The forging–healing of the internal porosity defects affects the tensile, impact and fatigue properties of heavy forgings. In the present work, the effect of deformation process on the microstructure in the joint area as well as the tensile strength, impact toughness and fatigue strength was studied experimentally. It is shown that the tensile strength is restored once the porosity defects were healed, and the impact toughness is recovered when the flat grain band is eliminated. The fatigue strength can be restored if a uniform grain structure can be achieved in both the joint area and the matrix, whereafter precipitate become the key factor affecting the fatigue strength. A complete healing of the porosity defects, a uniform grain structure in the joint area and the matrix, and a fully controlled precipitate are essential to guarantee the mechanical properties and in-service performance of the heavy forgings. As load-bearing components, forgings are widely used in energy equipment manufacturing, shipbuilding and other important fields. The quality and performance of the forgings determine the reliability of the equipment. The mechanical properties of the forgings depend on their chemical composition and structures. Therefore, it is very important to study the influence of plastic deformation processes on the structures as well as their corresponding effects on the tensile, impact and fatigue properties, which is essential for providing guidelines to formulate appropriate forging processes to guarantee the comprehensive mechanical properties and service life of the forgings ,2,3,4,5.Nowadays, ingots produced using double vacuum smelting process are used to produce heavy forgings, where inclusions are normally well controlled. However, due to the solidification shrinkage or the thermal expansion of the materials, cavity or porosity defects exist in the ingots inevitably, and with the increase in the size of the ingots, it becomes more difficult to control the porosity defects. The forging–healing of the porosity defects has remarkable influences on the tensile, impact and fatigue properties of the forgings, especially for super heavy forgings. Accordingly, a precise control of the healing quality becomes the key factor ensuring the internal quality, load bearing capacity and service performance of the forgings. A508.3 steel is generally used to manufacture pressure vessel forgings for nuclear reactors. The comprehensive mechanical properties of forgings, such as tensile strength, impact toughness, fatigue strength, etc., play an important role in reactor safety, which has attracted great attention.It is the most feasible and efficient method of hot plastic deformation for mechanical properties required in the actual production of heavy forgings. Han et al.  found thHowever, the study about the recovery of mechanical properties under dynamic load, which is important for actual service condition of heavy forgings, is still not enough, and it is essential to carry out further research on crack healing methods to improve mechanical properties recovery. The effects of deformation modes and quenching and tempering (Q&T) process on internal crack healing in SA 508-3 steel were studied, which laid the foundation to further study optimum internal crack healing strategy.For a certain steel grade with a particular chemical composition, the forging process parameters including deformation amount, temperature and deformation rate determine the microstructure of the forgings. It is of significance to investigate the relationship among the process parameters, microstructures and the mechanical properties under static and dynamic loads, i.e., tensile strength, impact toughness and fatigue strength, which is a knowledge base for exploring approaches to increase the load bearing performance and improve the in-service safety of the forgings.In the present work, the forging–healing of the internal porosity defects was studied as well as the effect of the deformation process parameters and microstructures on the tensile strength, impact toughness and fatigue strength of the forgings.An experimental method called butt-joint and high-temperature compression method was employed to simulate the forging–healing process, by which the effect of the process parameters and microstructure on the tensile strength, impact toughness and fatigue strength was studied. SA508.3 forged steel was chosen as the experimental material and cylindrical pieces with a diameter of 120 mm and a height of 60 mm were cut from the forged material. The chemical composition  of the material cut from the SA508.3 steel forging is C 0.19, Si 0.22, Mn 1.4, P 0.006, S 0.006, Cr 0.12, Mo 0.53, Ni 0.53 and the balance is Fe. The high-temperature forging experiment was conducted by using two cylindrical pieces welded at the edges. The geometry of the cylindrical piece and the forging experiment process are shown in In order to evaluate the forging–healing effectiveness, experiments for the purpose of comparison were designed and conducted, which means that for any experiment, a counterpart forging experiment was carried out under the same deformation condition. We may consider the joint interface between the two cylindric pieces to be an artificial crack, and in the counterpart experiment, the work piece is a single bulk of the material without any artificial crack. In addition, a term ‘percentage of recovery’ was defined as the ratio of the property value measured in the experiment to that measured in the counterpart experiment.The cylindric work pieces were forged under designed processes, held at a certain temperature and cooled to room temperature. Specimens for mechanical property test were taken from the forged work piece by using wire cutting along the axial direction of the piece.The geometry and dimensions of the tensile test specimens are shown in According to the China National Standard GB/T229 ‘Metallic Materials Charpy Impact Test Method’ , the siz−1, and the load ratio Rs is −1. The specimen was broken when the load dropped to 25% of the stable load.According to the China National Standard GB/T 15248-2008 ‘Axial Constant Amplitude Low Cycle Fatigue Test Method for Metallic Materials’ , the speThe specimens were heated to 950, 1050 and 1150 °C for upsetting deformation, and the upsetting reduction rates were 10%, 20% and 30%, respectively. After the forging is completed, keep it at the deformation temperature for 60 min, and then air cool it to room temperature. According to the method and location in The tensile strength of the specimen decreases with the increase in deformation temperature. When the deformation temperature is the same and the upsetting reduction rate is 20%, the tensile strength of the material can basically recover to the initial structure state. Under the experimental temperature conditions, the effect of temperature on the recovery of tensile strength is not significant.The cylindrical piece was heated to 1050 °C and held at this temperature for 60 min, followed by upsetting deformation and natural cooling to room temperature. As shown in The comparison of the structure state shows that the tensile strength basically recovers after the elimination of internal porosity defects. Temperature and plastic deformation are important factors that affect the forging of internal porous defects to improve the tensile strength. The effect is mainly reflected by the existence of porous defects.The cylindrical piece was compressed by the reduction rate of 30% at 1050 °C with different strain rates, and the microstructure of the joint area is shown in −1, the joint interface area is uneven, and there are many small pores in it, as shown in −1, the unevenness in the joint area was significantly reduced, as shown in When the strain rate is 1 sUnder the condition of the same reduction, a decrease in the strain rate means an increase in the deformation time, which allows a longer time for recrystallization nucleation and growth in the joint area. The recrystallized grains fill the cracks and pores, thus reducing the unevenness in the joint area.It was found that the thermoplastic deformation resulted in the healing of the joint interface; however, microstructure differences still exist between the joint area and the matrix. Therefore, for the steel SA508.3, a treatment of holding at 1050 °C for a period of time is necessary to promote homogenization and reduce the microstructure difference between the joint area and the matrix, though the tensile strength does not have a noticeable change in this process.Based on the above results shown in At this time, typical defect repair tissues were cut for impact test, as shown in The cylindrical pieces were heated to 950, 1050 and 1150 °C, respectively, followed by holding at the temperatures for 60 min, and then the pieces were treated with the following processes: upsetting, upsetting–drawing–upsetting and upsetting–drawing–upsetting–drawing, respectively. The reduction rate of the upsetting is 20%, and the reduction sequence in each drawing cycle is 0°→180°→90°→270°. One drawing operation is composed of four reduction cycles, and a full anvil reduction was applied with reduction rate of 20% for each pressing. After the forging process, the pieces were holding at the deformation temperature for 60 min, and then were cooled naturally to room temperature.According to the test, the recovery of the impact properties of the section bonding zone under different deformation modes is shown in When the deformation mode is the same, the impact property of the control specimen decreases with the increase in deformation temperature. When the deformation temperature is the same, the impact absorption energy of the comparison specimen after two upsetting and one drawing deformation is significantly increased compared with that of the first upsetting, and the impact absorption energy of the simulated defect specimen returns to the initial state at 1150 °C. The impact absorbing energy of the specimen after two upsetting and two drawing deformation is basically the same as the impact performance of the comparison specimen, which indicates that the impact performance of the defect is basically recovered.It can be seen from this that temperature and plastic deformation are important factors that affect the forging of internal porosity defects to improve the impact performance. Obviously, it is more difficult to repair the dynamic mechanical properties such as impact than the static mechanical properties such as tension and compression.It can be seen from By comparing with the reported work , we may When the deformation mode is the same, the impact absorption energy of the specimen decreases with the increase in the deformation temperature. When the deformation temperature is the same, the impact absorption energy of the specimen processed by upsetting–drawing–upsetting was increased significantly as compared with that by only one upsetting. When the deformation temperature is 1150 °C, the impact absorption energy of the specimen was fully restored. When the upsetting–drawing–upsetting–drawing process was applied, the impact absorption energy of the specimen was also fully restored, and it was noticed that the microstructure of the joint area is almost the same as that in the counterpart experiments.The deformation temperature, amount and time are the important factors affecting the healing of internal porosity defects and changing the impact property, and the microstructure of the joint area, a bridge of the process and property, accounts for the difference in the recovery mechanism in the tensile and impact properties.In the early stage of the deformation, the strain was small and the grain boundaries began to bend, which created conditions of energy and structure for the nucleation of dynamic recrystallization.With the increase in the strain, a large quantity of sub-grains were formed near the original grain boundaries, and the sub-grains formed the nuclei of dynamic recrystallization.In the consequent deformation process, the recrystallized nuclei grew up through the migration of large angle grain boundaries and finally replaced the original grain structure.It implies that sufficient plastic deformation and time are required to achieve a similar grain size in both the matrix and the joint area. In other words, the recovery of the impact property requires sufficient plastic deformation and holding time for homogenization.The microstructure of the joint surface of the two upsetting and two drawing forgings is shown in The cylindrical pieces were heated to 950, 1050 and 1150 °C, respectively, followed by holding at the temperatures for 60 min, and then the pieces were treated with the processes of upsetting–drawing–upsetting–drawing. The reduction rate of the upsetting is 20%, and the reduction sequence in each drawing cycle is 0°→180°→90°→270°. One drawing operation is composed of four reduction cycles, and a full anvil reduction was applied with reduction rate of 20% for each pressing. After the forging process, the pieces were holding at the deformation temperature for 1 h, and then were cooled naturally to the room temperature. Counterpart experiments were conducted for evaluating the recovery percentage of the fatigue property.For the pieces deformed at 950 °C and 1150 °C, the fatigue cycles show a remarkable difference under the test condition with the strain amplitude of 0.0064. The number of fatigue cycles of the pieces deformed at 950 °C is 3566, while that of the pieces deformed at 1150 °C is 26,522. It was noticed that the fracture does not always appear in the joint area, which indicates that the fatigue property of the joint area has no difference from that of the matrix. Inclusions and carbides were found in the fracture and there is an apparent difference in microstructure of the joint area of the pieces deformed at 950 °C and 1150 °C.By using the same conditions as the impact toughness test, the recovery of fatigue stress cycles in the junction area of the section under different deformation modes is shown in When the deformation mode is the same, the fatigue performance of the control specimen increases with the increase in deformation temperature. When the deformation temperature is the same, the number of fatigue stress cycles of the comparison specimen after two upsetting and one drawing deformations is significantly higher than that of the first upsetting, and the number of fatigue stress cycles of the simulated defect specimen returns to the initial state at 1150 °C. The number of fatigue cycles of the specimen after two upsetting and two drawing deformations is basically the same as that of the reference specimen, indicating that the fatigue strength of the defect is basically recovered. By comparing with the reported work , the numIt can be seen that temperature and plastic deformation are important factors that affect the forging of internal porosity defects to improve the fatigue strength. The effect is mainly reflected by the degree of grain structure uniformity, which also explains the difference in recovery mechanisms of static load properties such as tension and dynamic load properties such as impact and fatigue. The main reason is the difference in grain structure uniformity and micro holes. Obviously, it is more difficult to repair the dynamic mechanical properties such as impact and fatigue than the static mechanical properties such as tension and compression.3 and SiO2. The inclusions significantly decrease the fatigue strength as normally cracks initiate at the boundaries of inclusions and propagate in the cyclic loading process, resulting in fatigue failure. The inclusions at the joint interface and the unhealed micro pores are the major factor leading to low fatigue strength of the specimen after thermoplastic deformation.Energy spectrum analysis and chemical composition analysis were conducted for particular positions in the fatigue fracture, and the results are shown in The fracture and microstructure analysis demonstrated that the microstructure of the joint area is almost the same as that of the matrix, and the ‘mixed crystal’ phenomenon of the joint area and the matrix is eliminated. The tensile, impact and fatigue properties were fully restored. The fracture presents typical fatigue characteristics. Although the morphology of the inclusions in the fracture was not changed noticeably, its impact on the property under dynamic loads such as fatigue strength and impact toughness is greatly reduced.By referring to the data reported in literatures ,13,14, iBased on the above discussion, it is concluded that the deformation temperature, holding time and the deformation amount are important factors determining the healing effect of internal porosity defects, the evenness of grain structures and morphology of inclusions, thus affecting the mechanical properties of the forgings.In the process of high-temperature plastic deformation, the healing of the porosity or crack defects can be divided into six stages. The first is the formation and contact of the protrusions on the inner surfaces of the crack. The second is the growth of the protrusions, changing the initial crack into discrete crack segments, followed by the transition of the crack segments into micro hole. In the fourth stage, the micro holes disappears, followed by the fifth stage in which the grain growth happens in the joint area. The final is the stage in which the microstructures in the joint area and the matrix are homogenized. The first to the fourth stages can be achieved only by a single upsetting or drawing, while repeated upsetting and drawing are required if a microstructure adjustment in the last two stages is desired.The tensile property can be restored after the porosity defects are eliminated in upsetting process, while the recovery of the impact and fatigue properties requires not only the elimination of the porosity defects but also the homogenization of the grain structures in both the joint area and the matrix. The mechanical properties under dynamic loads show recovery only after the healing of porosity defects and the homogenizing of the grain structures, which means that the conditions for recovering the fatigue property are more stringent than those for recovering the tensile property. Although the adverse effects such as inclusions and chemical segregation in the material cannot be completely eliminated by the forging process, porosity defects in the material can be eliminated and microstructure can be homogenized by forging, which reduces the risk of crack propagation greatly. Sufficient plastic deformation in multiple directions under high-temperature conditions with guaranteed time for recrystallization helps to achieve fine grains with desired orientations; however, its technical difficulty undoubtedly increases.By studying the influence of plastic deformation process on the tensile strength, impact toughness and fatigue strength, relationship between the recovery of the mechanical properties under static and dynamic loads and the healing of the porosity defects is established, as shown in The performance under static loads such as tensile strength and that under dynamic loads such as impact toughness and fatigue strength are a pair of contradictions. Generally, a material with a higher tensile strength has lower impact toughness and fatigue strength. A process window can be determined to achieve a reasonable balance between the static and dynamic properties based on a thorough understanding of the critic microstructure for the property transition.The mechanical properties under static and dynamic loads must meet certain requirements. The deformation amount must be higher than the critic value at which the flat grain band can be eliminated and a homogenized grain structure becomes possible. Grain structure that is fine and uniform or orderly distributed is the key for the full restoration of the comprehensive mechanical properties of the forgings. The multi-directional plastic deformation achieved by the combined process of upsetting and drawing is the effective way to restore the dynamic mechanical properties.The tensile strength, impact toughness and fatigue strength can be recovered after the porosity defect is healed and the microstructure is made uniform.The microstructure feature of tensile property recovery is the disappearance of porosity defects, and the microstructure feature of impact toughness recovery is that the joint surface is not on the same line, and the microstructure feature of fatigue strength recovery is uniform. The deformation conditions for various performance recoveries vary greatly.Precipitate becomes the key factor affecting the mechanical properties under dynamic loads once the porosity defect is healed and the microstructure is homogenized.


Filter pruning is widely used for inference acceleration and compatibility with off-the-shelf hardware devices. Some filter pruning methods have proposed various criteria to approximate the importance of filters, and then sort the filters globally or locally to prune the redundant parameters. However, the current criterion-based methods have problems: (1) parameters with smaller criterion values for extracting edge features are easily ignored, and (2) there is a strong correlation between different criteria, resulting in similar pruning structures. In this article, we propose a novel simple but effective pruning method based on filter similarity, which is used to evaluate the similarity between filters instead of the importance of a single filter. The proposed method first calculates the similarity of the filters pairwise in one convolutional layer and then obtains the similarity distribution. Finally, the filters with high similarity to others are deleted from the distribution or set to zero. In addition, the proposed algorithm does not need to specify the pruning rate for each layer, and only needs to set the desired FLOPs or parameter reduction to obtain the final compression model. We also provide iterative pruning strategies for hard pruning and soft pruning to satisfy the tradeoff requirements of accuracy and memory in different scenarios. Extensive experiments on various representative benchmark datasets across different network architectures demonstrate the effectiveness of our proposed method. For example, on CIFAR10, the proposed algorithm achieves 61.1% FLOPs reduction by removing 58.3% of the parameters, with no loss in Top-1 accuracy on ResNet-56; and reduces 53.05% FLOPs on ResNet-50 with only 0.29% Top-1 accuracy degradation on ILSVRC-2012. Deep neural networks(DNNs) have become one of the most widely used algorithms in image classification  Due to the different distributions of the values of the convolution kernels in different layers, the abovementioned pruning methods based on global or local criteria for sorting filters may ignore filters with smaller values in the sorting but extract edge features. Huang et al.  comparedThis paper focuses on the above problems and aims to improve the network performance under the same compression ratio. Therefore, we propose a channel pruning framework based on filter similarity, and optimize the pruning redundancy criterion, pruning strategy, pruning structure and pruning process, as shown in We propose a novel method for estimating filter redundancy based on filter similarity, which does not rely on precise criteria to evaluate the importance of filters.The algorithm adaptively obtains the pruning rate of the layers according to the redundancy degree of each layer, which is difficult to determine in previous methods.The algorithm optimizes the channel pruning strategy of the special network structure, allowing the input and output channels of the residual block to be removed, further increasing the pruning space.The algorithm prunes the filters of the entire network at one time, and adopts two different pruning processes, hard pruning and soft pruning, which greatly reduces the large amount of fine-tuning caused by layer-by-layer pruning.The typical work of network pruning is weight pruning and filter pruning. Weight pruning prunes individual parameter in the network to obtain a sparse weight matrix. Different from weight pruning, filter pruning removes the entire filter according to a certain measure. Filter pruning significantly reduces storage usage and decreases the computational cost of online inference. The key to filter pruning is the selection of filters, which should yield the highest compression ratio with the lowest compromise in accuracy. Based on the design of the filter importance criterion, we empirically divide the filter pruning into the following categories.The simplest heuristic is to evaluate importance according to the absolute value of the parameter (or feature output) and then prune the part below the threshold by the greedy method, which is called amplitude-based weight pruning. Li et al.  proposedThe assumption based on absolute value judgment is that the smaller the absolute value of a parameter is, the smaller the influence on the final result. We call this the “smaller-norm/less-important” criterion, but this assumption is not necessarily true  after each linear convolution, then:Considering two consecutive convolutional layers and using non-linear activation nl-dimensional weight of the nl+1-th convolution kernel, corresponding to the nl+1-th input feature map. We explore and analyze the loss of representational power brought about by removing one of two similar feature channels and its filter. Suppose that where We use mean squared error (MSE) to quantify the loss of the two feature maps before and after pruning:al+1 = Hl+1×Wl+1×B. For each feature map l+1-th convolutional layer, the loss caused by removing the feature map l-th convolutional layer, as defined in Equation (4), admits the following upper bound:where K2 corresponds to the size of each filter L2-norm of the convolution kernel and its weights. In experiments, −2, which means that the loss of removing one of the similar channels is negligible, as long as there are sufficiently similar channels to replace it.where lth convolutional layer:In practice, our goal is to find similar channels and remove one of them. However, computing the similarity of channels directly has two apparent limitations. First, the activations of feature maps are affected differently by different batches of data. Second, calculating the similarity between all channels is inefficient for current large CNN architectures. To solve these issues, we use the convolution kernel as a unit for comparison. It can be seen from Equation (2) that when the input feature maps are the same, the feature maps obtained by similar convolution kernels are also identical, and the parameters of the kernels are not affected by the data batch. Intuitively, we quantify the similarity of two kernels by Euclidean distance, which is more commonly used in the analyses that need to reflect a difference in dimensions. In addition, Euclidean distance measures the distance between points in multidimensional space and can remember the absolute difference of characteristics. Therefore, for the whereFl, j) of a Gaussian distribution, the probability of falling within  is 0.68:we set a scaling factor α such that λ = μ−ασ∈,P = 0.5; and α → +∞, λ → −∞, P → 0. If where Φ(•) is the distribution function of the standard normal distribution, it can be obtained from checking the Standard normal distribution table: when α = 0, λ = μ, lth layer, we count the number of times of the jth appears in jth filter and all other filters, then r ∈  to represent the frequency of the jth filter,It can be seen in the experiment that a filter satisfies similar conditions simultaneously with multiple filters, but how can we determine the final deleted filters in the candidate set. For the If In addition to the judgment method of network parameter redundancy, the pruning strategy, implementation and network structure are also essential factors that affect the compression performance. As the pruning rate increases, network performance loss increases, and the redundant judgment of parameters is also prone to deviation when the network parameters deviate from the optimal point. Previous work uses layer-by-layer pruning and fine-tuning strategies or retraining to reduce the judgment error caused by performance loss and iterates this process until the target compression rate is achieved. However, when the iteration parameter setting is small and the target compression rate is significant, the pruning period will greatly increase, and the training time cost will be very high. Therefore, we prune all layers at once instead of layer-by-layer pruning and fine-tuning, significantly reducing the pruning cost. After complete pruning, the computation and parameter quantity of the whole network are calculated. If the set pruning requirements are met, the pruning is completed; otherwise, the redundant filters will continue to be searched for further pruning on the network structure of the last pruning until the set pruning requirements are met , as shown in In the implementation of pruning, He et al.  proposedDl) for one forward propagation. For the H and W are the length and width of the input feature map, respectively, and Cin, Cout are the number of input and output channels of the lth convolutional layer, which correspond to the number of filters Nl and Nl+1.We evaluate the performance of the convolution kernel pruning method by using the method of parameter quantity or the drop rate of computation, and different performance indicators can be used according to the requirements of different scenarios:Different pruning methods use pre-trained models or self-trained models as the baseline network. Due to the different training parameters  and different experimental frameworks , the Top-1 and Top-5 accuracies of the baseline network reported in the original papers are different. To make a fair comparison, we evaluate the effectiveness of pruning using the drop rate of Top-1 and Top-5 accuracy on the test set, which is the accuracy difference between the baseline network and the compressed network. Under the same compression rate, the smaller the difference, the better the pruning effect. All the comparison results in this paper are directly quoted from the original paper of the related method or the official code reproduction. All experiments are implemented on four NVIDIA TITAN Xp GPUs using PyTorch.−4, and an initial learning rate of 0.1. The learning rate is set to 0.001 when updating parameters or fine-tuning. For VGG16 and DenseNet-40, the baseline network is trained for 300 epochs with a batch size of 256. And for ResNet, the baseline network is trained for 200 epochs with a batch size of 256.We analyze the performance on the CIFAR datasets with VGG16, DenseNet-40, and ResNet-32/56/110. All the networks are trained using SGD with Nesterov momentum  all extract the overall outline of the cat. Our algorithm prunes the filters  and keeps the other two similar filters , as shown in We evaluate the actual speedup of our proposed method on the intelligent edge accelerator Jeston nano, as shown in In this article, we propose a novel strategy for judging the redundancy of filters based on similarity. To obtain the redundant filters, we analyze the similarity distribution law for filters in a convolution layer, and obtain a compact network by pruning the redundant filters with certain strategies. A large number of experiments proved the effectiveness and flexibility of the method under the same experimental parameters and the performance does not depend on a large number of fine-tunings.Although the pruning method we proposed does not need to specify the pruning rate of each layer, it still relies on the values of two hyperparameters. If different hyperparameters are specified for each layer according to the redundancy of each layer, the network will be further compressed. We plan to combine this method with reinforcement learning to automatically adjust the required parameters and improve the performance to a higher level. We performed simple statistical tests on similar filters to provide a basis for further pruning, which is far insufficient for complex CNNs. We will further analyze the filters' similarity data and combine the visual analysis of each layer to provide guidance for pruning.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.TW: writing—original draft. CS and PZ: writing—review and editing. All authors have read and agreed to the published version of the manuscript.


The nonreceptor tyrosine kinase (NRTK) Ack1 comprises a distinct arrangement of non-catalytic modules. Its SH3 domain has a C-terminal to the kinase domain (SH1), in contrast to the typical SH3-SH2-SH1 layout in NRTKs. The Ack1 is the only protein that shares a region of high homology to the tumor suppressor protein Mig6, a modulator of EGFR. The vertebrate Acks make up the only tyrosine kinase (TK) family known to carry a UBA domain. The GTPase binding and SAM domains are also uncommon in the NRTKs. In addition to being a downstream effector of receptor tyrosine kinases (RTKs) and integrins, Ack1 can act as an epigenetic regulator, modulate the degradation of the epidermal growth factor receptor (EGFR), confer drug resistance, and mediate the progression of hormone-sensitive tumors. In this review, we discuss the domain architecture of Ack1 in relation to other protein kinases that possess such defined regulatory domains. Activated Cdc42-associated kinase Ack1, TNK2) is a nonreceptor tyrosine kinase (NRTK) that belongs to the Ack family of kinases. The gene encoding Ack1, TNK2, is located on chromosome 3q29 in humans and is activated in response to growth factors and integrin-mediated signaling pathways , TNK2 is. The AckGiven the unique domain composition of Ack1 among NRTKs, this review aims to analyze the distinct features and domain architecture of Ack1 considering the current knowledge of other proteins that possess these domains. Recent reviews have extensively discussed the regulation and signaling pathways of Ack1 , its invCaenorhabditis elegans, Ack-like and DACK in Drosophila, bovine Ack2 and Ack1/Pyk1 and Kos1 in mice , DACK and Ack-like  are two Ack family kinases that are also effectors of the small GTPase Cdc42 . T. T88]. TIn addition to their role in controlling self-assembly, the SAM domains also mediate several signaling cascades through hetero interactions with the SAM domains of other proteins. For example, EphA5 and SAMD5 interact with each other through their SAM domains C 90]. T. T90]. TThe SAM–SAM domain interactions do not follow a universal blueprint. Given that the SAM domain of Ack1 is required for full activity and that formation of homodimers activates Ack1, the SAM domain of Ack1 could likely adopt a dimerization mechanism similar to that of EphA4, as evidenced by their similarity in secondary structure and sequence.All Ack family kinases possess a SAM domain. The sister kinase of Ack1 in humans, TNK1, also has a predicted SAM domain. The SAM domain of TNK1 is postulated to have a similar role to that of Ack1 as deletion of TNK1 SAM leads to a significant decrease in TNK1 autophosphorylation (Y277) in cells . This obIn addition to the Ack family, the two other protein kinases known to possess SAM domains are the Eph family RTKs and the sterile alpha motif and leucine zipper-containing kinase ZAK, a member of the STK/MAP3K family. The SAM domain of ZAK aids in the formation of homodimers or oligomers in mammalian cells . AlthougAnalogously, the hydrophobic core of the Eph receptor SAM domains is similar in all members of the Eph family. Although the SAM domains of these receptors are similar in their secondary structures, each of them interacts with a different set of SAM domains from other proteins, owing to the diversity in their primary structure. This exemplifies the wide range of interactions SAM domains can provide even within the same TK family. It should be noted that the SAM domains of Eph RTKs diverge from that of Ack1 regarding their location. While the SAM domain of Ack1 falls at the N-terminus of the kinase, Eph receptors carry C-terminal SAM domains. These features add an extra level of complexity to putative SAM–SAM dynamics in Ack1 and underline additional mechanisms that may be present that surpass the limitations of models based on primary and secondary structures.The kinase domain of Ack1 is sandwiched between the SAM domain at the N-terminus and the SH3 domain at the C-terminus. At the structural and sequence levels, the kinase domain has characteristics that are highly conserved among other NRTKs. The Ack1 KD adopts a bi-lobed structure , which iThe D270 of the DFG motif and the H250-R251-D252 comprising the catalytic triad are highly conserved A. Other The ATP-binding site is a highly conserved binding site for ATP and ATP-competitive inhibitors; a gatekeeper residue is often located within this pocket. Gatekeeper residues regulate the binding of nucleotides and influence the specificity of small-molecule inhibitors. This residue is often threonine, as evidenced in Abl and Ack1. Mutation of the gatekeeper residue is often linked to drug resistance, and T315I of Abl is a well-known example.Another structural hallmark of a TK is presence of two hydrophobic spines called the catalytic spine (C-spine) and regulatory spine (R-spine). The R-spine is made up of two residues in the N-lobe and two in the C-lobe, which consist of the His of the HRD motif (H250), the Phe of the DFG motif (F271), M181 at the C-terminus of the αC-Helix, and L192 at the beginning of β4 A,B. The The “catalytic spine” incorporates the adenine ring of ATP and establishes a connection between the N-lobe and the C-lobe upon nucleotide binding . The resC. elegans Ark-1 and D. melanogaster Ack-like, but in human Ack1 this motif has been replaced with an APQ motif (A298-P299-Q300)  and ends with an APE motif. The APE motif is present in human TNK1, mouse Ack1, and in the most distant  Ack1 rel99-Q300) A.Of the 12 structures available on the PDB, 11 capture Ack1 KD in its active form. Among these structures, 4EWH is the only structure with a resolved loop before the αC-helix , 4HZR haSimilar to other kinases, there are two hallmarks of Ack1 activation: a change in the (1) DFG motif conformation and (2) αC helix position. In the active state of Ack1, the conserved DFG motif is positioned such that D270 points toward the ATP-binding pocket, serving as a coordinator for divalent cations along with N257, with F271 facing toward the hydrophobic core. Meanwhile, the activation loop extends outward, creating a cleft for the incoming substrate. Upon substrate binding, N257 orients the catalytic residue D252, which serves as a nucleophile that targets the hydroxyl group of the substrate. The αC-helix exhibits an inward position, where a conserved E177 of the αC-helix forms a salt bridge with the activating lysine, K158, on the β-strand. The salt bridge orients K158, allowing it to form hydrogen bonds with the incoming ATP . A conseDisruption of one of the two hallmarks deactivates Ack1. In the inactive crystal structure of Ack1 (PDB: 4HZS), the KD is autoinhibited by the SH3 domain, with four Ack1 KD molecules forming an asymmetric unit. Like other NRTKs, inactive Ack1 features the αC-helix moving outwards, breaking the salt bridge between K158 and E177. The E177 points away from the kinase core and forms a salt bridge with R275, stabilizing the outward conformation of the αC-helix. In addition, the β-sheet is rotated relative to the C-terminal lobe pivoted around the gatekeeper residue T205. The A-loop of inactive Ack1 flips, positioning the DFG motif to point away from the ATP-binding site. Repositioning of the αC helix with respect to the β-sheet can potentially affect Ack1 dimerization ability . The two ligand-bound structures of Ack1 KD, one to debromohymenialdisine (PDB: 1U4D)  and anotOf all the TKs, the kinase domain of Ack1 is most closely related to EGFR KD. The two kinases share 39% sequence identity and have an RMSD of 1.1 Å with 237 of 252 possible Cα pairs superimposed . NeitherSimilar to the structure of EGFR KD , the unpThe SH3 domain-containing proteins are found in multiple organisms, including eukaryotes and viruses. These include signal-transducing adaptor proteins and proline-rich tyrosine kinases. The SH3 domains interact with proline-rich regions and play a role in substrate recognition, autoinhibition, and cellular localization through protein–protein interactions. The NRTK families with SH3 domains include the Abl, Ack, Csk, Frk, Src, and Tec families . The SH3Unlike typical NRTKs, the Ack family has a unique domain arrangement; instead of the typical N-terminal SH3 followed by the catalytic domain, the Ack family has a SH3 domain located at the C-terminus of the kinase domain. 10 loop) .In the PAK-Cdc42 complex, the intermolecular β-sheet between Y40-I46 of Cdc42 and I75-H83 of PAK contains Y40 of Ccdc42 interacting with F81/H83 of PAK. A few residues outside the CRIB domain also contribute to the binding; L543 and L541 of Ack1; L106 and L107 of PAK interact with L67 and L70 (switch II) of Cdc42 ,132,137.2, compared to ~2500 Å2 in PAK-CRIB. PAK-CRIB forms a β-hairpin structure that binds to the top of switch II, while Ack1-CRIB binds to switch II through a loop. This difference in binding behavior may contribute to the difference in affinity between PAK-CRIB vs. Ack1-CRIB for Ras-related Rho GTPase Rac [There are some minor differences in the binding behaviors of PAK-CRIB and Ack1-CRIB to Cdc42. Unlike PAK-CRIB, the α-helix of Ack1-CRIB forms a right angle to switch II upon binding, while in PAK-CRIB, the α-helix binds to residues 62–70 of Cdc42. In addition, Ack1-CRIB forms a more regular intermolecular β-sheet with the β2 strand of Cdc42. The buried surface of Ack1-CRIB is ~4200 ÅPase Rac .Drosophila DACK, Ack family kinases are clathrin-binding (CB) proteins. The Ack1 can directly associate with the heavy chain of clathrin and localizes to clathrin-containing vesicles [Except for TNK1 and vesicles ,20. OverThe clathrin-binding motif LIDFG drives an interaction involved in receptor-mediated endocytosis. The Ack1 participates in receptor trafficking through its CB motif and downregulates EGFR ,25,140. Clathrin-mediated endocytosis is the major internalization pathway for many cell surface receptors, including EGFR. The Ack1 has significant regulatory roles in EGFR endocytosis, trafficking, and sorting for lysosomal breakdown ,20,21. TOne of the unique features of Ack1 is its homology to the Mitogen-Inducible Gene 6 protein, Mig6, a negative feedback inhibitor of EGFR. The Mig6 protein inhibits EGFR activity by binding to its kinase domain and facilitating its endocytosis . The resOther than Ack1, no other protein has any homology to Mig6, suggesting that the high conservation of the residues might have functional significance. Strikingly, Ack1 and Mig6 are 80.6% homologous and 53.7% identical within the MHR A. SegmenThe MHR of Ack1 has been proposed to have an auto-inhibitory role; a phenylalanine residue (F352) in segment 1, which plays a key role in the Mig6-EGFR interaction  Figure A, is conAs a region rich in prolines, MHR serves as a site for protein–protein interactions, especially for those with SH3 domains. These include interactions between EGFR and Ack1, which are mediated indirectly in a Grb2-dependent (through its SH3) manner or directly in an MHR-dependent manner. Other binding partners for proline-rich sequences within Ack1 include the NRTK Hck , the HeaBased on the crystal structure of the EGFR kinase domain and phosphorylated Mig6 encompassing both segments 1 and 2 (PDB: 4ZJV), Mig6 segment 1 binds to the bottom of the KD C-lobe independently of segment 2 A. SegmenA model superimposing segment 2 of Mig6 onto the KD (mimicking segment 2 of MHR) has a binding behavior similar to that of Mig6 towards EGFR B–D. HoweThe phosphorylation of Y394 and Y395 is critical for the successful inhibition of EGFR , as unphThe K875 of EGFR plays a crucial role in acting as a hinge in orienting phosho-Y395 of Mig6, which is catalyzed by Src, priming Y394 for phosphorylation by EGFR. Phosphorylated Y394 then can be readily coordinated by K879. Therefore, the presence of two lysines is a key element in orienting the two tandem tyrosines in EGFR KD–Mig6 interactions. Though these tyrosines are conserved as Y859 and Y860 in Ack1, only one lysine is present in the Ack1 kinase domain. Interestingly, K875–K879 of EGFR are also conserved in Abl (K419–K423), a tyrosine kinase that binds the two tandem tyrosines of Mig6. In contrast to EGFR, however, Y394-Y395-phosphorylated Mig6 activates Abl kinase activity by relieving inhibitory intermolecular interactions.Polyubiquitylation and degradation are regulated by the interaction of the UBA domain with mono- or polyubiquitin chains . GeneralIn contrast to that of Ack1, the UBA domain of the other human Ack family kinase, TNK1, serves a positive regulatory role. The TNK1 is phosphorylated by microtubule affinity-regulating kinases (MARKs), resulting in the abolishment of its activity through the binding of 14-3-3 ,150. In There are two pathways controlling the stability of Ack1: (i) through lysosomes  and (ii)The Ack1 is involved in the transport of EGFR into a non-canonical degradative pathway . The AckKnockdown of Ack1 enhances EGFR trafficking to lysosomal compartments; the presence of Ack1 inhibits EGFR from rapidly translocating to lysosomes after EGF stimulation. It is important to note that deletion of the UBA domain of Ack1 has no effect on its association with EGFR, implying that ubiquitylation of EGFR is unnecessary for this association .The interaction of EGFR with the clathrin-binding domains of Ack1, as well as Ack1 association with ubiquitin and p62/SQSTM, are thought to be the fundamental mechanisms for EGFR non-canonical trafficking. The Ack1 acts as a shuttle between the p62/SQSTM1 compartments and the canonical endocytic pathway, preventing EGFR trafficking via the classical lysosomal pathway.The AlphaFold model for the UBA domain of Ack1 comprises three α-helices A. This gBecause the UBA domain of Ack1 is a target for mono-and polyubiquitin chains , it is pDuring ubiquitin binding, the surface of discontinuous helices forms a contact interface with the β-sheet of ubiquitin . Most UBBesides Ack1, AMPK-related serine/threonine kinases (MARKs) have UBA-like domains that play a key role in the autoinhibition and activation of the kinase by binding to the kinase domain ,157. UnlThe UBA domain of MARK adopts the traditional fold of an UBA domain with a conserved MGY motif D in the The kinase domains of Ack1 and MARK3 exhibit increased kinase activity upon binding to their UBA domains. While there are minimal structural data regarding the Ack1 KD-UBA interaction, the crystal structure of MARK3 reveals a possible kinase activation mechanism mediated by the UBA domain C, where Unlike Ack1, the UBA domain of MARK binds ubiquitin with minimal affinity . This coUsing the structures and AlphaFold-predicted models used in this review, we summarized the functions of domains in Ack1 and the protein–protein interactions they facilitate, indicating the partner domains responsible where possible .The Ack1 is regulated by intermolecular and intramolecular mechanisms. The Ack1 forms dimers (or multimers) in cells. The SAM domain-mediated dimerization of Ack1 is necessary, but not sufficient for activation . The MHRThe Ack1 is the only protein known in the literature to carry a region highly homologous to Mig6, a negative feedback inhibitor of EGFR. The EGFR-Mig6 and Ack1-MHR interactions differ fundamentally in that Mig6 is a separate protein that regulates EGFR via association and dissociation, whereas MHR is a domain on Ack1 that presumably binds via intramolecular interactions. The entropic effect of being present on the same polypeptide would increase the affinity of the MHR for the kinase domain in full-length Ack1. As a result, the affinity of the Ack1 KD for the isolated MHR domain (or of peptides derived from the MHR) would be expected to be much lower than those of Mig6 for EGFR KD, which presents a challenge for experimentally addressing the Ack1 KD-MHR interactions.The only other human tyrosine kinase known to carry an UBA domain in addition to Ack1 is its sister kinase TNK1. The UBA of TNK1 has been proposed to facilitate its oligomerization through interaction with other ubiquitylated proteins and locate TNK1 to ubiquitin condensates . AnderseOut of all the existing Ack1 structures, only one autoinhibited Ack1 KD-SH3 structure has been reported. In addition to the SH3 domain, multiple studies have suggested a possible autoinhibition mechanism involving the MHR ,104. HenThe association of Ack1 with the wide range of protein–protein interactions and signaling cascades discussed above underlines the value of Ack1 as a potential therapeutic target. Further investigation of the structural characteristics and non-catalytic domains of Ack1 could provide insights into the development of promising selective drugs.Given the correlation between Ack1 dysregulation and tumorigenesis, the mechanistic details underlying the cross-talk of EGFR and Ack1 remains as an outstanding research question. The Ack1 regulates EGFR degradation and shares sequence homology with an EGFR feedback inhibitor while the kinase domains of EGFR and Ack1 share ~ 40% sequence homology and have closely related structures. Because their interaction facilitates internalization and degradation of EGFR, the status of Ack1-mediated signaling could have implications for EGFR-driven cancers such as glioblastoma, nonsmall-cell lung cancer, and colorectal cancer.The Ack1 is involved in many biological functions including cell proliferation, migration, and apoptosis. Dysregulation of Ack1 is associated with multiple diseases such as cancer, neural disorders, and autoimmune diseases. However, more research is needed to elucidate the cellular functions of Ack1 distinct from those pertaining to cancer progression. For example, Ack1 was recently found to activate TLR signaling and promote the production of proinflammatory factors . Future A variant of Ack1 has been identified in three siblings with autosomal recessive infantile-onset epilepsy and intellectual disability , reveali


Citrus unshiu Marc. cv. Miyagawa-wase early) is one of the most widely cultivated citrus varieties on Jeju Island in Korea. Mutation breeding is a useful tool for the induction of genetic diversity for the rapid creation of new plant variants. We previously reported the use of gamma irradiation for the development of new citrus varieties. Here, we report a new mutant, Ara-unshiu, with a unique late fruit ripening phenotype. We investigated the fruit morphological characteristics including weight, vertical/transverse diameter, peel thickness, hardness, and color difference, as well as sugar and acid contents of the Ara-unshiu compared to wild-type controls. We then used whole genome re-sequencing and functional annotation by gene ontology to identify and characterize single nucleotide polymorphism (SNP) and insertion/deletion (InDel) variants in the Ara-unshiu, finding a greater abundance of annotated genes containing InDels compared to SNPs. Finally, we used allele-specific PCR to identify molecular markers among the homozygous SNPs detected from the Ara-unshiu genome sequencing. We report a primer set that effectively distinguishes the Ara-unshiu from the wild-type control and other citrus varieties. Our findings provide insights into the mechanisms controlling the timing of fruit ripening and tools for the molecular breeding of citrus varieties.‘Miyagawa-wase’ mandarin ( Miyagawa-wase’ mandarin (Citrus unshiu Marc. cv. Miyagawa-wase) has become one of the most widely cultivated varieties, accounting for more than 80% of the total citrus cultivation on Jeju Island in Korea. According to the 2023 Korea National Statistical Office report, Jeju Island’s citrus cultivation area is the largest at 15,818 ha, followed by plums and persimmons at 56 ha and 55 ha, respectively. The improvement of citrus varieties through conventional breeding is difficult due to several reproductive biological features, such as apomixis, male and female partial sterility, self- and cross-incompatibility, a long juvenile period, and a high level of heterozygosity [Miyagawa-wase cultivar by crossbreeding is further complicated by its seedless phenotype. Although this cultivar can be crossed with a seed-forming maternal citrus parent, one of the major problems that ensues is polyembryony, by which one seed may contain two or more embryos with zygotic or nucellar origin [Citrus is a fruit crop genus with significant worldwide economic importance. Among citrus fruit, ‘r origin . Thus, tAn alternative approach is mutation breeding, where mutagenesis using irradiation or chemical mutagens is used to induce genetic diversity for creating new varieties in a short period of time . MutatioJedae-unshiu, induced by gamma irradiation of C. unshiu Marc. cv. Miyagawa-wase scions. Jedae-unshiu has a unique fruit shape with vertical troughs on the flavedo, smooth albedo without rough protruding fibers, and good adhesion between peel and flesh [C. unshiu mutant, Ara-unshiu, which shows a fruit phenotype with delayed ripening compared to the wild-type (WT) control fruit. Fruit ripening, the final stage of fruit development, is a complex process involving a series of physiological and biochemical changes. The ripening of fruits is always accompanied by a rapid change in color as pigments accumulate and chlorophyll breaks down. Simultaneously, the fruit softens through cell wall remodeling, and flavor develops as acids, sugars, and volatile compounds accumulate. Fruit ripening programs are known to be mediated by the combined effects of plant hormones, transcription factors, and epigenetic modifications, all of which can affect fruit quality [Citrus sinensis L. Osbeck) derived from a bud mutation of the Fengjie 72-1 navel orange ripens one month later than its parental line [Citrus sinensis have also been reported as spontaneous late-ripening mutants [Tardivo, another late-ripening mutant of the Clementine mandarin, is associated with an altered perception and sensitivity to ethylene [Liuyuezaoyou mutant is an early-ripening cultivar selected from a bud mutation of Guanximiyou (Citrus grandis Osbeck) [Citrus sinensis L. Osbeck) [Ara-unshiu. In addition, we conducted whole genome re-sequencing to identify specific molecular markers for Ara-unshiu mutant selection. Our results contribute to the understanding of the process of fruit ripening and provide a foundation for revealing the molecular mechanisms of fruit maturation.We recently reported a new mutant, nd flesh . In the  quality ,17,18. Stal line . Fengwan mutants ,21. Tardethylene . Convers Osbeck) . The Gan Osbeck) . Tomato, Osbeck) ,25,26,27Ara-unshiu was produced by gamma irradiation of wild type (WT) C. unshiu Marc. cv. Miyagawa-wase. These materials were planted in the Research Institute for Subtropical Agriculture and Biotechnology of Jeju National University, Seogwipo, Republic of Korea. Young leaves and immature green fruits (late June fruit) were harvested and immediately frozen in liquid nitrogen for genome re-sequencing analysis. Other citrus varieties used in this study were as follows: Jedae-unshiu (J), Chung-Gyeon (CG), Gam-Pyong (GP), Hanla-Bong (HB), and Kara-Hyang (KH). Jedae-unshiu has an early maturity and the others have a late maturity trait.The citrus mutant Ara-unshiu and WT control, 10 or more fruits per tree from five or more trees each year, over the course of two years, were harvested and investigated.Fruit trait analysis was performed according to the method of Eun and Kim . Fruit wAra-unshiu and WT control was extracted using a cetyltrimethylammonium bromide (CTAB) method [C. unshiu Marc. Miyagawa-wase was obtained from the NCBI (assembly CUMW_v1.0) and the GenBank assembly (accession: GCA_002897195.1).Genomic DNA from leaves of the ) method  and sequ) method . The refAra-unshiu mutant, allele-specific PCR (AS-PCR) primers were designed according to Bui and Lit [Ara-unshiu and WT control. For the allele-specific primer containing the target SNP allele of Ara-unshiu, an additional nucleotide mismatch was introduced at the second base position upstream of the 3′ terminus. The other primer employed the same sequence for both the Ara-unshiu and the WT control , 5 µM of each primer, and 50 ng of DNA. PCR conditions were as follows: an initial denaturing at 95 °C for 3 min; 30 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 20 s; and a final extension at 72 °C for 5 min. PCR products were electrophoresed in 1.5% (w/v) agarose gels. Besides DNA samples from the Ara-unshiu and WT control, DNA samples from other citrus varieties were examined, including Chung-Gyeon , Gam-Pyong , Hanla-Bong , and Kara-Hyang .To identify specific selection markers for the  and Lit . Five ho control . For theC. unshiu (Marc. cv. Miyagawa-wase), scions were irradiated with gamma rays  followed by grafting on branches of Miyagawa-wase (C. unshiu Marc.) [Ara-unshiu. The following year, Ara-unshiu scions were used for veneer grafting on the rootstocks (C. unshiu Marc. cv. Miyagawa-wase) and continuously monitored for the maintenance of the late-ripening trait [To induce mutant lines of u Marc.) . Previoung trait ,32.Ara-unshiu. At the mature fruit stage (late November), control fruit were fully orange-colored; however, Ara-unshiu fruit were still green-colored on the peel (Ara-unshiu exhibited better adhesion between peel and flesh than the control fruit. Across two years of study (2021 to 2022), Ara-unshiu fruit were similar to WT in horizontal length, vertical length, fruit weight, and peel thickness, but had higher fruit hardness compared to the WT control  of the Ara-unshiu (13.76 ± 2.52 in 2021 and 6.84 ± 8.89 in 2022) was lower than the WT control (25.22 ± 0.72 in 2021 and 25.92 ± 2.01 in 2022); however, no significant differences in white (L) or yellow (b) values were noted. Sugar levels and acidity also showed similar values between Ara-unshiu and WT control fruit  [C. unshiu Marc. Miyagawa-wase (CUMW_v1.0) was used as a reference genome and had a length of 359.7 megabases (Mb) [Ara-unshiu and WT control, we obtained 80,976,944 and 80,693,250 of clean reads, respectively. The rate of mapped reads was 86.77% and 86.75% and the average coverage per sample was 29.50× and 27.49× for Ara-unshiu and WT, respectively (We recently reported that the citrus mutant g fibers . To furtA745525) . C. unshses (Mb) . After pectively .Ara-unshiu, respectively  and 712,344 were detected in intergenic regions. We also detected InDels in the WT and Ara-unshiu genome sequences after comparison with the reference genome  and 57,351  events, respectively. Classification by genome annotation  and 110,398 intergenic. Thus, the patterns of SNP and InDel detection and classification by genome annotation were similar between the two samples, with both SNPs and InDels mainly located in intergenic regions. The detection of a large number of SNPs and InDels in both the WT and Ara-unshiu compared to the reference genome indicates the published reference genome sequence is quite different from our WT sequence despite the two belonging to the same cultivar. Similarly, Eun and Kim [C. unshiu Marc. cv. Miyagawa-wase plants cultivated in different geographical region shows genome-wide DNA sequence variation including SNPs and InDels, and Torkamaneh et al. [Compared with the reference genome, 1,198,650 and 1,193,106 SNPs were present in the WT and ectively . Of thosa-unshiu . In the e genome . In the notation  showed tAra-unshiu, we compared the genomic sequences of the WT and Ara-unshiu and uncovered 3469 SNPs (46 homozygous and 3423 heterozygous) and 7162 InDels (45 homozygous and 7117 heterozygous)  responsible for the late ripening of the ozygous) . Of thesozygous) . Thus, bhan SNPs .Ara-unshiu compared to the WT control that were annotated in the GO database and performed GO analysis on them. GO analysis classified the variant genes by biological process (BP), cellular component (CC), and molecular function (MF) categories  were selected from the SNP variants between the Ara-unshiu and WT control. We then designed a pair of common primers and a specific primer with an introduced mismatch in the vicinity of each SNP site   or antepenultimate (−3) position of the 3′ end of the mismatched primer are generally used to introduce the mismatched base to maximize the specificity of detection and avoid false positives [AS-PCR is a method for SNP detection based on the extension of a PCR primer only when its 3′ end is perfectly complementary to the template ,36. AS-PSNP site . Of the  and KH) . The SNPositives ,35. Accoositives .Ara-unshiu, that carries a late fruit ripening phenotype. We report the comparison of several fruit morphological traits as well as sugar/acid contents between the Ara-unshiu and its WT control. We then used AS-PCR to identify a specific selection marker (Ara-SNP4) that distinguishes the Ara-unshiu from other citrus varieties.In summary, we report a new citrus mutant induced by gamma irradiation,


Results showed that the mean concentrations of heavy metals in mg/kg were Fe (11.60–34.45), Cu (0.50–8.10), Ni (0.50–1.60), Mn (0.70–25.40), Zn (4.40–15.90), Pb (0.53–10.20), Cd (0.51–0.85), Cr (0.50–1.53) and Co (0.50–1.51). The highest concentrations were found in flour milled using a traditional metallic mortar while the lowest levels were in those samples milled using a wooden mortar. The Fe, Pb and Cd contents of flours produced using the metallic mortar and some commercial mills was found to be higher than the permissible limits set by WHO/FAO. Human health risk assessment showed that there are potential carcinogenic health risks from adults’ intake of heavy metals in maize flour milled using a metallic mortar. Therefore, processing of maize flour needs to be monitored by the relevant statutory bodies in Uganda to minimize the possibility of heavy metal contamination of food products and animal feeds.Consumption of maize and maize-based products contributes a significant percentage to the total food energy intake in Uganda. However, the production of maize-derived foodstuffs is performed traditionally or by small- and medium-scale processors using different processing techniques. This can lead to differences in the quality of these products from processors, raising food safety concerns. In this study, the effects of mechanical processing (milling) methods on deposition of heavy metals into milled maize flour and the associated consumption health risks were assessed. Atomic absorption spectrophotometry was used to quantitatively establish the concentration of iron (Fe), manganese (Mn), zinc (Zn), cadmium (Cd), lead (Pb), chromium (Cr), copper (Cu), cobalt (Co) and nickel (Ni) in 100 samples of maize milled using a wooden mortar ( Food is nutritionally considered to be the primary source of essential human micronutrients such as copper (Cu), cobalt (Co), nickel (Ni), iron (Fe), molybdenum (Mo), manganese (Mn), selenium (Se) and zinc (Zn) ,2. HowevFood safety hazards are of biological, chemical and physical nature and may be present at the same time in a given food matrix . They maDietary exposure to HeMs has been extensively studied. However, most studies do not pay attention to potential introduction of these contaminants during food processing. A couple of empirical studies give credit that milling of cereals  expose consumers to leached HeMs ,19,20,21The study was limited to the quantification of HeMs  in maize flour from Mbarara City, Western Uganda . The HeMAll reagents and chemicals used in this study were of high analytical purity  or Sigma Aldrich , and were therefore used in the analytical work without prior purification. Unless otherwise indicated, all solutions were prepared using double distilled water.Dry maize grains (50 kg) were purchased from rural farmers in Kamwenge Sub County, Kamwenge District, Western Uganda. This is an agricultural area with a limited number of automobiles and no heavy industries that can pollute the environment via aerial deposition. In this area, farmers depend on the natural fertility of soils for growing their crops; thus, there is limited pollution through the use of synthetic fertilizers. Maize grains from this area are bought by the business community from Mbarara city who later transport, mill and sell the maize flour within the city.Measured grains (1 kg) were washed and rinsed with distilled water, spread on a plastic sheet to dry indoors to reduce possible aerial pollution (deposition of HeMs). The clean grains (0.5 kg) were pounded into flour using a wooden mortar and pestle without any metallic contact. Similarly, another sample of the washed grains (0.5 kg) was pounded into flour using a metallic mortar and pestle. The two samples generated were used as controls. The remaining grains were milled into flour using selected grain milling machines in Mbarara city, Western Uganda.The maize flour samples for laboratory analysis were collected between the months of December 2022 and March 2023. These were from four different diesel engine and electric powered hammer-type mills. The diesel mills were labeled A, B, C and D while the electric mills were coded E, F, G and H. The location of the mills was as follows: A and E in the Kamukuzi division, B and F in the Kakoba division, C and G in the Nyamitanga division and H and D in the Makenke division.Three samples were collected in polythene bags from each of the mills  at two different intervals during each milling phase, that is, within the first 5 min when the flour first exits on starting and the second interval of sampling was when the flour would be exiting before milling is stopped. A total of 48 samples was collected from diesel engine-powered mills. Similarly, 48 flour samples were collected from the electric motor-powered mills. Two flour samples each were collected for maize processed using wooden and metallic mortars and pestles. Therefore, a total of one hundred (100) maize flour samples was collected and kept in airtight plastic buckets at room temperature in the Chemistry laboratory at the Department of Chemistry, Mbarara University of Science and Technology, Mbarara, Uganda.v/v) in a fumehood for 30 min. They were transferred onto a hot plate for hot digestion which went on until brown fumes were given off from the samples. Then the contents were washed down using distilled water and left to boil to below 50 mL. The digests were cooled and filtered through Whatmann filter papers. The filtrates were transferred into 100 mL volumetric flasks and made up to the mark with deionized water.Analytical flour samples were obtained from pooled maize flour samples using the cone and quarter method. Each analytical sample (3 g) from the cone and quarter method was put on a porcelain crucible and dried in the oven for 24 h at 85 °C. The samples from the oven were removed and cooled in a desiccator to avoid reabsorption of moisture for dry basis analysis. Measured 2.0 ± 0.1 g of the samples were transferred into 250 mL beakers and cold digested with 100 mL of perchloric acid/nitric acid mixture  at 248.3, 279.5, 213.9, 324.7, 232.0, 228.8, 248.3, 238.3 and 357.9 nm for Fe, Mn, Zn, Cd, Pb, Cr, Cu, Co and Ni, respectively ,35.2 > 0.996). The assurance of the quality of the analytical results was addressed through analysis of procedural blanks and spiked samples, whose recoveries were found to be acceptable (range: 98% to 100%). The relative standard deviations of the experiments  varied from 3.5 to 5.0%.The elemental concentrations were established from calibration curves constructed from diluted working standards of 1000 ppm stock solutions of nitrate and chloride salts of the HeMs. The linearity of the curves fell within acceptable limits (RADD), hazard quotient, hazard index and cancer risk for both adults and children [ADD (mg/kg/day) was estimated to discern human exposure through direct ingestion of maize flour as bread or porridge (Equation (1)).C is the concentration of the heavy metal (mg/kg), IR is the ingestion rate = 100 mg/kg/day and 200 mg/kg/day for children and adults, respectively, Ef is the exposure frequency = 365 days/year, Ed is the exposure duration = 58.65 years for an adult Ugandan [ab is the average body weight; as per the clinical guidelines from the Ugandan government, children are those who are 6 years of age weighing 15 kg, while adults were defined as those who are 30 years or older weighing 70 kg [Tae is average exposure time for non-carcinogens = Ef × Ed.The carcinogenic and non-cancer health risk assessments involved the computation of average daily dose  was calculated to establish non-cancer risks from the HeMs (Equation (2)). Since the effects of HeMs can be augmentative, the hazard index (HI) or total HQ was consequently calculated (Equation (3)).e US EPA ,40,41,42−4 indicate unacceptable risks.The carcinogenic health risk (CR) was computed as the incremental lifetime cancer risk for the carcinogenic elements  (Equation (4)). The total cancer risk (TCR) was estimated as the sum of all the cancer risks due to the ingestion of the four heavy metals in maize flour (Equation (5)).ectively . The CR p < 0.05 using GraphPad Prism for Windows .Quantitative data from experimental analyses performed in triplicate were captured in Microsoft Excel  where they were averaged and expressed as means ± standard deviations of replicates. The concentration of the HeMs was compared with the Ugandan standard for maize flour US EAS 44: 2019) [: 2019 4 and thosp < 0.05). The results indicate that the contribution of the metallic mortar was highest with regard to the HeMs Fe, Mn, Zn, Pb and Cu as compared to the other metals (p < 0.05) when compared to the other elements determined. Nevertheless, the concentration of Cd, Pb, Cr, Co and Ni were found to be higher than the WHO/FAO food standards [The effect of different mechanical grinding methods on the deposition and concentrations of some selected HeMs in milled maize from Mbarara city, Uganda was investigated. The concentrations of HeMs in maize flour samples were higher in those ground using the metallic mortar than the wooden mortar . Samples milled using electric motor−powered mills had higher HeMs than those produced using diesel engine−powered mills. However, these differences were not statistically significant (p < 0.05). In comparison to the Ugandan standard (US EAS 44:2019) [On the other hand, maize flour obtained using hammer mills had higher levels of HeMs than that in samples from the wooden mortar  to 1890 × 10−6 for Cd in maize milled with the metallic mortar. For adults, the hazard quotients span from 0.95 × 10−6 (for Cu from maize flour milled by use of the wooden mortar) to 8325.71 × 10−6 for Pb in maize milled with the metallic mortar. Accordingly, the hazard quotients from Pb and Cd, as well as the total hazard quotient, were less than 1, showing that there are no potential health risks from the ingestion of milled maize flour  in the picture, e.g., “10000” should be “10,000”.otal cancer risk values were within the acceptable range of 1 × 10−6 to 1 × 10−4, implying that there are no potential carcinogenic health risks for children who consume the maize flour. For adults, cancer risk values ranged from 0.715 × 10−6 for Cr to 247.690 × 10−6 for Pb. In adults, the individual cancer risk value for Pb in flour milled using a metallic mortar was greater than the safe limit of 1 × 10−6 to 1 × 10−4.For the incremental life cancer risks, the risk values of the individual groups were computed . For theNutritional food quality plays an important role in maintaining human health. However, food and drinking water, along with occupational exposure, are the main routes of exposure to potentially toxic elements in humans. In this study, the concentrations of HeMs in milled maize flours were higher in samples obtained using the metallic mortar than the wooden mortar. However, relatively higher levels of Fe and Zn leached from the wooden mortar into the flour probably because the wooden mortar and pestle are made of natural solid materials which contain phytoavailable levels of geogenic HeMs. The highest concentrations of HeMs were recorded in flour samples obtained using the metallic mortar, though Cd, Cr, Co and Ni occurred as traces. The high levels may be attributed to the presence of these metals as impurities which are added or not removed during metal refining processes. In Uganda, metallic mortars are shared within the neighborhood for preparing small quantities of food and medicinal herbs which cannot be commercially milled. These metallic mortars are possible sources of food contaminants because they are fabricated from rusty and corroded metallic pipes that have been discarded from the Kilembe mines (Western Uganda) and later collected by scrap dealers for recycling. Without any tests for hardness and other chemical properties, local artisans fabricate these pipes into food equipment for use in small scale food industries.For the maize flour obtained using hammer mills, the higher levels of HeMs than that found in samples from the wooden mortar could be because hammer mills are fabricated using mild steel which contains Fe, Mn, Cr and Cu . DependiIn the human health risk assessment, the results of our study agreed with a previous study done in Kampala (Uganda)  in which−4 threshold provided by the US EPA. Another study in Ghana [For the cancer risk assessment, the individual and total cancer risk values for Pb in flour milled using the metallic mortar surpassed the safe limit suggested by US EPA, suggesting potential cancer risks in the exposed population. A similar study  of maizein Ghana  concludeThis study revealed that the milling method used for processing maize into flour had an effect on (enriched) the heavy metal content of the flours. The metals were plausibly deposited into the flour due to frictional wear of the mortars/mill plates during milling. The concentrations of the HeMs in maize flour produced by metallic mortar and commercial mills were found to be higher than the dietary tolerance limits set by the Uganda National Bureau of Standards (for Pb) and the WHO/FAO . Human health risk assessment showed that there are potential carcinogenic health risks from adults’ intake of HeMs in maize flour milled using a metallic mortar. Thus, the processing of maize flour needs to be monitored by the relevant government bodies such as UNBS which are mandated to control quality manufacturing (fabrication) of food processing equipment including mill plates to minimize the possibilities of heavy metal contamination of human food and animal feed.


We examined the effect of two different types of linker distribution—random or correlated distribution—on the pore size and shape within single-layers of three multi-component COFs. We reveal a relationship between linker distribution and the porosity of COF solid solutions. The methods presented in this paper are generalisable and could be used in further studies to examine the properties of disordered framework materials. We examined the effect of two different types of linker distribution—random or correlated distribution—on the pore size and shape within single-layers of three multi-component COFs. Therefore, the linker distribution is disordered within the structure. Forming COF solid solutions—homogenous mixtures of multi-component materials with a single crystal structure—unlocks the ability to tune the COFs properties by varying the stoichiometry of the linkers and balancing the monomers’ intrinsic properties.To introduce another level of functionality into COFs, the number of monomers can be increased. For example, introducing two types of linkers: a π-donor and a π-acceptor, creates arrays of donor and acceptor π-columns, facilitating intercolumnar interactions resulting in favourable conductivity.et al.10 showed that the distribution of the linkers is disordered, the arrangement of the linkers may not be random due to correlations between the linker positions. This can occur when there is a chemical reason for linkers of the same type to avoid being near each other e.g. due to differences in electronegativity or to minimise the strain introduced from different length linkers. For some topologies with a 1 : 1 ratio of two different linkers, this scenario leads to an ordered arrangement of the linkers , but for others, such as hexagonal COFs , but thround it . Correla0.5BDA0.5), TAPB-(BDA0.5TDA0.5), and TAPB-(PDA0.5TDA0.5). We find that the different linker lengths have a negligible effect on the pore size and shape when the ratio between the linker lengths is small (≈5 : 6), and a more significant effect for larger differences (≈5 : 7). These findings may have implications in using COF solid solutions as membranes because the distribution of the linkers affects the extent to which the pore size and shape can be selectively tuned for.16,17 Moreover, the methods reported in this paper provide a route to produce atomistic models of disordered COFs using our software, stk,18 that can be used to study the effect of disorder on other COF properties.In this paper, we analyse the effect of monomer distribution on the pore size and shape of COFs formed from the condensation of 1,3,5-tris(4-aminophenyl) benzene (TAPB) with a 1 : 1 mixture of two of the three dialdehydes: terephthalaldehyde (PDA), 4,4′-biphenyldicarbaldehyde (BDA), or -4,4′′-dicarbaldehyde (TDA) which have one, two, or three benzene rings respectively . These fSi = ±1 representing the linkers such that when S = +1(−1) the site was populated with a long(short) linker. The MC simulations used the Metropolis algorithm with periodic boundary conditions where sites were randomly selected and inverted. The energy of each move was calculated according to the HamiltonianJ is the interaction parameter and 〈i,j〉 denotes a sum over nearest neighbour sites i,j. J > 0 such that it is energetically preferable for the linkers around each node to be different. To produce configurations with random (correlated) distributions of linkers, the simulations were performed at high (low) temperatures where T/J = 25(0.25). Although setting the temperature instead of cooling the simulations can result in a metastable state, the configurations from the low-temperature simulations were examined to ensure there were no sites with three of one type of linker around each node. Each MC simulation ran for 100 000 × n3 steps where n is the size of the supercell and was repeated 5 times to average the results.We implemented Monte Carlo (MC) simulations to produce COFs with random and correlated linker distributions. A starting configuration corresponding to a 16 × 16 supercell of the underlying kagome unit cell , ESI20 Due to the smaller number of pores per configuration (16 vs. 256 for 16 × 16 supercells), we averaged the results over 10 configurations instead of 5. This produced comparable results , ESI21 with the c lattice parameter set to 5 Å to ensure that only the size of the pores within the layer was measured. The pore size distributions were calculated using the high accuracy flag with 50 000 MC samples, a probe diameter of 1.2 Å, and was plotted using kernel density estimation in Seaborn: bandwidth method Scott's, adjust is 1. As the pore size distribution overlooks the nuances in the structure that arise from changes in the shape of the pore due to different linker combinations, we measured “how hexagonal” the pores were by calculating the similarity of the pore shape to a perfect hexagon, HP-6, using the SHAPE software.22,23 Given a coarse-grained model, we extracted the atoms representing the monomers which defined each hexagon (i.e. TAPB), using the atomic simulation environment.24 For the 4 × 4 atomistic models which were relaxed further, we used the cheminformatics Python package, RDKit,25 to convert the TAPB molecule to a single atom at its centroid, where each centroid is bonded to its neighbouring TAPB centroid. The HP-6 measure was calculated for each six-member ring, where the closer HP-6 is to 0, the more hexagonal the pore is. Code for converting the coarse-grained models to atomistic structures using stk and performing shape analysis is open-sourced here https://github.com/andrewtarzia/cg-2d-cofs.For each COF, the pore size distribution was determined using Zeo++10,26,27 However, we have included a discussion of how the different pore size and shape distributions may affect or disrupt this eclipsed stacking.The MC simulations only considered the distribution of linkers within a single-layer of the COF, and as such, our results exclude the effect of stacking behaviour on porosity. As it has been reported that both the TAPB-PDA and TAPB-BDA COFs, as well as the solid solutions formed by them, have eclipsed stacking, we expect the single-layer pore behaviour to capture most of the features of interest.Our results  show thaFor the pore shape, the anisotropy increases with the ratio of linker lengths: BDA/TDA < PDA/BDA < PDA/TDA, evidenced by the increased average value of HP-6 . Similar28 the distribution between layers is likely to be uncorrelated. Therefore, the broad pore size distribution and anisotropic pore shapes will likely lead to worse alignment between the layers compared to ordered COFs. This may affect the COF's utility in molecular separations as the worse the alignment, the more staggered the stacking, decreasing the width of the pore channels. The extent of misalignment will likely be more pronounced for COFs with randomly distributed linkers due to the broader pore size distribution, possibly making them suitable for chromatography applications similar to gel permeation columns. In these systems, the misalignment between layers can lead to smaller molecules becoming entangled in the structure, having a longer path length than larger molecules, separating the two. A caveat to this is that a greater extent of misalignment likely results in weaker interlayer interactions and thus significantly more stacking disorder than in the correlated disordered case. Since stacking disorder results in less control over the size and shape of the pore channels, one would expect this to hinder the COFs application in molecular separations. However, 2D COFs with stacking disorder have recently proved useful in the separation of benzene and cyclohexane.29 On the other hand, the weaker interactions from the misalignment between layers also results in a higher propensity to form polycrystalline structures, hindering selectivity.29The anisotropic pore size and shape will effect the COFs layer alignment, impacting the material properties. As the monomers polymerise to form 2D sheets before stacking,30In summary, we have studied how linker distribution affects the properties of COF solid solutions. We show that the pore size and shape distribution is broader for randomly distributed linkers rather than for correlated distributions. For small ratios of linker lengths (≈5 : 6), this effect is negligible, but for a larger differences (≈5 : 7) this effect is larger, which may affect the application of COFs in membrane separation. Developing an understanding of the stacking behaviour in COF solid solutions will enable more accurate determination of the effect of disorder on the properties. However, layered COFs are notorious for containing stacking disorder, making studies on the structural behaviour of ordered COFs difficult, let alone structurally disordered COFs.Through this work we have presented a computationally inexpensive method of creating structural models of disordered COFs. Outside of porosity, the distribution of linkers in COFs can impact other properties such as the electronic and optical properties. The process outlined in this paper can help create atomistic models for further property prediction of disordered COFs, elucidating a disorder/property relationship.K.E.J acknowledges the Royal Society for a University Research Fellowship and Enhancement Award and the ERC through Agreement No. 758370 (ERC-StG-PE5- CoMMaD). We thank Becky Greenaway and Annabel Basford for helpful discussions.There are no conflicts to declare.CC-059-D3CC01111A-s001


Philadelphia chromosome (Ph)-like acute lymphoblastic leukemia (ALL) is a subset of ALL that demonstrated a high treatment failure rate. One of the hallmarks of Ph-like ALL is PDGFRB gene fusion, with fusion partner proteins often harboring dimerization domains and enhancing the kinase activity of PDGFRB. We determined a novel oncogenic PDGFRB fusion gene, NRIP1::PDGFRB, from a pediatric patient with ALL, encoding a protein with the carboxy-terminal kinase domain of PDGFRB, without the partner peptide. We confirmed the oncogenic potential of NRIP1::PDGFRB in vitro and the efficacy of all ABL1-specific inhibitor generations, including imatinib, dasatinib, nilotinib, and ponatinib, in suppressing this potential. PDGFRB activation mechanism may include juxtamembrane domain truncation in the predicted peptide. In conclusion, we determined a novel fusion gene pattern in Ph-like ALL. Risk classification contributed to adapting appropriate treatment options, such as intensified treatment and molecular targeting agents for patients at adverse risk, or treatment with reduced intensity for patients at favorable risk. Philadelphia chromosome (Ph)-like ALL, demonstrates a gene expression profile similar to BCR::ABL1-positive ALL2 and accounts for 15%–30% of B-cell lineage ALL (B-ALL) in children and adults3. Ph-like ALL is associated with high rates of treatment resistance and relapse4. The 5-year event-free survival rates are ~60% and 80% in Ph-like ALL and other childhood ALL subtypes, respectively5. Ph-like ALL often carries oncogenic fusions of tyrosine kinases, including PDGFRB fusions. Most PDGFRB fusions include amino (N)-terminal partner protein with a dimerization motif, such as EBF1, and carboxy (C)-terminal kinase domain of PDGFRB3. The dimerization motif facilitates homodimer formation of the kinase domain, causing autophosphorylation6. Herein, we report a novel truncated form of PDGFRB without a partner protein in B-ALL and confirm its oncogenicity and sensitivity to tyrosine kinase inhibitors (TKIs).The development of a risk classification strategy based on molecular subtyping has significantly improved the prognosis of childhood acute lymphoblastic leukemia (ALL) in recent decades10/L white blood cells with 81% blasts  domains  of leukemia cells revealed the presence of a novel FRB Fig. . Cytogenzed Fig. . Whole eion Fig. . Thus, tins Fig. . The codele Fig. . Consideoup Fig. . AdditioNRIP1::PDGFRB to validate the oncogenic potential of NRIP1::PDGFRB. Wild-type (WT) PDGFRB cDNA was also transduced as a positive control. Ba/F3 cells expressing NRIP1::PDGFRB and WT PDGFRB cDNA survived upon IL-3 withdrawal for 1 week , with a trend toward a lower IC50 in cells expressing NRIP1::PDGFRB than WT PDGFRB. Reduced phosphorylation of downstream targets of NRIP1::PDGFRB was achieved by ABL1 TKI administration, but not by other TKIs, supporting the results of the drug sensitivity assay  of JM domain in FLT3 causes a structural alteration of JM domain, resulting in constitutive activation of its enzymatic function and cell proliferation14. FLT3-ITD alterations occur in acute myeloid leukemia (AML), accounting for ~30% of AML cases15. Mutations in JM domain of KIT in gastrointestinal stromal tumors (GIST) demonstrated a similar activation mechanism16. TKIs are effective and clinically used for FLT3-ITD-positive AML and KIT rearranged GISTs18. FIP1L1::PDGFRA fusion, one of the major oncogenic fusion genes of myeloproliferative disorders, is another example of JM dysfunction. The translated protein contains truncated JM and kinase domains of PDGFRA19. Similarly, proliferative ability20 and response to TKI21 have been demonstrated.The characteristics of these fusion protein types are truncated JM domain, detected in our case. JM domain in tyrosine kinase receptors has been reported as an autoinhibitory domain that suppresses kinase activity through conformational proximityPDGFRB20, and Chen et al. reported the crucial role of W566 in maintaining JM domain assembly22. The protein encoded by NRIP1::PDGFRB, in our case, lacks JM domain  in JM region of ain Fig. ; the fusain Fig. . A recenons Fig. 23. The sPDGFRB rearrangement was 50%24. Accumulating reports indicated the efficacy of TKIs against Ph-like ALL, including those with PDGFRB rearrangement27, although they remained prospectively not validated. Currently, an ongoing prospective trial aims to confirm the efficacy of dasatinib in patients with Ph-like ALL with specific fusions . We and others23 confirmed the proliferative capacity and response to TKIs in JM dysregulated PDGFRB; thus, our data will be beneficial for future patient selection. In conclusion, our study identified a novel truncated PDGFRB fusion in Ph-like ALL without fusion partner peptides which can be targeted by TKIs.The 5-year event-free survival of Ph-like ALL with We used a bone marrow aspiration specimen for RNA sequencing (RNA-seq). The patient’s parents signed a written informed consent for genetic analyses and publication of the case report. The National Cancer Center Research Ethics Review Board approved this study (2015-059). We followed the ethical principles of the Declaration of Helsinki.28. We used Arriba to detect gene fusion29.We extracted total RNA from the bone marrow sample and prepared and subjected RNA-seq libraries to next-generation sequencing as previously describedNRIP1::PDGFRB fusion transcript by cDNA PCR from the patient sample using the following primer sets:We identified NRIP1 forward: TTGGATTGTGAGCTATTTCAGAACPDGFRB reverse: AGGGTTTGGGGCACAACACGTCAGPDGFRB cDNA and NRIP1::PDGFRB cDNA coding regions were inserted into the pMXS plasmid. Ba/F3 cells were infected with the generated retroviruses from each plasmid.The wild-type Cells were seeded into 96-well plates at a 100 μL volume. After overnight incubation, cells were treated with each drug, including imatinib (Selleck), dasatinib (Selleck), nilotinib (Selleck), ponatinib (Selleck), and vemurafenib (Selleck), at doses ranging from 0.1 nM to 1 μM, incubated for 72 h. Subsequently, 10 μL of PrestoBlue (Thermo Fisher Scientific) was added to the plates, and the fluorescence was measured after 3 h of incubation.8, which is hosted by the DNA Databank of Japan.Sequencing data of Japan Adult Leukemia Study Group (JALSG) B-ALL clinical samples were obtained from the Japanese Genotype–Phenotype Archive (accession JGAS00000000047)Standard protocols were used for protein detection by immunoblot analysis, using primary antibodies PDGFRβ , phospho-PDGFRβ (Tyr751) , Akt , phospho-Akt (Ser473) , Erk1/2 , phospho-Erk1/2 , and β-Actin  purchased from Cell Signaling Technology. Uncropped immunoblots blots of each Figure are included in Supplementary Fig. Further information on research design is available in the Supplementary InformationReporting Summary