Respiratory complications are common in anaesthetised rabbits , and theThe technique for ST insertion described in the present study allowed correct positioning as confirmed by the diagnostic imaging. The positioning, however, required multiple attempts (up to 4), which is a higher rate than that reported for the laryngeal mask and endotracheal tube placement . HoweverCompared with other devices, one of the advantages of the ST is that it is equipped with an oesophageal cuff which seals the oesophagus, which would help in correct positioning. Previously, other authors have described that oesophageal cannulisation could be used in rabbits as guidance for preventing endotracheal tube dislodgement in the oesophagus . The following complications were recorded in humans during the insertion of the ST: air leak, laryngospasm, obstruction after insertion, the presence of gastric insufflation and sore throat upon removal . In the Another important advantage is associated with the use of the ST. In fact, in humans, the ST is designed to be used not only as an extraglottic airway device. In fact, as soon as patient stabilisation is achieved, the same device can be advanced into the trachea under laryngoscopic or fibreoptic guidance and can be used as a regular endotracheal tube. In addition, in rabbits, the fiberoptic or rigid (1.9 to 2.7 mm) endoscopes can be used for enabling ET insertion into the trachea by direct visualisation on the endoscopic screen ,19. Howe®) in rabbits. There are several limitations regarding the present study. First of all, the airway seal was not evaluated. Therefore, additional studies are needed to evaluate whether the ST provides an adequate airway seal for allowing controlled ventilation. In addition, in rabbits, the laryngeal mask emits 33% more waste isoflurane emission as compared with the cuffed endotracheal tube when sampled at the oral commissure [Overall, in humans, the application of a laryngeal mask is associated with a greater incidence of complications (19.1%) when compared with the insertion of an ST (6.0%) . Furthermmissure . In the In conclusion, the results of the present study allowed designing an ST-specific for rabbits which was quickly positioned and was successfully used as a supraglottic airway device for maintenance of isoflurane anesthesia in spontaneously breathing rabbits.Euorpean patent application for the Spritz tube: EP3880282A1.


NF1, NRAS, KRAS, and PTPN11 genes). In 79 homogeneously treated AML patients from this cohort, RAS+ status were associated with higher white blood cell count, higher LDH, and reduced survival. In AML models of oncogenic addiction to RAS-MEK signaling, the MEK inhibitor trametinib demonstrated antileukemic activity in vitro and in vivo. However, the efficacy of trametinib was heterogeneous in ex vivo cultures of primary RAS+ AML patient specimens. From repurposing drug screens in RAS-activated AML cells, we identified pyrvinium pamoate, an anti-helminthic agent efficiently inhibiting the growth of RAS+ primary AML cells ex vivo, preferentially in trametinib-resistant PTPN11- or KRAS-mutated samples. Metabolic and genetic complementarity between trametinib and pyrvinium pamoate translated into anti-AML synergy in vitro. Moreover, this combination inhibited the propagation of RA+ AML cells in vivo in mice, indicating a potential for future clinical development of this strategy in AML.Despite recent advances in acute myeloid leukemia (AML) molecular characterization and targeted therapies, a majority of AML cases still lack therapeutically actionable targets. In 127 AML cases with unmet therapeutic needs, as defined by the exclusion of ELN favorable cases and of FLT3-ITD mutations, we identified 51 (40%) cases with alterations in RAS pathway genes (RAS+, mostly  FLT3, IDH1, or IDH2 mutations , heated at 90 °C for 5 min and resolved by SDS-polyacrylamide gels electrophoresis, transferred to nitrocellulose membranes, and probed with primary antibodies. Protein signals were revealed by chemoluminescence  and detected using a CCD camera . The following antibodies were used: phospho-ERK 1/2 T202-Y204 (#4377), ERK (#9108), NF1 (#14623), Cleaved caspase 3 (#9661), and PARP (#9542) from Cell Signaling Technology, RAS (05-016) from Merk Millipore and β-actin (A1978) from Sigma Aldrich.6 cells were lysed and active RAS was pulled down after interaction with a RAF1-RBD motif conjugated with agarose beads. Beads were then solubilized in Laemmli buffer and RAS detection—proportional to its activity quantified by the RAS-RAF interaction—was performed by immunoblotting.RAS activity was assessed by a GST-RAF1-RBD pull-down assay according to manufacturer’s instruction . Briefly, 5 × 107 viable cells in 200 μl sterile PBS. In other experiments, mice were injected via tail vein with 0.2 × 106 viable luciferase-expressing HL-60 cells in 100 μl sterile PBS. The day after cells transplant, animals were randomly assigned into treatment groups. Trametinib and pyrvinium were given by oral gavage in 4% DMSO corn oil, and by intraperitoneal injection in 5% DMSO/PBS, at 0.25 mg/kg/d and 0.25 mg/kg/d, respectively, 5d/week. Bioluminescence analysis was performed using PhotonIMAGER OPTIMA  following addition of endotoxin-free luciferin (30 mg/kg).All animal experiments were performed in compliance with the laws and in agreement with the French Guidelines for animal handling and approved by animal ethics committees. Healthy 6–9-weeks-old NOD.Cg-Prkdc scid/J mice (NSG) were maintained under sterile conditions with sterilized food and water provided ad libitum and maintained on a 12 h light and 12 h dark cycle. In experiments with TF-1 cells, mice were preconditioned with 20 mg/kg Busulfan  and injected via tail vein with 2 × 105 cells in 100 µL of PBS in the tail vein of each mice. After the detection by flow cytometry of human CD33+AML cells in peripheral blood, mice were treated with Vehicle (N = 8), Trametinib (N = 9), Pyrvinium (N = 9) or trametinib and Pyrvinium combination (N = 9) by oral gavage 5 days a week. Doses used were 0.25 mg/kg/d trametinib (in 5% DMSO PBS), 0.5 mg/kg/d pyrvinium (in 4% DMSO corn oil), combination of 0.25 mg/d trametinib and 0.25 mg/kg/d pyrvinium , or combination of the vehicles in the control group. Disease propagation was monitored by hCD33 detection in peripheral blood, and AML tumor burden was quantified after sacrifice by hCD33 detection in whole bone marrow and spleen of each mice. Investigators were not blinded to the group allocation during experiment, but were blinded when assessing the outcome.We xenografted a RAS+ patient-derived AML sample to NSG mice. We injected 5 × 104/ml and 106/ml for cell lines and primary AML cells, respectively. The UptiBlue® viable cell-counting reagent  was then added for 4 h and fluorescence was measured with a Typhoon 8600 scanner .Cells were seeded in 100 μl of culture medium in 96 well plates for 48 h. Ideal cell density was determined to be 2 × 105/ml in 40 μl culture medium in 384-well plates for 24 h, then 10 μl of culture medium without or with compound were added for 72 h. At the end of incubation, 25μl of the CellTiter-Glo® 2.0 Assay reagent  was added in each well. The contents were mixed for 2 min at 300 rpm on an orbital shaker  and plates were incubated for 10 min at room temperature to stabilize luminescent signals. Units of luminescent signal generated by a thermo-stable luciferase are proportional to the amount of ATP presented in viable cells. Luminescence was recorded using a CLARIOStar  reader at a gain of 3600.Cells were seeded in at 1.25 × 10® reagent. Experiments were repeated three times separately and, after background noise subtraction, outliers removal and data normalization, results were presented as a percentage of each condition relative to the control (DMSO) condition.Compounds from the target selective inhibitor library were solubilized in DMSO, and distributed at the final concentration of 10 μM to cells seeded in 96-wells plates. Cell viability was assessed after 48 h using the Uptiblue3 for CTR and NF1KO_1 and 6 × 103 for NF1KO_2 TF-1 cell lines using T4 Cellometer (Nexcelom). Detailed procedures and data processing methods are provided in the Supplementary Material section.Cells were seeded in 384-well plates  using a MultiDrop combi (ThermoFisher Scientific), in 40 μL of cell media at 37 °C for 24 h. Optimal cell densities were determined as 5 × 105 cells were seeded in 96-well XF96 well plates coated with BD Cell-Tak  and loaded with XF Dulbecco’s Modified Eagle’s Medium. After 1 h incubation at 37 °C without CO2, cells were transferred to the XF96 analyzer. Oligomycin (1 µM) was added after 20 min, followed by FCCP (2 µM) after 40 min and Antimycin A/Rotenone (1 µM) after 59 min.Oxygen consumption rate (OCR) was measured using a Seahorse XF96 extracellular flux analyzer . Briefly, 1.5 × 105/ml in H4230 medium  supplemented with 10% of conditioned medium harvested from cultured 5637 cells. At day 7, L-CFU (colony of >20 cells) were scored under an inverted microscope.L-CFU assays were performed as previously described . Brieflyt test or a Mann-Withney test for parametric and nonparametric data, respectively. Two-by-two comparisons between colonies ratio of matched-pairs samples were made by using a Wilcoxon matched-pairs signed rank test. Statistical analyses were performed using Prism software 8.1.1 (GraphPad). Vertical bars indicate standard deviations. *P < 0.05, **P < 0.01, ***P < 0.001.In event-free survival (EFS) analysis, events were defined as induction failure, relapse or death of any cause and data were not censored at the time of stem cell transplantation . Death oFLT3 internal tandem duplication (FLT3-ITD) mutations, as mutually exclusive of RAS pathways alterations at diagnosis , while NRAS, KRAS, PTPN11, CBL, and BRAF mutations were detected in 13 (10.4%), 11 (8.6%), 9 (7.2%), 5 (4%), and 2 (1.6%) cases, respectively  were detected in the absence of complex cytogenetic abnormalities, two by fluorescence in situ hybridization (FISH) and one by NGS , we detected at least one RAS pathway gene alteration in 51 (40%) AML samples of 127 samples tested Fig. . NF1 mutons Fig. . However cancers  in 104 sWe performed RNA sequencing (RNAseq) in 52 samples (26 RAS+ and 26 RAS−). The distribution of mutations commonly found in myeloid neoplasm was similar between RAS+ and RAS− samples , and expressed mutant NRASG12D and PTPN11D61Y in TF-1 and Ba/F3 cells. In following experiments, the genetic background from these isogenic cell lines remained stable and the differences between parental and modified cells could be directly attributed to RAS dependency. After lentiviral transduction, cells were starved from cytokines, and NF1KO and NRASG12D cells grew readily from a bulk population in both TF-1 and Ba/F3 cell lines in contrast to control cells, while PTPN11D61Y expression led to cytokine-independent growth in TF-1 cells only  by increased ERK phosphorylation and active RAS form detection in RAS−RAF1 pulldown assays  mice using CTR and NF1activity , 24. XenKO cells were more sensitive to different MEK inhibitors compared to control cells , while these cells were equally sensitive to the majority of the compounds including p38 inhibitors  were less sensitive and even showed an increased colony formation in four cases  assays in 69 primary samples of AML patients from our cohort, among which 37 (53.5%) had a significant colony formation  ex vivo. In these samples, RAS status had no impact on the clonogenic potential of AML samples  over the same cell lines. Based on median dose–effect  and drug sensitivity score (DSS), we selected 6 compounds as having the strongest cytotoxic activity against NF1KO cells, within the same range than cytarabine and daunorubicin, two key AML chemotherapies  was higher, and that the mitochondrial electron transport chain complex I inhibitor IACS-010759 was preferentially cytotoxic in NF1ism Fig. . Moreoveism Fig. . In Ba/FG12D TF-1 cells, trametinib induced apoptosis even in the presence of glucose, and this effect was not modified by glucose starvation or addition of galactose  leukemic cells flow cytometry detection in mice blood until total tumor burden measure after sacrifice at day 57  RAS+ HL-60 AML cell line to immunodeficient NOD-SCID gamma-null (NSG) mice Fig. . We foll 57 Fig. . We obse 57 Fig. . While t 57 Fig. . CollectRAS was the first oncogene identified in human cancers, and its implication in oncogenesis has been widely studied since . While tt, inv(16), bi-allelic CEBPA, and NPM1 without FLT3-ITD mutations) [FLT3-ITD cases that generally do not harbor RAS pathway mutations at diagnosis  . We alsoNF1, NRAS, KRAS, and PTPN11, but we also observed variants affecting CBL, BRAF, RASA1, SOS1, MAP2K2, and RAF1 genes. We did not detect mutation in RAS pathways negative regulators, including DUSP6, SPRED, and SPRY family members. However, we identified an increased frequency of PRC2 alterations in RAS+ samples, as reported in JMML [IDH2 mutations more frequently detected in RAS− samples, as reported [From the 127 patients of our cohort, we identified at least one RAS pathway alteration in 40% of AML cases. The proportion of RAS mutations range between 25% and 40% in large AML genomic studies and may depend on the depth of sequencing technique employed allowing the detection of small-sized subclones , 39, 40. in JMML , 42 and  in JMML , 43, whireported .From RNAseq in 52 samples from the cohort, we did not detect a clear clustering of RAS+ samples, as reported , suggestNRAS and KRAS mutations in the ELN favorable core-binding factors AML subtype, and showed that the presence of subclones carrying RAS gene mutations identified, among these seemingly good prognostic patients, those having a reduced survival probability [NRAS and/or KRAS mutations were reported as having a neutral impact on survival in AML [NRAS or KRAS mutations had an independent negative impact on survival [NF1 mutations were associated with reduced survival probability among the adverse cytogenetic subgroup of AML [KRAS mutations had prognostic significance when considered individually, but that the RAS+ group aggregating RAS pathway mutation cases had a reduced survival probability, particularly within the ELN intermediate prognostic group. From our analysis of the BEAT AML database using the same filters as in our study, we also observed a near-significant reduced survival probability in RAS+ ELN intermediate AML patients, even if those mutations were reported in only 19.5% of this category, as compared to 32.1% in our cohort. Interestingly, RAS mutations were significantly associated with increased cell proliferation markers including elevated white blood cell count, blast cell percentage, and LDH levels. Our data suggested that the presence of RAS activating mutations might define a group of patients having a similar clinical course upon intensive induction chemotherapy.We investigated the prognostic impact of RAS pathway alterations in 79 AML patients from our cohort homogeneously treated with intensive anthracycline- and cytarabine-based induction regimens. Recent studies found frequent bability . While Nl in AML , 46, a rsurvival . Moreovep of AML , 49. In NF1 by CRISPR/Cas9, and expressed NRAS G12D or PTPN11 D61Y mutant forms in these cells, which grew in culture independent of cytokines, as reported [NF1 KO cells. Moreover, these RAS−activated cells demonstrated an increased sensitivity to the MEK inhibitor trametinib in vitro, and in vivo in NF1 KO cells, indicating that these cell line models became addicted to oncogenic RAS signaling [We took advantage of growth-factor-dependent cell lines to model the most frequent RAS pathways alterations detected in AML patients. We deleted reported . We confKRAS- or PTPN11-mutated compared to NRAS or non-RAS−mutated samples, as reported [NRAS G12D-mutated AML patient, illustrating the dependency of AML cells to RAS mutations, results of early phases MEK inhibitors clinical trials found a limited efficacy of these molecules [However, the antileukemic activity of trametinib appeared heterogeneous in primary samples from AML patients when assayed ex vivo in colony formation assays, with a significantly lower efficacy against reported . In contreported , suggestolecules , 35, 52.olecules .NF1 KO AML cells using a large FDA-approved compound library and identified pyrvinium pamoate, an oral anthelminthic drug employed in pinworm infection [FLT3-mutated AML [To repurpose molecules potentially targeting RAS−activated cells, we screened nfection . Other infection , this efnfection , 54. Thenfection . Particunfection –60. Mechated AML –63, and ated AML . Beside ated AML . In contated AML , 66.PTPN11 and KRAS than in NRAS mutants, while the opposite was seen for trametinib and interestingly, all but one sample were more sensitive to the combination of the two compounds compared to single-agents. These results suggested that vulnerability or resistance to pyrvinium or trametinib could correlate with RAS mutational status in AML, while combination therapy may overcome these limitations. The recent development of mutation-specific KRAS G12C inhibitors highlighted the heterogeneous structural and functional consequences of specific NRAS and KRAS mutations [The complementary metabolic profile of trametinib and pyrvinium in AML cells led to combination experiments, and we observed synergy between these two compounds in vitro in RAS−activated cell lines models. In primary samples from AML patients, pyrvinium appeared more active in utations , 68 and Direct pharmacological targeting of activated RAS remains one of the most challenging problem of cancer drug discovery . TargetiSupplemental Figures and Methods


Solving in his PhD thesis the one-dimensional version of a certain lattice model of ferromagnetism formulated by his supervisor Lenz , Ising cThe present Special Issue, “Ising Model: Recent Developments and Exotic Applications”, consists of eight original research papers that contribute greatly to our understanding of the Ising model and suggest its possible new applications.Early research on the Ising model was restricted mainly to ferromagnetic interactions and regular lattices. Gradually, however, interest shifted towards more complex models that incorporate, for example, competing interactions. Recently, there has been a growing interest in studying Ising models of heterogeneous lattices such as random graphs or scale-free networks. In the article  publisheThe Ising model, or its more general version—the Potts model—can be used to examine the anomalies of the IT network infrastructure, as demonstrated in the paper by Paszkiewicz  that alsThe next paper in our Special Issue by Valle et al.  demonstrA number of NP-optimization problems can be translated into energy minimization problems of a certain Ising model . Such anIn the next paper of our Special Issue, Žukovič et al. argue that the Ising model can be used for the interpolation of spatial data . Such teThe idea that an image can be represented as a certain configuration of the Ising spins appears also in the paper by Choi et al. . They shUp and down spins in the Ising models can be also interpreted as left and right enantiomers of chiral molecules. Using such an analogy, Dutta and Gellman developed adsorption thermodynamics of chiral molecules . They ard [In yet another paper of the Special Issue, Kryzhanovsky et al. developed an analytical method to examine the Ising model on lattices of large dimensions d . Their a


Many cancers are organized as cellular hierarchies sustained by cancer stem cells (CSC), whose eradication is crucial for achieving long-term remission. Difficulties to isolate and undertake in vitro and in vivo experimental studies of rare CSC under conditions that preserve their original properties currently constitute a bottleneck for identifying molecular mechanisms involving coding and non-coding genomic regions that govern stemness. We focussed on acute myeloid leukemia (AML) as a paradigm of the CSC model and developed a patient-derived system termed OCI-AML22 that recapitulates the cellular hierarchy driven by leukemia stem cells (LSC). Through classical flow sorting and functional analyses, we established that a single phenotypic population is highly enriched for LSC. The LSC fraction can be easily isolated and serially expanded in culture or in xenografts while faithfully recapitulating functional, transcriptional and epigenetic features of primary LSCs. A novel non-coding regulatory element was identified with a new computational approach using functionally validated primary AML LSC fractions and its role in LSC stemness validated through efficient CRISPR editing using methods optimized for OCI-AML22 LSC. Collectively, OCI-AML22 constitutes a valuable resource to uncover mechanisms governing CSC driven malignancies. Acute myeloid leukemia is a heterogeneous disease –6 drivenA number of key biological processes distinguish LSC from leukemia cells lacking stem cell activity. These include proteostatic responses, epigenetic pathways, immune escape ability and metabolism , 19–23 wHere, we report the development of a patient-derived primary AML model, called OCI-AML22, that reflects the functional, transcriptional and epigenetic cellular hierarchy common to most primary AML samples, providing a powerful resource that permits investigation of the molecular basis of the stemness in LSC. OCI-AML22 can be efficiently modified with lentivectors and CRISPR-based methods, yielding a novel model that allows interrogation of stemness features that are critical for the future development of more efficient LSC-targeted therapies.Please, refer to Supplementary Material for detailed methodsn = 34) for their ability to grow and expand in culture  was undertaken on the primary donor sample (day 0), the cultured OCI-AML22 model ~day 30), and on xenografts generated from CD34+CD38− cells sorted from the cultured sample (~day 120) Fig. . Genomic0, and onTo determine if the transcriptional signature of the CD34+CD38− fraction was maintained over time, we performed deep RNA sequencing (RNA-seq) of CD34+CD38− cells from the donor sample and four immunophenotypic fractions harvested at multiple time points of culture  Fig. . PrincipTo determine whether OCI-AML22 CD34+CD38− fraction recapitulates the stem cell transcriptional programs shared with primary LSCs, we used the LSC104 signature, a stem cell tool whose clinical relevance has been demonstrated in multiple cohorts . The sten = 60) or abnormal karyotype (n = 55) samples -driven anti-tumor immunity. LSCs upregulate PARP1 whose overexpression downregulates the activity of NKG2D ligands that are recognized by NK cells . As expeCollectively, these data validate that the OCI-AML22 hierarchical structure recapitulates key hallmarks previously reported for primary AML samples, both in vitro and in vivo. Thus, OCI-AML22 represents a good model to study LSCs and interrogate stemness properties.n = 41) and LSC depleted  fractions that were isolated from 25 uncultured AML patient samples and functionally validated as described previously [We next set out to uncover potential new stemness regulators associated with LSC in primary AML, as a basis to demonstrate the power of OCI-AML22 to provide new insights into LSC biology. Chromatin accessibility of non-coding genomic regions has greater power in predicting cell state identity as compared to gene expression . We estaeviously  Fig. 5An = 41 anFunctional studies require an experimental model, so we first mined ENCODE. No cell lines showed a similar pattern of accessibility peaks as found in our set of 93 primary AML sorted fractions Fig. . By contTo functionally test if LSCs depend on individual CREs found within the LSC+ CORE locus, we specifically targeted 2 CREs in the LSC fractions of OCI-AML22 using CRISPR/Cas9 methods we optimized. The OCI-AML-22 LSC fraction was used to either individually knock-out (KO) CRE3 or CRE6 or delete the entire flanking region Fig. . EffectiHere, we address the long-standing need for a tool to overcome the challenges of identifying, extracting, and obtaining high numbers of LSC from primary AML samples to enable functional, genetic/epigenetic, biochemical and metabolic studies. OCI-AML22 is the first human AML model that not only faithfully recapitulates the hierarchically organized LSC and non-LSC hallmark states of primary AML samples , 20, 21,CRISPR editing strategies have opened new avenues beyond gene interrogation including editing non-coding elements, which are increasingly being recognized as being regulators of stemness and/or cancer . Non-codSupplementary FiguresSupplementary Figures LegendsSupplementary methods


Management reasoning is distinct from diagnostic reasoning and remains incompletely understood. The authors sought to empirically investigate the concept of management scripts.In November 2020, 4 investigators each reviewed 10 video clips of simulated outpatient physician-patient encounters, and used a coding form to document observations about management reasoning. The team used constant comparative analysis to integrate empirically-grounded insights with theories related to cognitive scripts and Type 1/Type 2 thinking.Management scripts are precompiled conceptual knowledge structures that represent and connect management options and clinician tasks in a temporal or logical sequence. Management scripts appear to differ substantially from illness scripts. Management scripts varied in quality  and generality. The authors empirically identified six key features (components) of management scripts: the problem (diagnosis); management options; preferences, values, and constraints; education needs; interactions; and encounter flow. The authors propose a heuristic framework describing script activation, selection, instantiation with case-specific details, and application to guide development of the management plan. They further propose that management reasoning reflects iterative, back-and-forth involvement of both Type 1  and Type 2  thinking. Type 1 thinking likely influences initial script activation, selection, and initial instantiation. Type 2 increasingly influences subsequent script revisions, as activation, selection, and instantiation become more deliberate (effortful) and more hypothetical .Management scripts constitute a key feature of management reasoning, and could represent a new target for training in clinical reasoning (distinct from illness scripts).The online version of this article (10.1007/s40037-022-00714-y) contains supplementary material, which is available to authorized users. It Type 1 thinking supports non-analytical clinical reasoning, often manifest in rapid generation of diagnostic hypotheses and pattern matching  .“enables uniquely human facilities, such as … mental simulation and consequential decision making” e presume that management reasoning reflects a balance of non-analytical processes … and analytical processes …, yet the relative contributions remain unknown. … It seems plausible that management reasoning may be inherently more analytic  than diagnosis” he script, or generic event sequence, maps onto the general clinical picture of a disease, whereas each individual patient can be considered an instantiated illness script, with both typical  or atypical  features, which appear in a certain order” xplicit consideration of treatment costs and benefits, use of rubrics to guide management decisions, and thoughtful shared decision-making all suggest a slow, deliberate process” [As an introductory investigation with nontrivial limitations, immediate applications of our findings are constrained. However, we believe that management scripts, and their interrelationship with cognitive processes as discussed above, have potentially profound implications for education research, theory, and practice. We previously speculated that process” . If trueprocess” , 18, 27,process” , teachinprocess”  for breaprocess” –32, and process” , 34.Our findings suggest avenues for future research. Among these, we need more empirical evidence to test our model, including confirming the distinctions between management scripts and illness scripts Tab.  and recoe‑Fig. 1. Pattern matching in management script activation, selection, and instantiatione‑Fig. 2. Relationship of type 1/type 2 thinking in management reasoninge‑Box 1. Key features of management reasoninge‑Box 2. Management reasoning video coding forme‑Box 3. “Paper trail” (audit trail): Evolution of insights regarding management scripts and dual process thinking


FEV was re-expressed in 30% of primary AML samples and in almost all relapsed AML samples, and FEV expression levels were significantly higher in relapsed samples compared to primary samples. Interference of FEV expression in AML cell lines delayed leukemic progression and suppressed homing and proliferation. Moreover, FEV directly activated integrin subunit alpha 4 (ITGA4) transcription in a dose-dependent manner. Inhibition of integrin α4 activity with natalizumab (NZM) reduced the migration and colony-forming abilities of blasts and leukemic-initiating cells (LICs) in both primary and relapsed AML. Thus, our study suggested that FEV maintains the homing and expansion of AML cells by activating ITGA4 transcription and that targeting ITGA4 inhibits the colony-forming and migration capacities of blasts and LICs. Thus, these findings suggested that the FEV-ITGA4 axis may be a therapeutic target for both primary and relapsed AML.Acute myeloid leukemia (AML) is an aggressive hematological malignancy that recurs in approximately 50% of cases. Elevated homing and uncontrolled expansion are characteristics of AML cells. Here, we identified that Fifth Ewing Variant (FEV) regulates the homing and expansion of AML cells. We found that  Acute myeloid leukemia (AML) is an aggressive hematological malignancy in which immature cells accumulate and expand uncontrollably. Nearly 50% of patients relapse after induction chemotherapy , 2. AlthEnhanced homing and migration abilities are features of AML cells, which rapidly home to bone marrow (BM) and hijack the normal hematopoietic niche to aid extensive expansion of leukemic initiating cells (LICs) –7. Cell-FEV was initially reported to regulate the synthesis of 5-hydroxytryptamine (5-HT), and FEV deficiency results in the differentiation arrest of the majority of 5-HT–producing neurons and a 70–80% decrease in 5-HT  is an E26 transformation-specific transcription factor , 13. FEV in 5-HT , 15. In  in 5-HT . In our aft mice . HoweverFEV was re-expressed in 30% of primary and in almost all relapsed AML samples with high expression levels, and we found that FEV maintains the homing and expansion abilities of AML cells by directly activating integrin subunit alpha 4 (ITGA4) transcription. Integrin α4 blockade inhibited the migration and colony-forming abilities of blasts and LICs in both primary and relapsed AML. Our results demonstrated the role of the FEV-ITGA4 axis in homing and expansion maintenance, providing a potential therapeutic target for primary and relapsed AML.In the present study, we demonstrated that Human bone marrow (BM) aspirates of patients with leukemia from 2018 to 2020 were obtained from the First Affiliated Hospital of Soochow University, and the studies were approved by the Medical Ethical Committees of the hospital in accordance with the Declaration of Helsinki protocol. All human participants signed a written informed consent form. In total, 69 AML samples containing 16 relapsed AML samples were included. The detailed information for the clinical samples is shown in via intraperitoneal injection. The mice were injected with genetically modified MV4-11 cells at a dose of 1 × 105 through the caudal vein.NOD-SCID mice (aged 6–8 weeks) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China. The mice were bred under pathogen-free conditions at the Laboratory Animal Center of Soochow University. All animal experiments were performed in accordance with the protocols approved by the Experimental Animal Ethical Committee at Soochow University. The NOD-SCID mice were irradiated with 2.0 Gy of X-rays and subsequently treated with CD122 antibody at a dose of 200 μg per mouse ACTB expression levels. FEV positivity was defined in accordance with a previous report  in accordance with the manufacturer’s instructions. All experiments were performed in triplicate with ABI QuantStudio 3 Real-time PCR System . The primer sequences are summarised in s report .ChIP assays were performed using the Magna ChIP A/G kit  in accordance with the manufacturer’s instructions. MV4-11 cells with ectopically expressed flag-FEV were cross-linked in 1% formaldehyde and then sonicated to create soluble chromatin. Antibodies against flag  were added to precipitate the DNA fragments. The recovered DNA was amplified using PCR or quantitative PCR.6 transduced MV4-11 cells were injected into sublethally irradiated NOD-SCID mice by the caudal vein. 16 hours later, bone marrow (BM) cells of the recipients were stained with human CD45-PC7 and analysed using a flow cytometer . All antibodies were obtained from BD Bioscience.Homing assays were performed as previously reported . In thisP value < 0.05 and fold changes ≥ 1.5 were recognized as differentially expressed genes between the two samples (Dataset 1). Pathway analysis  of these differentially expressed genes was conducted using KOBAS 2.0, and the analysed results are provided in MV4-11 cells transduced with NC or shFEV were cultured for 3 days and sorted by BD FACS Melody . The cells were collected and high-throughput RNA sequencing (RNA-seq) was performed by Illumina HiSeq 2500  at CapitalBio Corporation . Genes with a FEV expression. The definition of FEV positivity was based on a previous report  and GraphPad Prism software .The Mann–Whitney U test was used to estimate the differences in s report . The FEVFEV expression in AML, BM aspirates from 53 primary AML patients were examined using reverse transcription-polymerase chain reaction (RT–PCR), and BM mononuclear cells (MNCs) and CD34+ cells from healthy donors were also examined as controls. According to the definition of FEV positivity from a previous report  and CD34+ cells (7/7), and 30.2% (16/53) of primary AML samples were FEV positive , which indicated that 37.0% (67/181) of primary AML samples were FEV positive  and cytogenetics. However, statistical analysis showed a worse risk category in FEV+ patients compared with the FEV- group (P=0.0017), according to the guideline of ELN risk stratification. Also, a significantly lower rate of complete remission was observed after two courses of induction chemotherapy in FEV+ group  and relapse-free survival (RFS) than the FEV- patients   of relapsed samples than in the diagnostic samples , and the most effective shRNAs (FEV expression in AML leukemic cell lines was examined by RT–PCR and immunoblotting (e shRNAs .FEV shRNA1 (sh1) or shRNA2 (sh2) were flow-sorted according to GFP positivity, and colony-forming cell (CFC) assay and cell proliferation assay were conducted. We found that FEV knockdown markedly reduced CFCs , bserved. –J, sugg+) were transplanted into immunodeficient NOD-SCID mice treated with CD122 antibody (NS122 mice)  , 20, 21.n the BM –D, whicn the BM . FEV knoFEV knockdown was examined. Transduced MV4-11 cells were injected into sublethally irradiated NOD/SCID mice followed by analysis of human CD45+GFP+ cells in the spleen and BM 16 hours after the injection. The AML cells that homed to the BM or spleen were significantly decreased in the shFEV group  were downregulated (MV4-11 cells transduced with NC or shFEV (sh1) were flow-sorted and subjected to gene transcriptome examination to investigate the underlying mechanisms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis highlighted an intensive expression alteration of genes involved in cell adhesion molecules (CAMs), transcriptional misregulation in cancer and the PI3K-Akt signaling pathway . Consistent with FEV knockdown, the number of CFCs was reduced  or mutant FEV-binding sites (ITGA4-P mut). The results suggested that FEV positively regulated wild-type ITGA4 expression in a dose-dependent manner but not mutant ITGA4 , a therapeutic antibody approved by the U.S. Food and Drug Administration (FDA) for multiple sclerosis   , we nextTo determine whether NZM affects relapsed AML samples, blasts and LICs from relapsed AML patients were treated with NZM. The abilities of relapsed blasts or LICs to form colonies ; we also demonstrated that FEV is essential for leukemia propagation of LICs . The expression of FEV was associated with worse risk category, CR rate after 2 course of induction chemotherapy and prognosis. Most importantly, we observed that FEV was expressed in almost all relapsed AML samples, and the expression levels of FEV were higher in relapsed samples than in primary samples. We further demonstrated that FEV was required for AML progression. FEV deficiency markedly reduced the homing and expansion abilities of AML cells. These findings revealed the mechanism underlying the function of FEV in the propagation of LICs.In previous work, we reported that  of LICs , but theFEV and ITGA4 (α4 subunit of VLA4) expression in AML samples, and ectopic ITGA4 expression elevated homing and expansion of shFEV MV4-11 cells and accelerated AML progression. ChIP and luciferase assays demonstrated that ITGA4 was a downstream target of FEV, which activated ITGA4 transcription by directly binding to the promoter in a dose-dependent manner. Thus, we identified a novel FEV-ITGA4 axis for homing and migration.Leukemic cells are retained in the BM microenvironment with elevated homing and migration abilities, which are mediated by cell-to-cell or cell-to-matrix interactions. CXCR4-CXCL12 signaling, VLA4 signaling and CD44 signaling have been reported to contribute to the homing of leukemic cells to the BM microenvironment –11. HoweITGA4 may be a potential target for both primary and relapsed AML. In agreement with Hsieh et al.  can be found below: The studies involving human participants were reviewed and approved by the Medical Ethical Committees of the First Affiliated Hospital of Soochow University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Experimental Animal Ethical Committee at Soochow University.JZ performed the experiments and wrote the manuscript. LQ and TW performed the experiments and analyzed the results. BZ and YF established the xenograft model. YL and MS collected the patient samples and clinical information. JA, DH, YX and DW discussed the data and contributed to the writing of the manuscript. TL supervised the project, designed the experiments and wrote the paper. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key Natural Science Foundation of China (81730003), the Excellent Youth Science Fund of Jiangsu Province (BK20211553), the Natural Science Foundation of China (81700139 and 81870120), the National Science and Technology Major Project (2017ZX09304021), the National Key R&D Program of China (2019YFC0840604 and 2017YFA0104502), the Key R&D Program of Jiangsu Province (BE2019798 and BE2019655), the Natural Science Fund of Jiangsu Province (BK20170360), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Jiangsu Medical Outstanding Talents Project (JCRCA2016002) and the Jiangsu Provincial Key Medical Center (YXZXA2016002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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