MDS/AML overlap has recently been introduced as novel myeloid disease entity by the International Consensus Classification (ICC) with 10–19% blasts in the absence of AML-defining recurrent genetic abnormalities, acknowledging the biologic continuum between MDS and AML ) where iRisk assessment is often considered for clinical trial inclusion criteria. In MDS patients, risk stratification is based on the revised International Prognostic Scoring System  and then = 626;  years and a median follow-up of 9.5 years  as well as an independently published unselected MDS cohort  compared to a bona fide MDS cohort  used as  = 2701; ; 0.7155)TP53 mutations  and/or MR-associated cytogenetic aberrations . Within MDS/AML, the intermediate risk group still showed longer OS than the adverse risk group . However, the survival of MDS/AML patients substantially differed from a bona fide AML control cohort . Likewise, the OS of MDS/AML patients classified as intermediate risk according to ELN 2022 was also longer than the corresponding intermediate risk AML patients   were classified as intermediate risk, and the vast majority  as adverse risk. Adverse risk classification was primarily driven due to MR-associated gene mutations , TP53. We found that for MDS/AML patients, MDS-based risk assessment according to IPSS-M is fully applicable with comparable OS data to a real-world MDS cohort despite a skewing towards high-risk categories. In contrast, AML-based risk classification according to ELN 2022 guidelines is not applicable for MDS/AML. The classification of nearly all patients as adverse risk due to their MR-associated genetic profile is not meaningful—in particular as it is not supported by OS data. If MDS/AML patients were to be included into AML studies, development of a specific risk assessment for MDS/AML other than ELN 2022 would be needed.In summary, we confirmed the prognostic significance within MDS/AML entities according to ICC, in particular MDS/AML with Limitations of our study include the retrospective design, the non-uniform treatment, and the low number of patients in some of the IPSS-M risk groups. We cannot exclude that an AML-specific treatment of MDS/AML patients would affect the risk stratification as the number of intensively treated patients in our cohort was too small for subgroup analysis. In this regard, it has to be noted that the ELN risk stratification is meant to be applied to intensively treated patients. The performance of the ELN risk stratification—or a modified version of it—in intensively treated MDS/AML patients remains to be studied. However, the substantially better survival of MDS/AML patients compared to adverse risk AML despite more intensive therapy in the latter and higher age in the former (74 vs. 69 years in our cohort) raises substantial concerns about a potential justification of a general inclusion of MDS/AML patients in a clinical trial designed for adverse risk AML. Our data clearly support the WHO that decided not to introduce this MDS/AML category arguing that this may lead to the risk of overtreatment in some patients . NeitherTaken together, the value of another arbitrary blast cell cut-off remains questionable. One could argue that blast cell thresholds remain inevitable in the field of MDS and AML. In contrast, we previously showed that a genetic-based MDS subclassification better reflects biology than blast counts and that the latter is rather a sequela of the former than an independent biological category . This coSupplementary Material


There is growing evidence of a strong association between SARS-CoV-2 and cancer prognosis and treatment outcome. However, there are no reliable SARS-CoV-2 assessment models to accurately predict prognostic and therapeutic effects in acute myeloid leukemia (AML). Here, differentially expressed genes associated with SARS-CoV-2 were detected, and multiple Cox regression methods were used to construct a SARS-CoV-2 risk index (SC2RI). Then, RT-qPCR was used to validate the gene expression levels in the AML samples. Finally, we explored how the SC2RI affected prognosis, immune infiltration, immunotherapy, and drug sensitivity in AML. We found that CYB5R3 and CLIP4 had been confirmed as hub genes in AML and were used to generate the SC2RI. The datasets indicated that the SC2RI had a superior predictive impact on the prognosis of AML. In addition, high expression of immune checkpoints and numerous immunological infiltrations were substantially correlated with a high SC2RI. However, it responded poorly to immune checkpoint blockade, which may be related to T-cell dysfunction, lack of effective antigens, and deficiency of synaptic capacity. Moreover, a high SC2RI was less susceptible to mTOR-related pathway medications but more sensitive to cell cycle suppressors. Therefore, categorization based on SC2RI could enhance the prognostic prediction of AML and help identify novel therapeutic approaches. AML is a highly heterogeneous disease associated with numerous gene mutations and cytogenetic abnormalities  and Bioland Laboratory, China  [grant number 2018GZR110105014].This work was supported by the All data presented in this study are included in the article/supplementary material.Jiaxin Guo: Writing – review & editing, Writing – original draft, Visualization, Software, Resources, Methodology, Investigation, Formal analysis, Data curation. Yiyi Wei: Writing – original draft, Visualization, Validation, Software, Methodology. Qingyan Cen: Writing – review & editing, Software, Resources. Jianyu Chen: Writing – review & editing, Validation. Yuhua Li: Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The acidic extracellular microenvironment has become an effective target for diagnosing and treating tumors. A pH (low) insertion peptide (pHLIP) is a kind of peptide that can spontaneously fold into a transmembrane helix in an acidic microenvironment, and then insert into and cross the cell membrane for material transfer. The characteristics of the acidic tumor microenvironment provide a new method for pH-targeted molecular imaging and tumor-targeted therapy. As research has increased, the role of pHLIP as an imaging agent carrier in the field of tumor theranostics has become increasingly prominent. In this paper, we describe the current applications of pHLIP-anchored imaging agents for tumor diagnosis and treatment in terms of different molecular imaging methods, including magnetic resonance T1 imaging, magnetic resonance T2 imaging, SPECT/PET, fluorescence imaging, and photoacoustic imaging. Additionally, we discuss relevant challenges and future development prospects. Malignant tumors are among the major diseases that threaten human life, health, and safety. Effectively diagnosing and treating them presents a challenge for the medical community. Most tumors cannot be located based on early symptoms and can only be diagnosed after a certain period, which creates great difficulties for treatment . TherefoMolecular imaging is an imaging technology with medical application value that visualizes, characterizes, and measures biological processes at the tissue level, cell level, and subcellular level in vivo using imaging methods. Molecular imaging is critical for the early diagnosis and treatment of diseases, especially tumors. Compared with traditional imaging, molecular imaging can be better used for early tumor screening and diagnosis at the cellular and molecular levels since it exhibits several advantages, including high sensitivity and specificity. Thus, molecular imaging plays an important role in improving the accurate clinical diagnosis and targeted treatment of diseases . MoleculAt present, the commonly used molecular probes  in clinipHLIP is a 36-amino-acid peptide from the third transmembrane helix of bacteriorhodopsin (sequence: NT-GGEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT-C). pHLIP exhibits no definite structure at a neutral pH and mainly uses the random coil conformation . However2+ and Mg2+ in pHLIP insertion, cell targeting, and drug delivery and found that the combination of pHLIP with monomethyl-auristatin-F, a cytotoxic compound, exhibited an increased ability to target HeLa cells  in the presence of Ca2+. Scott et al. [As research into pHLIP has increased, some researchers have found that different ions can enhance or weaken the effect of pHLIP insertion into the cell membrane. Vasquez et al.  studied t et al. , meanwhit et al.  found th64Cu and 18F) labeled with variants (var3 and var7) and wild-type (WT) pHLIP were used to study the biological distribution of Balb/c mice carrying tumors in situ in 4T1 [64Cu- and 18F-labeled var3 showed great tumor accumulation and significant tumor background contrast in images throughout the imaging studies.Many researchers have also tried to modify and adjust the original pHLIP structure, designing a pHLIP variant that exhibits better performance and improved insertion into the lipid membrane. Two positron-emitting radionuclides  is an imaging technique that uses the signal generated by the resonance of atomic nuclei in the magnetic field to reconstruct the image. Atomic nuclei containing odd-number protons, such as hydrogen nuclei, which are widely found in the human body, are positively charged, and their protons show spin motions and produce magnetic moments, like a small magnet. The spin axes of the small magnets are arranged irregularly. For example, in a uniform strong magnetic field, the spin axes of the small magnets are rearranged according to the direction of the magnetic field line of force. The axes are only arranged in two directions that are parallel or antiparallel to the external magnetic field. The magnets are excited by a specific radio frequency (RF) . As the 3+ must be prepared in a chelate form or loaded into 13X zeolite nanocarriers to reduce its toxicity [3+ has a particle radius, similar to that of Ca2+, which can interfere with calcium-mediated signal pathways; thus, Gd3+ is highly toxic [The T1 contrast agent is usually a paramagnetic or superparamagnetic metal ion-type contrast agent. Gd DTPA is the earliest and most widely used contrast agent in clinical practice, representing a nonselective extracellular agent. Gd DTPA functions by shortening the T1 relaxation time of tissue , increastoxicity . Gd DTPAtoxicity . Gd DTPAtoxicity . It has toxicity . Gd can toxicity ,38. In aly toxic . In summly toxic  developely toxic  prepared3O4) is among the three main iron oxides in the SPION category. Pershina et al. [3O4 magnetic nanoparticles and confirmed that pHLIP-anchored nanocomposites can bind to tumor cells in an acidic environment of HTC cells and LLC tumor models (Superparamagnetic iron oxide nanoparticles (SPIONs) are a new MRI contrast agent developed after gadolinium. In a key difference from paramagnetic contrast agents, SPIONs can interfere with the uniformity of the local magnetic field because of their high magnetic momentum, so adjacent protons are affected by an “outer region” or “susceptibility effect”, which works to accelerate the desynchronization phase of protons and shorten the T1 and T2 times. However, due to the large particles of these contrast agents, protons are difficult to approach and are affected by unpaired electrons in the inner region, which mainly affect T2 rather than T1 . The infa et al.  covalentr models . The bio99mTc, 111In, 123I, and 177Lu). With a dedicated gamma-ray detector and a camera or SPECT instrument, the gamma rays can be recorded and converted into an image through signal processing, to determine the location of the radiolabeled antibody. PET imaging requires positron-emitting radionuclides  for radioactive labeling [64Cu and 18F) to label the pHLIP variants (var3 and var7) derived from NOTA and NO2A and the wild-type (WT) pHLIP to study the biological distribution of 4T1 breast cancer in in situ tumor-bearing mice, and found that all variants formed high-level radioactive markers. Chen et al. [125I, observed the biological distribution of 125I pHLIP (var7)-P1AP in MDA-MB-231 cells in a breast cancer mouse model and SPECT imaging results in small animals, and found that in an acidic environment, pHLIP (var7)-P1AP could efficiently target MDA-MB-231 cells and inhibit their growth. Small animal SPECT with 125I pHLIP (var7)-P1AP could clearly show tumors. A high concentration of 125I pHLIP (var7)-P1AP was observed in the tumors, and small animal SPECT imaging showed obvious tumors 4 h after injection. These pHLIP-modified radionuclide variants are currently undergoing clinical transformation research and are expected to enter use as new nuclear imaging tracers for detecting acidic lesions.SPECT and PET are the most commonly used molecular imaging technologies in nuclear medicine. Both methods have inherent advantages, including their systemic, semiquantitative, and real-time nature, along with their high sensitivity. These methods are among the most important means for accurately diagnosing and treating tumors in clinical practice. SPECT imaging is based on gamma rays, the technique of X-ray development in which antibodies are conjugated to radionuclides  entered a phase 1 clinical trial for patients with breast cancer [Since the acidic microenvironment is a ubiquitous hallmark of various diseases , pHLIP-labeled platforms show good potential for early diagnosis, tumor boundary detection, and evaluation of the therapeutic impact. Recently, a pHLIP-based imaging agent ([t cancer , moving Despite a series of advances, pHLIP-based theranostics still faces many challenges. First, given that hypoxia is primarily involved in causing an acidic tumor microenvironment, an answer to the following must be sought: what is the earliest state of hypoxia that the pHLIP-functionalized imaging agents/probes can detect? Emerging evidence has reported that pHLIPs accumulate in the hypoxic region in correlation with the corresponding microvascular density, that is, when there are more microvessels remaining, there is lower accumulation of pHLIP . HoweverSecond, the question remains: how can this pHLIP imaging technique distinguish early- from late-stage tumors? Numerous reports have demonstrated that the highly acidic microenvironment is a significant feature in the transition from an avascular preinvasive tumor to a malignant invasive carcinoma ,76. HencThird, we must also ask: How effectively are the pHLIP-functionalized probes cleared from the body? Safety considerations necessitate a metabolism study of pHLIP-modified probes. Extant studies pointed out that radiolabeled pHLIP probes can remain intact for at least 1 h in the blood of rats after intraperitoneal injection . The detOnce these limitations are overcome, we will be in a position to determine whether pHLIP-based alternatives truly have broad application prospects in the field of molecular imaging.


This article presents the development and implementation of the Delamination Plug-in, an open-source tool for modeling delamination tests in the ABAQUS software. Specifically designed for stochastic modeling of 3D printed composites, the plug-in combines the benefits of the graphical user interface (GUI) and the programming of commercial finite element (FE) software. The Delamination Plug-in offers an effortless alternative to the time-consuming analytical modeling and GUI work involved in delamination tests and includes algorithms for several tests, such as the double cantilever beam, end-loaded split, end-notched flexure, and modified end-loaded split tests, solved using the virtual crack closure technique and the cohesive zone method. It enables the user to develop simulations for both simple symmetric laminates and generally layered laminates with additional thermal stresses. The applicability of the tool is demonstrated through its use in two distinct delamination problems, one for conventional and one for 3D printed composite laminates, and its results are compared to analytical models and experimental data from the open literature. The results demonstrate that the Delamination Plug-in is efficient and applicable for such materials. This establishes the tool as an important means of automating delamination analysis and for the development and testing of 3D printed composites, making it a valuable tool for both researchers and industry professionals. Composite laminates are widely used in various industries , includiNumerous studies have been conducted on delamination mechanisms, and various standard organizations, such as ASTM and ISO, have developed representative test methods for characterizing the fracture toughness of unidirectional polymeric composites ,10,11,12Common delamination tests include the double cantilever beam (DCB), the end-loaded split (ELS), the end-notched flexure (ENF), and the modified end-loaded split (MELS) test. The DCB test is a common delamination test used for measuring mode I (opening mode) SERR . The ELSDelamination tests are commonly conducted on symmetric laminates through standardized processes. Nevertheless, they can also be performed to measure the SERR of generally layered  laminates ,17. HoweUsing simple theories in the standards is inadequate to address such complex cases, and specific analytical handling utilizing innovative bi-layer interface theories is required to accurately capture the SERR ,19. ThesA more effortless solution to modeling delamination problems can be achieved by using commercial finite element (FE) software, such as the widely used ABAQUS software . The sofStochastic models are important in the study of the delamination of 3D printed composite laminates ,23. The In software such as ABAQUS, the modeling of such structures can be achieved via scripting in object-oriented Python, which allows for full parametrization and optimization of the software’s capabilities. However, this task is non-trivial and requires programming experience with concurrent knowledge of the background FE algorithm. Furthermore, scripting lacks the ease of use of the GUI.Plug-ins combine the efficiency of scripting and the practicality of the GUI by developing a tailored algorithm for the solution of distinct problems, with the aim of automatizing the process. The implementation of this algorithm is achieved via a custom GUI panel (the plug-in) that aids the user’s interaction with the background script. The user can then provide parameters to the script via the plug-in and create, solve, and process results much faster and without investing in studying or developing the background script.Various plug-ins have been developed for solving problems in micromechanics ,25,26, vDelamination Plug-in, an accurate and practical means of modeling delamination tests in the ABAQUS software. The innovative tool combines the advantages of the GUI and the programming of commercial FE software and offers an effortless alternative to the time-consuming analytical modeling and GUI work needed when performing delamination tests. The plug-in allows for simulations of delamination tests for generally layered laminates with additional thermal stresses and, furthermore, drastically simplifies the stochastic modeling of 3D printed laminates. The background algorithms of the open-source tool are provided for specialized individual applications and modifications. Thus, the Delamination Plug-in can be considered as a foundation stone of the automatization of delamination analysis and can accompany the analytical and experimental work of researchers and industry professionals in the domain.Considering the above, this paper aims to develop the This work is structured as follows. Several studies have utilized the FE method to investigate delamination problems in both one and two dimensions . This reG), which is equal to the J-integral, is given as a function of the stress  is used. The linear softening part is then introduced when the stress reaches a maximum value  and displacements . However, this requirement can lead to computationally intensive models with dense meshes. For the VCCT, the user defines the connected regions where the crack will propagate, and the nodes between the two surfaces are rigidly attached and released when the computed SERR meets a failure criterion. The B–K criterion ,38 is tyNumerous analytical theories have been created to study delamination tests , which gAdvanced delamination theories, such as the conventional composite beam theory, the shear deformable bi-layer beam theory, and the interface deformable bi-layer beam theory, assume that the sub-laminates are Timoshenko beams and are modeled using first-order shear deformation theory (FSDT). These bi-layer beam theories are described as the rigid, semi-rigid, and flexible joint deformation models, respectively. Unlike the standards-based theories ,14,15, tThe three delamination theories assume different boundary conditions between the two sub-laminates. The conventional composite beam theory considers that both the relative displacement and rotation are restricted on the interface between the two sub-laminates. The shear deformable beam theory releases the rotational degree of freedom and considers that only the relative displacement is restricted. This improves the accuracy of the theory and mitigates significant errors caused by the zero slope restrictions at the beam root . FinallyThe bi-layer theories describe the displacement, rotation, and stresses  at the bonded section. The displacement and rotation of the bonded section are then utilized in a J-integral  equationThe Delamination Plug-in offers a practical and effective 2D simulation of delamination tests. The long GUI process, from geometry preparation until the post-processing, is automated, requiring only the necessary parameters for a correct delamination simulation. The parameters will be introduced stepwise in the following sections. The software is completely written in Python 2.7 utilizing the built-in ABAQUS Python application programming interface (API). The simulation is separated into three main phases: the pre-processing, simulation, and post-processing. The parameters of the main phases are inserted using five tabs in a straightforward process. Graphical representations of the required input parameters are illustrated to aid the understanding of the user. A flowchart describing the main phases of the software is presented in The Delamination Plug-in requires an active model and materials (one or more) with elastic constants defined as ‘engineering constants’ and ‘orthotropic’ thermal expansion coefficients.The plug-in is initialized simply by placing it in the directory ‘abaqus_plugins’. The user can choose to initiate the plug-in either with CZM or VCCT . The twoThe background of the three phases is elaborated in the following section, addressing the most important modeling points.In the pre-processing phase, the user creates and meshes one of the four parts available in the library: the DCB, the ELS, the ENF and the MELS. The ‘Part’ tab  allows tn number of plies. Different materials , thicknesses (column thickness), and temperature differences (column temperature) can be given for each ply. A thin cohesive layer is defined for the CZM. Furthermore, a thin ply  is created on both sides of the user-defined crack. The refined artificial ply addresses the issue of convergence of the components of SERR for VCCT as described in The laminate stacking sequence is defined in the ‘Laminate’ tab . The useThe mesh parameters are defined in the ‘Mesh’ tab. In ‘Refined region’, the user specifies the length of the refined region, which is symmetric around the crack tip. The refined region is used to densify the mesh close to the crack tip, offering high accuracy and lower computational cost . Four meP) or displacement (k) and the maximum interfacial strength . Furthermore, a file containing all the input parameters used for the analysis, as well as the load, displacement, and energy values, is exported. Finally, a file containing the location of the crack tip in every increment is exported, in addition to the coordinates of each of the bonded nodes using a custom crack detection algorithm. The crack detection algorithm, compares the bonding status of the nodes and compares the time of opening. Then, it assigns the coordinates of the opening nodes to the crack tip.For the CZM, similar information is extracted concerning the cohesive elements. The debond status, the stress, and the strain of each integration point are given for each element. Here, the ERRT is estimated using Equation . The infFinally, information of the first node (for the VCCT) or the first element (for the CZM) in the crack direction can be conveniently exported and printed in the ‘Message Area’. The information includes the label of the first node or element, the SERR components, and the active increments, and can be used to identify whether the crack propagates immediately from the GUI. The CSV files can be used to perform separate post-process analyses and visualize the information in separate software. The output files contain the most crucial information required for delamination tests.It is important to mention that during the use of the Delamination Plug-in, critical mistakes for the geometry creation are reported, aiding the user.Two case studies are given here to demonstrate the abilities of the Delamination Plug-in. Firstly, considering generally layered laminates with residual thermal stresses, a case study is conducted for a fiber metal laminate (FML) containing layers of aluminum and e-glass fiber reinforced polymer (EGFRP). This case study is of particular interest since it has been used before as a comparison of novel analytical methods ,17 and iSecondly, an example is taken from the field of 3D printed composite laminates. The performance of 3D printed laminates is highly influenced by uncertainties related to the manufacturing process. In particular, large scattering can be observed in their maximum load capacity when tested in mode I or mode II delamination, which is an indication of an inconsistent bond that forms between the laminates while printing. Furthermore, the tolerances of the 3D printing lead to uncertainties in the dimensions of the final part, which can also influence the outcome of delamination tests. The above uncertainties are characterized as dimensional and performance-related uncertainties and have been addressed for 3D printed nylon reinforced with carbon fibers in .The Delamination Plug-in can be used to capture the aforementioned uncertainties and facilitate an effective stochastic simulation that would otherwise require time-consuming, repetitive work. The example used here is taken from , where tThe FML, illustrated in −4, 2.5 × 10−5, 2.5 × 10−4, 5 × 10−6 for the Hl, Hs, Vl, and Vs, respectively. All simulations are performed using a ‘Force’ driving field and the critical values of SERR  and the flexible joint model (FJM) of Qiao and Wang from their pioneering analytical work . These mThe SRJM is the implementation of the shear deformable beam theory see  and consG) versus the load (P) is illustrated in The measured data of the SERR are extracted and compared with the analytical models. The total SERR (The implementation of the PCE method is similar to the Monte Carlo (MC) method. Both methods require the generation of points and the solution of a deterministic model (here the FE model of the delaminating specimen) on each point. These points can be, for example, groups of material and geometry properties that are generated via the probability distribution of each property. The generation of points for the PSPM method can be achieved using open-source libraries such as chaospy  in PythoConsidering the above, performing a stochastic simulation necessitates first generating the points and then creating an FE model for each point. However, if performed manually, this process is time-consuming. Hence, it is automatized here using the Delamination Plug-in.The process to develop each deterministic model is identical to the one presented in ith point argument. Finally, an iterative ‘for’ loop is used to call the two functions and solve the delamination problem for all points. This process allows one to successively solve all stochastically generated FE models. The solutions are stored in separate folders which can be accessed later to visualize and process the results. In view of the above, using the Delamination-Plug in, the process of performing stochastic simulations can be largely simplified, since the generation and the solution of the numerous FE models reduces to calling a single function, the ‘delamination_model’ function.The stochastic modeling is applied here for the DCB and the ELS test of symmetric 3D printed laminates of nylon reinforced with carbon fibers. The stochastic parameters  exhibits differences. These differences are attributed to the artificial stiffening of the laminate due to fiber-bridging, as explained in . For theUnderstanding the delamination process of composite laminates is an important task, since delamination is one of the most prevalent failure modes. The task can be tackled either analytically or numerically. However, complex geometries with general stacking sequences and residual thermal stresses are difficult to express analytically due to the complexity of the analytical solutions. Focusing on the numerical modeling, this article addresses the development of the Delamination Plug-in, a functional and practical tool for performing simulations of common delamination tests using the ABAQUS commercial FE software.The plug-in combines the advantages of the GUI of ABAQUS with a powerful custom kernel which drastically accelerates the pre-processing and post-processing phases. The Delamination Plug-In contains built-in geometry creation modules, adaptive user-defined meshing, and automatic generation of loading and interaction conditions. The simulation process is conducted in a straightforward manner using the VCCT or CZM. The user is offered the capability of creating customized models of simple and complex stacking sequences and visualize the data in separate software with powerful plotting tools. Stochastic modeling is also simplified, since the generation and the solution of the numerous FE models reduces to calling a single function in an iterative loop. Finally, distributing the algorithm of the Delamination Plug-in gives the opportunity to extend the capabilities of the plug-in and allows for custom development.In conclusion, the open-source Delamination Plug-in can accompany analytical or experimental work and aid the field of delamination analysis by providing a practical and effective alternative to complex analytical models or time- consuming GUI development in FE software.The latest version of the Delamination Plug-in can be acquired from the corresponding author following an email request.


Average Dorsiflexion/Plantarflexion ROM values increased significantly post-surgery: 21.7° preoperative to 45.6° postoperative (p < 0.001). The postoperative AOFAS scores were significantly greater than the preoperative values: 47.7 points preoperative, 92.3 points postoperative (p < 0.001). The sports activity improved from preoperative to postoperative where, preoperative, none of the patients were able to perform sports. Postoperative, eight patients were able to be sports-active again. The overall average postoperative level of sports activity was 1.4. The postoperative average patient’s satisfaction was 9.3 points. Conclusions: In painful talar component aseptic loosening of a three-component mobile-bearing TAA, H-TAA is a good surgical solution for reducing pain, restoring ankle function, and improving patients’ life quality.Introduction: Revision Total Ankle Arthroplasty (TAA) surgery due to TAA aseptic loosening is increasing. It is possible to exchange the talar component and inlay to another system for isolated talar component loosening in a primary mobile-bearing TAA: Hybrid-Total Ankle Arthroplasty (H-TAA). The purpose of this study was to analyze the results of the revision surgery of an isolated aseptic talar component loosening in a mobile-bearing three-component TAA with a H-TAA solution. Methods: In this prospective case study, nine patients  with symptomatic isolated aseptic loosening of the talar component of a mobile-bearing TAA were treated with an isolated talar component and inlay substitution. In all nine cases, a hybrid TAA revision surgery was performed by implanting a VANTAGE TAA talar and insert component . The patients were reviewed with the pain score (VAS Pain Score 0–10), Dorsiflexion/Plantarflexion (DF/PF) Range of Motion , the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle/Hindfoot Score (0–100 points), Sports Frequency Score (Level 0–4), and subjective Patients’ Satisfaction Score (0–10 points). Results: The average Pain score improved significantly from preoperative 6.7 points to postoperative 1.1 points ( Ankle Osteoarthritis (OA) is a condition characterized by pain, swelling, and inflammation associated with chronic, progressive, and irreversible damage to articular cartilage . The mosAnkle OA leads to severe functional limitations in everyday life and sports activity. The level of physical and psychological disability associated with ankle OA is at least as severe as that of hip OA .Surgical treatments of ankle OA can be divided into joint preserving and joint sacrificing surgical procedures . PatientThe two main therapeutic options for end-stage ankle OA are Total Ankle Arthroplasty (TAA) and ankle arthrodesis. Ankle arthrodesis, once considered the gold standard, was previously recommended in most patients because of its predictable outcomes and decreased complication rates . This thA recent meta-analysis showed that at a 10-year follow-up there is a TAA survival rate of 70 to 90% and an average 10-year TAA revision rate of 22% ; STAR shAseptic loosening is the most frequent complication following TAA, accounting for 55% of failures along with subsidence of the talar component into the talar body ,17,18,19Four surgical options are feasible for managing a TAA failure.Two-staged revision is where the loose TAA is removed during the first stage while simultaneously restoring the bone stock. This is followed by verification of a successful restoration of bone stock via CT-scan and while proceeding to the second stage of implanting a new TAA .Primary Hybrid TAA (H-TAA) is where a one-stage revision is carried out in selected cases with isolated talar or tibial component loosening. Only the loosened component is changed, and, simultaneously, the bone stock is restored if needed. This is possible only for the loosened talar component if a mobile-bearing TAA system has been used.Explantation of the failed primary implant and implantation of a TAA Revision System or patient-designed revision TAA is another option. Currently, the only systems available on the market are the INBONE prosthesis and the Salto Talaris XT Revision system. The INBONE device utilizes a lengthy modular tibial stem with intramedullary referencing and fixation , while tIn cases of severe prosthesis failure, loosening, subsidence, or a substantial structural bone post-TAA-arthrodesis  can be performed. The outcome of the structural bone transplant arthrodesis is unpredictable, and the success rate is, at best, low . The goaComplete extraction of implants from both joint surfaces, especially in cases where the problem resides on only one component of the prosthesis, would result in unnecessary bone loss that increases the difficulty of the surgery and decreases the likelihood of success. When faced with similar situations in hip and knee arthroplasty, the solution is to use cross-compatible components, possibly from different brands, to protect a well-fixed implant and to form a hybrid hip/knee joint system through partial component revision ,25.This paper aims to evaluate clinical and radiological outcomes of a TAA revision surgery by a hybrid total ankle arthroplasty (H-TAA) in a failed talar component of a mobile bearing-TAA at a one-year follow-up.In this prospective case series with a minimal 1-year follow up, 9 patients  with symptomatic aseptic loosening of the talar component of a mobile-bearing TAA were treated with isolated talar component and polyethylene insert substitution.The inclusion criteria of the study were painful, isolated, aseptic loosening of the talar component of a mobile TAA with a stable tibial component . The exclusion criteria were as follows: loosening of the tibial component, significant non-reconstructable bone defect of the loosened talus, and septic loosening. In all the nine cases, the hybrid TAA was performed through the implantation of a VANTAGE TAA Talar Component  with Flatcut Talar Component (six cases) and Standard Talar Component (three cases) . All surAll the patients were scored clinically and radiologically before the revision H-TAA surgery. Clinically, pain (VAS Pain Score 0–10), Dorsiflexion/Plantarflexion (DF/PF) Range of Motion , the American Orthopaedic Foot and Ankle Society (AOFAS) Ankle/Hindfoot Score (0–100 points), and Sports Frequency Score  level 0. A CompuAt a minimum 1-year postoperative follow-up after H-TAA implantation, the following data were documented: pain score, DF/PF-ROM, AOFAS Ankle/Hindfoot score, Patient’s Satisfaction (points 0–10), Sports Frequency Score.Weight-bearing radiographs were taken at the 1-year follow-up to document the talar component osteointegration and to rule out any possible sign of tibial component loosening.t-test. The cutoff for statistical significance was p < 0.05. Radiographic and statistical analysis were performed by the author S.S.To determine whether functional outcomes and scores had improved one year after surgery, data from preoperative and postoperative periods were compared using a 2-sample All the procedures  were performed in one stage. To exclude a possible infection/septic loosening, a Synovasure alpha defensin test (sensitivity of 69% and a specificity of 94%) with the joint fluid was performed . If the A standard anterior approach was made on the previous surgical scar. To preserve the anterior neurovascular bundle, which typically lies behind the tendon of the extensor hallucis longus muscle (EHL) or between the tendons of the extensor hallucis longus and extensor digitorum longus muscles (EDL), depth preparation is carried out lateral of the tibialis anterior tendon.In most cases, periarticular ossifications (PAO) removal was needed to free up the joint. This PAO autologous bone was collected for possible grafting of talar cysts. Polyethylene (PE) wear-induced synovitis and osteolysis were often detected. The mobile PE insert was removed and then the talar component was explanted. The stability of the tibial component was verified intraoperatively. A meticulous curettage of the aseptic loosening cysts of the talus was performed. At this point, an on-table decision was made to determine whether the bone loss was significant enough to use a VANTAGE Flatcut Talus or acceptable to use a VANTAGE Standard Talar component. The procedure is still possible even if there is substantial central talar bone loss, as the talar cortical ream is available . The talp < 0.001). Dorsiflexion/Plantarflexion ROM values increased significantly post-surgery on average: 21.7° preoperative to 45.6° postoperative (p < 0.001). The postoperative AOFAS scores were significantly greater than the preoperative values: 47.7 points preoperative, 92.3 points postoperative (p < 0.001). The sports activity improved from preoperative to postoperative where none of the patients could perform sports preoperatively. Postoperatively, eight patients were able to perform some sports activity. The postoperative overall average level of sports activity was 1.4. The postoperative average patients’ satisfaction was 9.3 points.The postoperative H-TAA X-rays at follow-up showed a good talar bone reconstruction and good osteointegration of the new VANTAGE talar component in all cases . The existing tibial components were unchanged and showed no loosening. No complications or reoperations for any of the included patients were noticed.In the present study, all revision Hybrid Total Ankle Arthroplasty (H-TAA) patients showed better results in the Vas pain Score, Dorsiflexion/Plantarflexion Range of Motion, AOFAS Ankle/Hindfoot Score, and sports activity at the 12-month follow-up. The H-TAA solution also noticed high personal satisfaction .Aseptic prosthetic implant failure is frequently due to abnormal tissue reactions (bone and soft tissue) induced by implant-derived material components of wear debris ,28. AccoPolyethylene wear, rate of wear, joint size, micromotion, high fluid pressure, and genetics are the main factors responsible for periprosthetic osteolysis .While the danger of periprosthetic osteolysis brought on by the wear of particles is decreased by the use of highly cross-linked polyethylene , it may In a 2019 study, Lachman et al. identified talar component subsidence/loosening as responsible for 36% of metal component failures in total ankle arthroplasty . There aNotwithstanding, when possible, a standard talar component has been used to preserve as much talar bone stock as possible. In mobile-bearing, three-component TAA systems, it is possible to keep the old tibial component and implant a talar component and an inlay of another system: Hybrid Total Ankle Arthroplasty (H-TAA). This is possible as the tibia component and the PE insert are both flat in all mobile-bearing three-component systems. This unique advantage of cross-brand hybridization is only possible in mobile-bearing three-component TAA systems. Suppose that a fixed-bearing, two-component TAA has an isolated talar component failure and the stable tibial component and the TAA system are no longer available in the market or the system has a lower quality. In that case, a H-TAA revision surgery is not possible, and the whole TAA, including the good tibial component, must be explanted.The present study’s limits are the small cohort of patients and the relatively short-term follow-up. However, this paper is the first paper with the biggest cohort of H-TAA. We believe a one-year follow-up is strong enough to show bone integration to a TAA . HoweverIn conclusion, Hybrid-Total Ankle Arthroplasty (H-TAA) is an excellent and feasible solution for mobile-bearing TAA revision with isolated talar aseptic loosening. H-TAA spares the patient the use of more invasive TAA revision systems or ankle arthrodesis. H-TAA improves pain, range of motion, function, and sports activity, with high patient satisfaction.


The mechanisms by which T2DM affects Ca2+ handling in VSMCs still remain poorly understood. Therefore, the objective of this study was to determine whether and how T2DM affects Ca2+ homeostasis in VSMCs. We evaluated intracellular Ca2+ signaling in VSMCs from Zucker Diabetic Fatty rats using Ca2+ imaging with Fura-2/AM. Our results indicate that T2DM decreases Ca2+ release from the sarcoplasmic reticulum (SR) and increases the activity of store-operated channels (SOCs). Moreover, we were able to identify an enhancement of the activity of the main Ca2+ extrusion mechanisms  during the early stage of the decay of the ATP-induced Ca2+ transient. In addition, we found an increase in Ca2+ entry through the reverse mode of NCX and a decrease in SERCA and PMCA activity during the late stage of the signal decay. These effects were appreciated as a shortening of ATP-induced Ca2+ transient during the early stage of the decay, as well as an increase in the amplitude of the following plateau. Enhanced cytosolic Ca2+ activity in VSMCs could contribute to vascular dysfunction associated with T2DM.Type 2 Diabetes Mellitus (T2DM) is a rapidly rising disease with cardiovascular complications constituting the most common cause of death among diabetic patients. Chronic hyperglycemia can induce vascular dysfunction through damage of the components of the vascular wall, such as vascular smooth muscle cells (VSMCs), which regulate vascular tone and contribute to vascular repair and remodeling. These functions are dependent on intracellular Ca Diabetes mellitus (DM) comprises a group of metabolic diseases characterized by an increase in plasma glucose levels (hyperglycemia) due to defects in the secretion (type 1 DM or T1DM) or action (type 2 DM or T2DM) of insulin . T2DM is2+ handling in vascular smooth muscle cells (VSMCs)  pathway and transient receptor potential canonical channels (TRPC) on the sarcolemma  Ca2+ release channels in the sarcoplasmic reticulum (SR)  . Remodelogenesis . Alterathenotype , the celrcolemma , and inolum (SR) . For inslum (SR) . In addile cells .2+ removal mechanisms, which significantly contribute to maintain intracellular Ca2+ homeostasis under physiological conditions, in VSMCs during T2DM. A study carried out on cultured A7r5 cells showed that hyperglycemia causes an increase in the activity of Plasma Membrane Ca2+-ATPase (PMCA), which extrudes Ca2+ across the sarcolemma, as well as a decrease in the activity of Sarco-Endoplasmic Reticulum Ca2+-ATPase (SERCA), which sequesters cytosolic Ca2+ into the SR (2+ clearance has not been directly assessed (+/Ca2+ exchanger (NCX) represents an alternative pathway for Ca2+ extrusion across the plasma membrane in VSMCs, but it is still unknown whether it is affected by T2DM. Therefore, it is mandatory to gain further insights about the remodeling of the Ca2+ handling machinery in an experimental model that effectively recapitulates the pathophysiology of T2DM, such as ZDF rats. The present investigation sought to exhaustively assess whether and how T2DM alters the intracellular Ca2+ signaling in VSMCs from the aorta of ZDF rats, loaded with the Ca2+-sensitive indicator, Fura-2-acetoxymethyl ester (Fura-2/AM).Only scarce information is available about the alteration of Cao the SR . Similarassessed . Na+/Ca2Norma Oficial Mexicana  for the care and handling of laboratory animals. The protocols were reviewed and approved by the Animal Care and Use Committee of the Benemerita Universidad Autonoma de Puebla, identification code: BERRSAL71, 18-05-2017. Experiments were carried out in male ZDF rats (3 months old) from Charles River Laboratories, California, U.S.A. Throughout the text, diabetic-obese ZDF rats (ZDF-Leprfa/fa) will be designated as OZDF rats, and lean controls, non-obese non-diabetic ZDF (ZDF-Lepr+/+) as LZDF. The rats were kept at the University Animal Core Facilities under controlled environmental temperature and, exposed to light-dark cycles of 12 h, with ad libitum consumption of water and high energy diet, Purina 5,008 chow.All the experiments were carried out according to the ® system . The day of the experiment, we proceeded to measure the body mass (weight), length (distance from the tip of the nose to the base of the tail) and the abdominal circumference using a measuring tape.To determine if OZDF rats did indeed develop T2DM, 5 days prior to the experiment, a glucose tolerance test was performed. The animals were fasted for 6 h in metabolic cages with free access to water. Immediately after, a glucose dose (2 g/kg of weight) was administrated intraperitoneally. Two hours later, a blood sample was obtained by puncture of the caudal vein to measure glucose levels using the AccucheckThe body mass index (BMI) was calculated using the following equation:Finally, all the epididymal fat surrounding both testes was accurately removed and weighed.2+ physiological salt solution . After de digestion, VSMCs were resuspended by gently triturating the tissue with a fire-polished Pasteur pipette. To stop enzymatic digestion, 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) was added. VSMCs were centrifugated for 10 min at 300g. The supernatant was discarded, and the resulting cell pellet was re-suspended in 1 mL complete medium (DMEM with 10% FBS and 1% antibiotic antimycotic solution (100x)). VSMC were plated on 35 mm culture dishes under a humidified atmosphere of 5% CO2 - 95% O2 at 37°C. Culture medium was changed on days 4 and 7 and Ca2+ imaging experiments were performed on subconfluent cultures on days 7 and 8. The aortic VSMCs identity was confirmed by α-actin labelling, as shown in  was monitored by measuring, for each ROI, the ratio of the mean fluorescence emitted at 510 nm when exciting alternatively at 340 and 380 nm (shortly termed “Ratio (F340/F380)”. An increase in [Ca2+]i causes an increase in the Ratio (F340/F380). Ratio measurements were performed and plotted on-line every 3 s. Ratio (F340/F380) values are expressed as arbitrary units (a.u.). Images were stored on the hard disk and converted offline to Ratio (F340/F380) images by ImageJ software . The experiments were carried out at controlled room temperature (22–23°C) to limit time-dependent decreases in the intensity of the fluorescence signal.Cultured VSMCs were loaded with 3 µM Ca2+ has been shown to quench Fura-2 fluorescence. Since Mn2+ and Ca2+ share common entry pathways in the plasma membrane, Fura-2 quenching by Mn2+ is regarded as an index of divalent cation influx. Experiments were carried out at the 360 nm wavelength, the isosbestic wavelength for Fura-2, and in Ca2+-free medium supplemented with 0.5 mM EGTA, as previously described  had the following composition (in mM): 140 NaCl, 5.36 KCl, 0.34 Na2HPO4, 0.44 KH2PO4, 10 HEPES, 1.2 MgCl2, 10 D-glucose and 0.05 CaCl2. Physiological salt solution (PSS2) composition (in mM) was: 140 NaCl, 5 KCl, 1.2 NaHPO4, 5 NaHCO3, 10 HEPES, 1.4 MgCl2, 1.8 CaCl2 and 11.5 D-glucose. In Ca2+ free solution (0Ca2+), 0.05 EGTA was added. In Mn2+-quenching experiments, 200 µM MnCl2 was added to the 0Ca2+ external solution. Solutions were titrated to pH 7.2 for PSS1 and 7.4 for PSS2 with NaOH. The osmolality of PSS as measured with an osmometer  was 338 mmol/kg.Low-Ca2+ response to either cyclopiazonic acid (CPA) or adenosine-trisphosphate (ATP) was measured as the difference between the Ratio (F340/F380) at the peak and the mean Ratio (F340/F380) of 500 s baseline before the peak (340/F380) 600 s after adding the agonist and the mean Ratio (F340/F380) of 200 s baseline before the peak of the Ca2+ response  (2+ peak amplitude (considered as 100%), shorty termed as “decay time” (DT) . For mean traces, fluorescence levels (F) were normalized to resting fluorescence (F0) to compare the height of the Ca2+ responses produced by cells displaying different basal fluorescence levels (F/F0). The rate of Mn2+ influx was evaluated by measuring the slope of the fluorescence intensity curve after Mn2+ addition (p < 0.05)), were statistically analyzed using an unpaired Student’s t-test. A p-value < 0.05 was considered statistically significant.For each protocol, data were collected from at least four rats under each condition. The amplitude of the peak Cathe peak . Amplituresponse . The dur (early) , 60% (in (early)  and 30%  (early)  of the ime” (DT) . The areaddition . Data arSEA0400 was obtained from Tocris Bioscience . Collagenase type 2 and FBS were obtained from Gibco . All other chemicals were purchased from Sigma-Aldrich.p ≤ 0.05) and of around 6.5% of the nose-tail length (p ≤ 0.05) compared with their control, LZDF rats. Given that the increase in weight could be attributed to an increase in the rat size (to its length), we proceeded to calculate the body mass index (BMI) to rule out that the differences seen in the weight were due to the difference in length between both groups. Likewise, the mean BMI value was statistically higher (46.6%) in the OZDF rats compared to the LZDF group (p ≤ 0.05), suggesting that the weight gain is due to obesity that has been widely reported in the literature in this strain of rats (p ≤ 0.05) and epididymal fat weight (p ≤ 0.05), respectively, in OZDF vs. LZDF rats. To confirm T2DM presence in OZDF rats, a glucose tolerance test was performed as described in Materials and Methods section. The mean ± SE of blood glucose levels measured 2 h after the intraperitoneal administration of glucose (2 g/kg of weight), was approximately 90% higher (p ≤ 0.05) in the OZDF rats compared to the LZDF rats  in VSMCs from OZDF rats (p ≤ 0.05)  . The fas2+ signals recorded from cultured VSMCs obtained from LZDF (2+) and absence  of Ca2+ in the extracellular medium. Removal of extracellular Ca2+ (0Ca2+) caused a decrease in peak amplitude, AUC, and decay time, as well as the disappearance of the plateau phase, in ATP-induced Ca2+ transients in both LZDF (2+ release through ER-resident IP3Rs and extracellular Ca2+ entry via store-operated channels (SOCs) support the Ca2+ response to ATP in proliferating rat aortic VSMCs  prevented the subsequent Ca2+ response to ATP (300 µM)  enhanced in rat aortic VSMCs from OZDF rats (rom LZDF  and OZDFrom LZDF  rat aortoth LZDF  and OZDFoth LZDF . These fic VSMCs . In acco(300 µM) . Of note(300 µM)  failed tZDF rats , which iZDF rats . Therefoal cells  and VSMCal cells , showed ells , in the absence of extracellular Ca2+ (0Ca2+) in rat aortic VSMCs from LZDF and OZDF rats  increased in VSMCs from OZDF rats  the Ca2+-transporting mechanisms clearing the initial Ca2+ peak are altered by T2DM, although this dysregulation is unmasked by removing extracellular Ca2+. The fastest decline of the initial Ca2+ peak recorded in the presence of external Ca2+ . In addition, enhanced SOCE is also likely to compensate for the lower IP3-induced SR Ca2+ release and bring the amplitude of the initial Ca2+ peak to the same value as that measured in LZDF rats.In order to assess whether IPZDF rats   in the presence of a selective SERCA inhibitor, CPA (10 µM).We first evaluated whether and how T2DM alters SERCA activity by measuring the Ca2+ did not cause any significant change in the amplitude of the initial Ca2+ peak evoked by ATP (300 µM), although there was a trend towards a reduction in LZDF rats and a trend toward an increase in OZDF rats (p < 0.05) increase in ATP-induced Amp600 in both LZDF and OZDF rats (p < 0.05) larger in VSMCs from LZDF as compared to OZDF rats (p < 0.05) increase in the decay time to 90% and 60% in VSMCs from both LZDF and OZDF rats (p < 0.05) larger in aortic VSMCs from OZDF rats (2+ response to 300 µM ATP was maintained at such a high plateau level (p < 0.05) in the AUC value in VSMCs from both LZDF and OZDF rats (p < 0.05) larger in diabetic VSMCs  reduced the Ca2+ peak amplitude of ATP-induced Ca2+ transient as compared to the control Ca2+ trace recorded without the inhibitor (p < 0.05) increasing the decay time to 90%, 60% and 30% and the AUC  increased as compared to the control Ca2+ trace obtained in the absence of CPA  to 90% and 60% (p < 0.05) larger in LZDF rats. These findings support the notion that, in rat aortic VSMCs from OZDF rats, SERCA activity is reduced during the late stage of the decay phase . In line with this hypothesis, blocking Ca2+ clearance with CPA converted the Ca2+ transient into a biphasic Ca2+ elevation in LZDF but not OZDF rats  to inhibit this mechanism during the stimulation with ATP in the presence and absence of extracellular Ca2+ (2+ peak amplitude (2+ response to ATP in VSMCs from LZDF rats (p < 0.05) increased in the presence of OV. As to the OZDF group, the inhibition of PMCA activity with OV caused a significant (p < 0.05) reduction in the Ca2+ peak amplitude (p < 0.05) increased  larger in VSMCs from LZDF rats as compared to the OZDF group. However, the increase in the decay time to 90% and 60% (ΔDT) was significantly (p < 0.05) larger in OZDF rats  caused a significant (p < 0.05) reduction in the Ca2+ peak amplitude (p < 0.05) reduced. In VSMCs from OZDF rats, pretreatment with OV also caused a significant decrease in the peak amplitude of the Ca2+ transient  were significantly larger in OZDF as compared to LZDF rats (p < 0.05) larger in the OZDF group compared to LZDF .Finally, in order to assess whether and how T2DM alters NCX activity in rat aortic VSMCs, we inhibited this mechanism using the selective inhibitor, SEA0400 (3 µM), in the presence and absence of extracellular Cap < 0.05) reduced the Ca2+ peak amplitude  reduced. In OZDF VSMCs (p < 0.05) affect the Ca2+ peak amplitude (p < 0.05) reduced the Amp600 (p < 0.05) increased (p < 0.05) difference in the ΔAmp600  larger in LZDF VSMCs (p < 0.05) difference in the ΔAUC between OZDF and LZDF rats  in the presence of CPA (10 µM) + OV (500 µM) + SEA0400 (3 µM) presented a significant (p < 0.05) decrease in peak amplitude  lower than in LZDF VSMCs  increase only in VSMCs from OZDF rats  (2+ response elicited by ATP (300 µM) in the presence of CPA (10 µM) + OV (500 µM) + SEA0400 (3 µM) still presented a significant (p < 0.05) decrease in peak amplitude (2+]i failed to fully recover to the baseline (p < 0.05) lower than in LZDF VSMCs  increase only in VSMCs from LZDF rats (In the absence of extracellular Ca (0Ca2+) , the Ca2mplitude  in both baseline , therebyDF VSMCs . This fiZDF rats .As shown in 2+ handling is altered in VSMCs from OZDF rats. In particular, T2DM causes two important effects on the ATP-evoked biphasic elevation in [Ca2+]i: 1) a significant reduction in decay time to 90% and 60% of the initial Ca2+ peak amplitude (early stage of the decay of [Ca2+]i to resting levels) and 2) a significant increase in the amplitude of the plateau (late stage of the decay of [Ca2+]i and SOCE activation). Shortening of the early phase of decay could be due to alterations in the activity of the Ca2+-clearing mechanisms, such as SERCA, PMCA and NCX.The experiments carried out in this study demonstrate that Ca2+ upon agonist stimulation in aortic VSMCs has extensively been reported. However, few studies are available above the effect of DM on the activity of these Ca2+-clearing mechanisms VSMCs. In A7r5 cells, hyperglycemia was reported to induce a reduction in SERCA expression and activity  activation, lower IP3R and RyR expression and/or activity, and a decrease in SR Ca2+ content. However, CPA-induced intracellular Ca2+ release, which is a widely employed readout of SR Ca2+ content, was not altered in OZDF VSMCs, as also reported in VSMCs from diabetic mice , was significantly reduced in OZDF rat aortic VSMCs. This finding suggests that either P2Y receptors are downregulated or their downstream signalling pathways, such as those culminating in PLC engagement and IP3R activation, are compromised by T2DM. A study performed in systemic arterial smooth muscle cells deriving from rat models of T1DM, as well as in VSMCs exposed to high glucose, showed a reduction in IP3R expression that attenuated vasopressin-induced Ca2+ response  whereby T2DM alters the Camulation . Ca2+ retic mice . In contresponse . Conversproteins . The disle VSMCs , dediffele VSMCs . Consistlar Ca2+ . The evi2+ elongated the decay phase of the Ca2+ transient induced by ATP in both experimental groups. However, this effect was greater during the early phases of decay  in OZDF rats. This observation suggests that Ca2+ sequestration by SERCA is enhanced during the initial stage of the Ca2+ transient and affects the early phase of decay in OZDF VSMCs. Furthermore, SERCA inhibition by CPA caused a larger increase in Amp600, which is a readout of plateau amplitude, in LZDF as compared to OZDF rats. This finding indicates that SERCA activity could decrease during the later stages of the decay phase, which would result in less removal of Ca2+ during the plateau phase. Therefore, the larger Amp600 recorded in OZDV VSMCs could be underlain by the combination of enhanced SOCE and reduced SERCA activity. Both an increase and a decrease in SERCA activity in the presence of DM have been reported. Particularly, in dyslipidemic diabetic pigs, Hill et al. found an increase in Ca2+ buffering by SERCA2 in coronary artery VSMCs isolated from dyslipidemic diabetic pigs (2+ and attenuating the Ca2+ response to vasopressin (2+ response to phenylephrine (2+] i can stimulate the production of reactive oxygen species (ROS) via the Ca2+-dependent recruitment of NADPH oxidase 5 (NOX5)  . Additio5 (NOX5) , such as2+-clearing mechanism evaluated in the present investigation was PMCA. In both experimental groups, OV caused a larger increase in Amp600 in LZDF as compared to OZDF rats. This finding indicates that also PMCA activity is reduced during the plateau phase of the Ca2+ response to ATP in diabetic animals. It turns out that blocking Ca2+ extrusion via PMCA with OV causes a larger accumulation of cytosolic Ca2+ in LZDF as compared to OZDF VSMCs. It is, therefore, plausible to conclude that the increase in plateau amplitude observed in diabetic rat aortic VSMCs is also due to a decrease in PMCA activity. As to the early phase of the Ca2+ signal, the decay time to 90% and 60% of the initial Ca2+ peak were significantly increased in OZDF, but not in LZDF VSMCs. This finding suggests that Ca2+ extrusion through PMCA is increased by T2DM at the onset of ATP-evoked increase in [Ca2+]i. Notably, OV reduced the Ca2+ peak amplitude in diabetic but not lean VSMCs. This finding suggests that enhanced PMCA activity is somehow required to propagate the rise in [Ca2+]i following agonist stimulation, as described in human platelets  NCX operates in the forward  mode during the late phase of the Ca2+ response to ATP in lean VSMCs and 2) NCX switches from the forward to the reverse-mode  during the decay phase of the Ca2+ response evoked by ATP in diabetic VSMCs. The Ca2+-clearing activity of NCX during the early phase of the ATP-evoked increase in [Ca2+]i could contribute to explain its sharper decline in OZDF VSMCs. Conversely, under 0Ca2+ conditions, Ca2+ extrusion via NCX seems to be decreased in diabetic VSMCs. In accord, blocking NCX activity accelerated the decay rate, which means that NCX is unlikely to clear cytosolic Ca2+ in the absence of Ca2+ entry. On the other hand, SEA0400 slows down the clearing rate in LZDF VSMCs, which indicates that in lean animals NCX drives the recovery of the Ca2+ transient to the baseline only under 0Ca2+ conditions.The last CaFive Similar to SERCA, it is still unclear how T2DM affects NCX activity in VSMCs. A study performed by El-najjar et al. showed that, in VSMCs exposed to high glucose, there were no alterations in NCX expression or activity . Convers2+-transporting machinery is deranged in aortic VSMCs from diabetic obese rats. The Ca2+ pumping activity of SERCA and PMCA is increased by T2DM during the early phase of the Ca2+ transient, whereas it is remarkably slowed down during the following plateau phase. Similarly, NCX contributes to buffer the initial increase in [Ca2+]i by operating in the reverse-mode, but it then reverses into the Ca2+-entry mode during the plateau phase. Therefore, the larger plateau observed in rat aortic VSMCs from OZDF rats is not only due to SOCE upregulation, as shown by others and confirmed in the present study, but also to the lower Ca2+ extrusion via PMCA and SERCA and to the activation of the reverse-mode of NCX. This latter observation suggests that a Na+-permeable channel is recruited or enhances its activity in diabetic VSMCs challenged with ATP. The non-selective cation channel, TRP Canonical 6 (TRPC6), can be gated by diacylglycerol produced upon PLC activation and generates local cytosolic Na+ transients beneath the plasma membrane that drive NCX-mediated Ca2+ entry in purinergically stimulated rat aortic VSMCs (2+]i during the plateau phase. Preliminary evidence showed that TRPC6 protein was upregulated in caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki rats (Herein, we provided the first demonstration that the Caic VSMCs . Future aki rats , whereasaki rats .2+ entry is required to engage PMCA and the forward mode of NCX during the decay phase. The functional coupling between Orai1, i.e., the pore-forming subunit of SOCs, and PMCA has been firmly established, while a straight-forward relationship between SOCE and NCX-mediated Ca2+ extrusion is yet to be reported. The faster decay of [Ca2+]i observed in diabetic rat aortic VSMCs challenged with ATP in the absence of extracellular Ca2+ and upon inhibition of either PMCA or NCX activity suggests the involvement of an alternative Ca2+-clearing mechanism. Mitochondrial have been shown to rapidly buffer Ca2+ at high [Ca2+]i in rat systemic arterial smooth muscle cells (2+ uptake could be increased by T2DM (2+ results in a plateau-like elevation in [Ca2+]i that presents a higher Amp600 in VSMCs from LZDF rats. This finding implies that an additional Ca2+-removal mechanism intervene to buffer, at least partially, the initial rise in[Ca2+]I elicited by ATP in rat aortic VSMCs from OZDF rats. These alterations in the Ca2+ clearing machine in aortic VSMCs could exacerbate vascular dysfunction in diabetes. For instance, the aberrant increase in [Ca2+]i observed could enhance VSMC proliferation and migration, thereby favoring atherosclerotic plaques and vascular calcification. In addition, cytosolic Ca2+ overload in VSMCs could exacerbate oxidative stress within the vascular wall (Intriguingly, extracellular Cale cells . Althoug by T2DM . Intrigular wall .


We investigated whether polyamine synthesis inhibition induced by DFMO may reverse this remodeling in CRC and, if so, the molecular basis for this phenotypic reversal. To this end we used calcium imaging and transcriptomic analysis in both normal and CRC cells. We found that CRC cells showed enhanced resting Ca2+ and store-operated Ca2+ entry (SOCE) but decreased Ca2+ store content relative to normal cells. Polyamine synthesis inhibition reversed not only this Ca2+ remodeling but also reversed the changes in the transcription of a dozen genes involved in Ca2+ transport in CRC cells, including genes modulating Ca2+ entry into the cells as store-operated channels and TRP channels as well as Ca2+ extrusion systems in the plasma membrane and mitochondria. These results provide a molecular basis for the role of polyamine synthesis in Ca2+ remodeling in cancer.The common activation of the c-Myc oncogene in colorectal cancer (CRC) induces the overexpression of ornithine decarboxylase (ODC), the limiting step in polyamine synthesis, which is a process blocked by α-Difluoromethylornithine (DFMO), an ODC suicide inhibitor and potential CRC treatment. We showed previously that intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer.Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca Colorectal cancer (CRC) is one of the most common types of cancer and causes of cancer deaths worldwide, with nearly 1,250,000 new CRC cases every year and a mortality rate as high as 50% of all cases . Substan2+ signaling induced by changes in the expression of TRPC1 channels as well as an increased ratio of STIM1 to STIM2  measurement from that ROI, as detailed before [2+] measurements, we used the median 340 nm/380 nm ratio of the first 30 s from each cell. After that, to obtain a measure of the Ca2+ store content from each cell, cells were treated with the reversible sarcoplasmic and endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor cyclopiazonic acid (CPA) at 10 μM in a calcium-free SEM. Accordingly, the Ca2+ store content from each cell was measured as both the increment in and area under the curve (AUC) of the 340 nm/380 nm ratio for the signal corresponding to the rise in intracellular [Ca2+] induced by CPA in Ca2+-free medium. Finally, after store depletion, cells were perfused with SEM containing CPA and 1 mM Ca2+ to induce SOCE. Then, the SOCE corresponding to every single cell was measured as both the rise in and the area under the curve (AUC) of the F340 nm/F380 nm ratio for the signal corresponding to the rise in intracellular [Ca2+].Fluorescence calcium measurements were conducted in colon cancer HT29 and SW480 cells as well as normal NCM460 cells, all treated and non-treated with DFMO. First of all, cells were washed with a standard external medium (SEM) containing (in mM) NaCl 145, KCl 5, CaCld before . For resAfter RNA isolation , microarrays were carried out in Centro Andaluz de Medicina Regenerativa , and RNA-seq assays were conducted by Sistemas Genómicos S.L . Then, data analysis based on the microarrays was performed by our group using .CEL files, whereas data analysis for RNA-seq was performed using count-matrix, provided by Sistemas Genómicos S.L. .p value = 0.1961) and homoscedasticity with the Bartlett test . The sample size was equal to 6:3, corresponding to HT29 cells without treatment and HT29 cells treated with DFMO, respectively; p < 0.01.HT29 cells were treated with vehicle or DFMO  and used for western blotting. Total protein was extracted from cells and used to quantify the expression of MCU. Whole-cell lysate was obtained using RIPA buffer  supplemented with the Halt™ Protease and Phosphatase Inhibitor Cocktail (100×) from ThermoFisher Scientific (ref #1861281) . Protein concentrations were determined by a Bradford protein assay. Proteins were fractionated by SDS-PAGE; electroblotted onto PVDF membranes; and probed with the antibodies at a dilution of 1/200, except for the anti-β-actin, which was used at a dilution of 1/5000. The antibody against MCU (SC-246071) had been previously characterized and was visualized by the addition of goat anti-rabbit IgG or rabbit anti-mouse IgG. Detection was performed using Pierce ECL Western Blotting substrate (Thermo Scientific) and a VersaDoc Imaging System . The quantification of protein expression was carried out using Quantity One software . The datasets were analyzed by adjusting a linear model to fit the data, the model assumptions were evaluated, the response variables were transformed with Box–Cox family transformations because the normality assumption was violated, and outlier detection was carried out by an analysis of the Cook’s distance and the studentized residual. After transformation, the model assumptions were fulfilled as follows: residual normality with the Shapiro–Wilks test  following the manufacturer’s instructions. A confluent 60 mm Petri dish per condition was employed for each assay. The quality of the RNA was determined by optical density measurements at 260 and 280 nm and by electrophoresis on agarose gels. After DNAse I treatment with a RapidOut DNA removal kit (Thermo Scientific), 1000 ng of RNA was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific) at 37 °C for 120 min to obtain cDNA.Total RNA from HT29 and NCM460 cells was isolated with a GeneMatrix Universal RNA purification kit  in a LightCyler® 480  thermocycler. The primers used are shown in Quantitative PCR was carried out on equal amounts of cDNA in triplicate for each sample using a Kapa Sybrp < 0.05, ** p < 0.01, *** p < 0.001The qPCR data were analyzed using the threshold cycle (Ct) relative quantification method (ΔΔCt). The gene expression levels were normalized by the housekeeping gene RP18S. The relative abundance of the genes was calculated as 2ΔCt, where ΔCt = Ctgene–Ct18S. Differences between cancer (HT29) and control (NCM460) cells were calculated from 2ΔΔCt, where ΔΔCt = ΔCt(HT29)–ΔCt(NCM460), using ΔCt(NCM460) as the calibrator. In this analysis, a value of 0 indicated no change, negative values indicated decreased expression, and positive values indicated increased expression relative to the calibrator. The statistical analysis method used was a Student’s t test with Bonferroni’s correction for two independent samples (ΔCt(HT29) and ΔCt(NCM460)) obtained with RP18S as endogenous control. * 2+], Ca2+ store content, and SOCE. Thus, as response variables, at the single-cell level, we considered the median from the absolute value of the signal across the first 30 s for resting intracellular [Ca2+], and the corresponding increase in [Ca2+] levels and the area under the curve (AUC) for both Ca2+ store content and SOCE. We evaluated the effect of two factors, treatment and cellular line, on the response variable, each of them with just two levels , so there was a total of 4 experimental conditions: control-NCM460, DFMO-NCM460, control-HT29, and DFMO-HT29. Due to the experimental design, the dataset showed a hierarchical structure, since observations were clustered into, or organized at, different levels: the experiment was carried out on 4 different days; each day we performed 2 replicates corresponding to each of the 4 experimental conditions; and within each replicate, we measured several cells from the same experimental conditions. Furthermore, both replicates and day were random factors.For the calcium imaging experiments, we were interested in testing which experimental conditions were different from each other in terms of resting intracellular  and Orai1 activation independently of store depletion in CRC, a process that is limited by polyamine synthesis inhibition. In addition, it has recently been reported that the epigenetic modulation of SPCA2 reverses the epithelial-to-mesenchymal transition in breast cancer cells . This gene returned to normal levels after DFMO treatment, thus probably contributing to decreased resting Ca2+ levels in treated cells.Regarding Cay to CRC , was dow2+ transport systems were differentially expressed in CRC cells. Most changes involved the decreased expression of the molecular players involved in the control of the mitochondrial Ca2+ uniporter (MCU) and the Ca2+ channel involved in mitochondrial Ca2+ uptake. Once more, polyamine synthesis inhibition had no effect on the transcription levels of the mitochondrial Ca2+ transport systems in normal cells. However, in cancer cells, polyamine synthesis inhibition increased the expression of both MCU and VDAC3. qPCR analysis also confirmed the increased expression of VDAC3 in DFMO-treated HT29 cells. We used western blotting to confirm that MCU expression was also enhanced at the protein level in HT29 cells treated with DFMO , the plasma membrane Ca2+ ATPase PMCA4 Ca2+ pump, and the mitochondrial Ca2+ channels MCU and VDAC3, contributing to dysregulated SOCE and enhanced resting Ca2+ and mitochondrial Ca2+ uptake in CRC. Conversely, polyamines could also induce the overexpression of TRPV6 channels, the Orai1 activator SPCA2, and the exchange of TRPP1 channels by TRPP2, thus likely contributing to enhanced Ca2+ entry and decreased Ca2+ store content in CRC. These findings may provide new insights into the role of polyamines in Ca2+ remodeling in cancer. Further research in animal models and/or tumor samples from patients is required to validate our findings.In summary, our results indicated that a few genes involved in Ca2+ and store-operated Ca2+ entry, as well as an increase in Ca2+ store content. These effects were observed in CRC cells but not in normal colonic cells. Analogously, polyamine synthesis inhibition induced the differential expression of 25% of the whole transcriptome in cancer cells but only about 0.25% of the transcripts in normal colonic cells.We conclude that polyamine synthesis inhibition may partially reverse changes in intracellular calcium homeostasis hypothetically associated with CRC, including a decrease in resting intracellular Ca2+ influx in DFMO-treated tumor cells. In addition, polyamine depletion enhanced the transcription of the plasma membrane PMCA4 Ca2+ pump and the mitochondrial channels MCU and VDAC3, thus probably contributing to enhanced Ca2+ extrusion in DFMO-treated cells. Collectively, these results may provide a transcriptional basis for the hypothetical calcium remodeling in CRC and its reversal by DFMO. Further research in animal models and/or tumor samples from patients is required to validate our findings.We also conclude that polyamine synthesis inhibition reversed the changes in the differential expression of transcripts associated with cancer. Specifically, polyamine synthesis inhibition decreased the expression of the TRPV6 and TRPP1 channels, as well as the Orai1 positive modulator SPCA2, probably contributing to decreased Ca


Diagnosis classification and risk stratification are crucial in the prognosis prediction and treatment selection of acute myeloid leukemia (AML). Here, we used a database of 536 AML patients to compare the 4th and 5th WHO classifications and the 2017 and 2022 versions of ELN guidance.AML patients were classified according to the 4th and 5th WHO classifications, as well as the 2017 and 2022 versions of the European LeukemiaNet (ELN) guidance. Kaplan–Meier curves with log-rank tests were used for survival analysis.NPM1, RUNX1, SH2B3, and TP53), and fusions (CBFB::MYH11 and RUNX1::RUNX1T1) were included, and our model could help divide the patients into favorable, intermediate, and adverse groups.The biggest change was that 25 (5.2%), 8 (1.6%), and 1 (0.2%) patients in the AML, not otherwise specified (NOS) group according to the 4th WHO classification, were re-classified into the AML-MR (myelodysplasia-related), KMT2A rearrangement, and NUP98 rearrangement subgroups based on the 5th WHO classification. Referring to the ELN guidance, 16 patients in the favorable group, six patients in the adverse group, and 13 patients in the intermediate group based on the 2017 ELN guidance were re-classified to the intermediate and adverse groups based on the 2022 ELN guidance. Regrettably, the Kaplan–Meier curves showed that the survival of intermediate and adverse groups could not be distinguished well according to either the 2017 or 2022 ELN guidance. To this end, we constructed a risk model for Chinese AML patients, in which the clinical information (age and gender), gene mutations (These results affirmed the clinical value of both WHO and ELN, but a more suitable prognosis model should be established in Chinese cohorts, such as the models we proposed. KMT2A/MECOM/NUP98 rearrangement and CEBPA mutation, as well as the AML, myelodysplasia-related (MR) subgroup.Acute myeloid leukemia (AML) represents the most common type of acute leukemia in adults worldwide . AccumulIn the present study, we used a dataset of 536 consecutive subjects with AML initially diagnosed using the 2016 WHO criteria to compare how these subjects would be classified using the 2022 WHO criteria. In addition, we compared the prognostic classification of AML according to the 2017 and 2022 versions of the European Leukemia Net (ELN) guidelines  were collected and shown as A total of 536 patients with primary AML were included in this study between September 2013 and February 2021 from the First Affiliated Hospital of Xi'an Jiaotong University. The patients were diagnosed with AML according to the 4th or 5th version of the WHO guidance  on the Novaseq  sequencing platform. The original sequencing was aligned with the human reference genome GRCh37. Single nucleotide variations (SNVs) and insertion and deletion (Indels) were screened by Shanghai Rightongene Biotechnology Co., Ltd.  based on the filtering conditions: (1) SNVs or Indels with a mutation allele frequency (MAF) of ≥0.001 in databases of 1,000 genomes project, 1,000 genome East Asian, ExAC all, or ExAC East Asian were removed; (2) SNVs or Indels with a variant allele frequency (VAF) of ≥ 1% were retained; (3) dbSNP (v147) sites present in COSMIC database were retained; and (4) SNPs or Indels including stopgain, stoploss, frameshift, non-frameshift, and splicing sites were retained.Genomic DNA (gDNA) was extracted from the formalin-fixed paraffin-embedded (FFPE) or fresh BM samples, followed by the targeted sequencing of 38 genes , rearrangements , and clinicopathologic features  were included in the models. Conventional Cox regression was used to train the models for assessing survival with the selected variables by Lasso using the “Glmnet” package. The optimal cutoff values for risk score were determined using the X-tile software   and the test set (30%), respectively. The training set was subjected to 10-fold cross-validation to account for variability and provide risk estimates. The mutated genes . Kaplan–Meier curves with log-rank tests were used to analyze the OS and PFS of AML patients in different groups. A KMT2A, MECOM, and NUP98 rearrangements and NPM1 mutation were diagnosed with AML regardless of the percentage of blasts; (2) the definition of AML with CEBPA mutation was changed to include both biallelic (biCEBPA) and single mutation of CEBPA located in the basic leucine zipper (bZIP) region (smbZIP-CEBPA); (3) the previous classification of AML with mutated RUNX1 was abolished; (4) the classification of AML with myelodysplasia-related changes (AML-MRC) has been changed to AML-MR, and the mutations in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2 genes were newly defined as the defining cytogenetic abnormalities; (5) the classification of AML with other defined genetic alterations was newly added; and (6) the classification of AML, not otherwise specified (NOS) was replaced with AML, defined by differentiation.The 5th WHO classification made some changes in the diagnostic criteria of AML, including (1) persons with CEBPA or the VAF of FLT3-ITD is not available. Six subgroups, AML with RUNX1::RUNX1T1 (n = 70), AML with CBFB::MYH11 (n = 29), AML with DEK::NUP214 (n = 5), AML with BCR::ABL1 (n = 4), AML with GATA2::MECOM (n = 2), and AML with NPM1 (n = 89), remained unchanged according to the 5th WHO, although the subgroup of AML with GATA2::MECOM was changed to AML with MECOM rearrangement. The group that changed the most was the AML, NOS (not otherwise specified) subgroup (n = 212); in detail, 8 patients were divided into the AML with KMT2A-rearrangement subgroup, 25 patients were divided into the AML-MR subgroup, and one case was divided into the AML with NUP98-rearrangement subgroup based on the 5th WHO classification  the previously defined favorable risk of biCEBPA was changed to bZIP in-frame mutated CEBPA; (3) KAT6A::CREBBP and mutated ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, or ZRSR2 were added as adverse events; and (4) VAF ≥ 10% was defined as an additional condition for the adverse event of TP53 mutation.Diagnostic criteria were largely unchanged in the new proposal, except for the following: (1) persons with FLT3-ITD and the lack of the bZIP in-frame mutated CEBPA. In addition, 13 patients in the intermediate group based on the 2017 ELN guidance were regrouped into the adverse group based on the 2022 ELN guidance due to the mutations of BCOR, SRSF2, U2AF1, and ZRSR2. Seventeen unclassified patients (the detailed information of CEBPA and FLT3-ITD unknown) based on the 2017 ELN guidance were regrouped into the intermediate group  according to the 2022 ELN guidance was consistent with the 2017 ELN. In contrast, 16 patients in the favorable group based on the 2017 ELN guidance were regrouped into the intermediate group based on the 2022 ELN guidance due to the low VAF value of te group , Table 3CEBPA, while the PFS and OS rates were lower for patients with MLLT3::KMT2A and GATA2::MECOM according to the 4th WHO classification  + 0.025315*age- 0.88687*CBFB:: MYH11-0.801312*NPM1+0.89387*RUNX1-0.757403*RUNX1::RUNX1T1+1.244844*SH2B3+1.120662*TP53, in which positive was defined as “1” and negative was defined as “0” in terms of gene mutation and fusion. Then, the patients were divided into three groups, favorable (risk score ≤ 1.80), intermediate (risk score < 1.8 < 2.37), and adverse (risk score ≥ 2.37). As shown in To establish a better prognosis model for Chinese patients with newly diagnosed AML, mutation, rearrangements, and clinicopathologic features were considered. For the PFS model, risk score was first calculated according to the following formula, risk score = 0.324134*sex  + 0.027509*age- 0.883355*CBFB::MYH11-0.84456*NPM1+0.870257*RUNX1-0.73479*RUNX1::RUNX1T1+1.240181*SH2B3+1.106195*TP53. After calculating the risk score of each patient, the patients were divided into three groups, favorable (≤ 1.94), intermediate (< 1.94 < 2.33), and adverse (≥2.33). The PFS time of the favorable group was obviously higher as compared with the intermediate and adverse groups, as well as the intermediate group vs. the adverse group in both the training set and test set , 8 (1.6%), and 1 (0.2%) patients in the AML, NOS group according to the 4th WHO, were re-classified into the AML-MR, KMT2A rearrangement, and NUP98 rearrangement subgroups, respectively. Still, 38% (185/486) of patients were divided into the AML defined by differentiation subgroup according to the 5th WHO classification due to the limitation of sequencing technology earlier in the years. However, it was decreased compared to the proportion of AML, NOS 44.5%, 216/485). With the advances in gene detection technologies, patients are divided into more precise categories with clearer treatment strategies and prognoses . For inss of AML –14. RUNXpatients . However/485. Wity et al.  showed tIn addition, we compared the risk stratification of AML patients according to the 2017 and 2022 ELN guidance. In total, 16 (3.3%) patients in the favorable group and 13 (2.7%) patients in the intermediate group were re-classified to the intermediate and adverse groups based on the 2022 ELN guidance, while 6 (1.2%) patients in the adverse group were re-grouped into the intermediate group based on the 2022 ELN guidance. Regrettably, both the PFS and OS curves of the intermediate and adverse groups were not very distinguishable in our cohort even after excluding the patients who received HCT, which significantly improved the prognosis of AML, as previously reported , 19. ThiNPM1, RUNX1, SH2B3, and TP53 genes, fusions of CBFB::MYH11 and RUNX1::RUNX1T1, and the clinical factors of sex and age were selected as the important influencing factors of both PFS and OS. Among them, CBFB::MYH11, RUNX1::RUNX1T1, and NPM1 mutations were the protective factors, while the mutations of RUNX1, SH2B3, and TP53 genes were adverse factors, which were consistent with the ELN guidance , and fusions (CBFB::MYH11 and RUNX1::RUNX1T1). This model could easily help divide the patients into favorable, intermediate, and adverse groups, which may be suitable for Chinese AML patients.Collectively, this study compared the 4th and 5th WHO, as well as the 2017 and 2022 ELN guidance in Chinese AML patients. Although the classification and risk stratification were improved and defined by the 5th WHO classification and 2022 ELN guidance, the risk models were not very suitable. Based on this, we established a risk model for Chinese AML patients, which included age, sex, mutations (https://ngdc.cncb.ac.cn/gsa-human/browse/HRA004529.The data presented in the study are deposited in the Genome Sequence Archive for Human (GSA-Human) repository, accession number “HRA004529”, The studies involving human participants were reviewed and approved by Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XW and JieW wrote the paper. SW, JuZ, JiZ, DW, ML, SZ, YC, HL, HZ, JinW, and WW provided the data. BX and GL analyzed the data. HW, HX, and PH reviewed the paper. All authors contributed to the article and approved the submitted version.


DLV2-6 and EIAVUK3, proviral DNA from peripheral blood mononuclear cells of artificially immunized horses, and virus nucleic acid from EIAV positive serum samples. The TG-qPCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the TG-qPCR assay was 1 copy/reaction for both viral RNA and proviral DNA based on the Poisson distribution. Compared to the qPCR, the TG-qPCR has better inclusivity and can detect not only Asian EIAV strains but also almost all the representative EIAV strains from other continents. The above results show that the TG-qPCR assay could serve as an effective tool for the early diagnosis of clinical EIA disease.Equine infectious anemia (EIA) has a worldwide distribution and causes severe economic losses to the equine industry. The EIA virus (EIAV) genome sequences from different countries are highly diverse, which poses a great challenge for pathogen identification with PCR. Phylogenetic analysis showed that although gag is the most conserved structural gene, it still has great genome variability. Currently, most existing PCR methods are designed based on the gag gene sequence and therefore do not cover all the viral strains, especially Asian EIAV strains. In this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes, which was relatively conserved in all the known EIAV strains. The performance of the TG-qPCR was evaluated against that of the standard qPCR (recommended by WOAH) by testing viral RNA extracted from viral supernatants of EIAVEquine infectious anemia (EIA) has a worldwide distribution and causes significant losses to the equine industry worldwide. A reliable detection method is necessary to control the transmission of EIA virus (EIAV). Currently, most of the available real-time PCR assays, including the qPCR of recommended by WOAH, are developed according to the sequences of European or American EIAV strains; however, the primers and probe sequences have low homology with Asian EIAV strains. To the best of our knowledge, no qPCR method capable of the well detection of Asian EIAV strains, especially Chinese EIAV strains, has been published to date. The development of a sensitive, specific, and rapid qPCR assay for the detection of the EIAV strains is therefore of great importance. Retroviridae family, which infects equines. EIA has a worldwide distribution (Equine infectious anemia (EIA) is caused by the EIA virus (EIAV), a lentivirus classified within the ribution  and causribution , 2. Finaribution . TherefoThe usual tests for this disease are pathological, serological, and molecular detection techniques. Pathological detection can diagnose EIA by immunohistochemical assays . HoweverIn this study, we developed a tat-gag-based real-time quantitative PCR (TG-qPCR) for the detection of EIAV by targeting the fragment between the tat and gag genes. We compared our TG-qPCR and the qPCR recommended by the WOAH by detecting Chinese and American EIAV in cell supernatants or synthesized plasmids containing the sequences of other EIAVs. The TG-qPCR performance was also evaluated in peripheral blood mononuclear cells (PBMCs) and serum samples.A phylogenetic analysis of gag sequences from 18 EIAVs was performed. The phylogenetic tree showed that although gag is the most conserved structural gene in the whole EIAV genome, it still has large genome variability . The gagThe sequences of the amplified DNA corresponding to the TG-qPCR and qPCR reactions were aligned . The aliH3N8, EIVH7N7, EHV-1, EHV-4, Salmonella abortusequi, and Streptococcus equi. The TG-qPCR detected only the EIAV, but none of the others, suggesting that the TG-qPCR has high specificity.The analytical specificity of the TG-qPCR was determined by testing the nucleic acids from EIAV, equine arteritis virus (EAV), EIV3–107 copies) of the standard plasmids were tested using TG-qPCR in duplicate for each dilution. The coefficient of correlation (R2) and amplification efficiency values were 0.996% and 98.6%, respectively  and 65% (13/20), respectively. For testing 1 copy of viral RNA from EIAVUK3 and EIAVDLV2-6 infected cells, the detection rates were 60% (6/10). Therefore, the detection limit of the TG-qPCR was found to be 1 copy/reaction based on the Poisson distribution.Serial 10-fold dilutions (10ectively . RNA andere 100% . For tes0 to 105 dilutions) of the proviral DNA and viral RNA from EIAVUK3 infected cells (The intra-assay and inter-assay reproducibility of TG-qPCR were tested with 10-fold dilutions (from 10ed cells . The intUK3 and EIAVDLV2-6 infected cells) were prepared and tested in duplicate using the above methods. As shown in DLV2-6 infected cells but those of EIAVUK3 infected cells. Meanwhile, the maximum detectable dilution at which the proviral DNA and viral RNA of EIAVUK3 infected cells could be detected (103) by qPCR was lower than that detected by TG-qPCR (105).Serial 10-fold dilutions of EIAV nucleic acids  and one American EIAVse EIAVs . To furtIt is currently almost impossible to obtain fresh PBMCs from horses suffering from EIA in China. The stored PBMCs collected 5 years ago from experimental horses were tested with our assay. Both the proviral DNA extracted from PBMCs and virus nucleic acid extracted from seropositive naturally infected horses can be detected by our assay. On one hand, the PBMC samples from the seven experimental animals immunized with attenuated vaccine all tested as EIAV proviral DNA positive, while only two of the corresponding sera tested positive for anti-EIAV antibodies . This imThe TG-qPCR has better inclusivity for the detection of EIAV. The TG-qPCR assay could successfully detect viruses from China and the United States, as well as synthetic plasmids of representative strains from other countries, including England, Italy, France, Japan, Ireland, and the recently discovered isolates from Serbia and Brazil Pantanal (2020) , 29. TheIn conclusion, we developed a TG-qPCR assay for the detection of EIAV with high specificity, sensitivity, reproducibility, and inclusivity. This assay could serve as a reliable tool for the rapid detection of EIAV in clinical PBMC and serum samples.2 in RPMI-1640 medium supplemented with 60% serum . Five days after the EIAV infection, macrophages were used to extract proviral DNA, and a cell culture supernatant was used to extract viral RNA.Equine macrophage cells were isolated from the whole blood of healthy horses and cultivated at 37°C in an atmosphere of 5% COUK3, EIAVDLV2-6, EAV, Equine influenza virus (EIV both H3N8 and H7N7 serotypes), equine herpes virus type 1 (EHV-1), equine herpes virus type 4 (EHV-4), S. abortusequi, and S. equi were provided by the Harbin Veterinary Research Institute.EIAVThe Chinese EIAV  and the America EIAV (UK3) were available in our laboratory, while the other viruses were not, so the specifically amplified sequences of those representative viruses were synthesized in order to evaluate the assay. The 18 whole-genome sequences of EIAV were downloaded from the National Center for Biotechnology Information (NCBI) and aligned, and the corresponding sequences of the amplicons corresponding to the TG-qPCR and qPCR were selected. These two fragments of each strain were linked with 10 bases and were synthesized by the Beijing Liuhe BGI Co. The P-Wyoming of synthetic plasmid was used as the standard plasmid for TG-qPCR. The concentrations of synthetic plasmids were measured on a NanoDrop 1,000 instrument, and the copy number of the standard plasmid was calculated using the standard formula .The gag gene sequences were identified from the 18 EIAVs strains from Genbank and were aligned using Clustal W in Mega version 7.0 and were used to construct a phylogenetic tree. The phylogenetic analysis was performed in the Mega program, version 7.0  using thhttps://www.idtdna.com/calc/analy zer  values.The analytical sensitivity of the TG-qPCR was evaluated using 20 replicates of the EIAV proviral DNA and 10 replicates of RNA extracted from EIAVGGAGCCTTGAAAGGAGGGCCACTAAA-3′, anti-sense primer: 5′-TTGTTGTGCTGACTCTTCTGTTGTATCGGG-3′, and probe [TaqMan probe: 5’-(FAM) ACGGGAAGCAAGGGGCTCAAGGGAGGCC (BHQ-1)–3’] –3’] . In addiTo evaluate the reliability and clinical feasibility of TG-qPCR assay, both PBMCs and sera samples were used for testing for virus nucleic acid. PBMCs samples were collected from the whole blood of experimental horses immunized with the EIAV vaccine  and storTo validate the authenticity of the TG-qPCR results, sequence analysis of the EIAV-positive amplicons from clinical samples was performed. For samples with a TG-qPCR Ct value of more than 30, the amplified products were diluted 500 times as a template and amplified again, and the amplified products from the second round of TG-qPCR were fractionated in a 2% agarose gel and purified using a FastPure Gel DNA Extraction Mini Kit  prior to cloning into the pMD18-T vector, and the resulting plasmids were sent to the biotechnology company for sequencing.