This study aimed to assess the cyclic load capacity of wooden chairs and subsequently categorize them based on their performance. A diverse selection of chair models was randomly procured from commercial markets. These chairs underwent performance testing, utilizing the cyclic stepped increasing loading method, with adherence to the standards set forth by the American Library Association Technology Reports (ALA). The study evaluated 315 chairs, encompassing 21 chair models. Each chair model underwent five replications of testing across three different loading directions. The resulting dataset of numerical values was subjected to statistical analyses, facilitating the categorization of chairs based on their strength under cyclic loads. Notably, the study revealed substantial variations in the load capacity among different chair models. As a consequence of this investigation, the study established acceptable design load thresholds. For instance, concerning front-to-back loading, it was determined that the chairs with cyclic load capacities ranging from 932 to 1449 N fell within the category of low-strength, between 1450 and 1968 N were classified as medium-strength (suitable for domestic use), and the chairs with cyclic load capacities exceeding 1968 N were considered to possess high strength . Similarly, for back-to-front loading performance, the study identified the chairs with cyclic load capacities between 625 and 895 N as low-strength, 896 and 1167 N as medium-strength, and the chairs with loads surpassing 1168 N as high-strength. The performance thresholds for side thrust loads were as follows: low-strength encompassed the cyclic load capacities ranging from 649 to 934 N, medium-strength spanned the cyclic load capacities between 935 and 1221 N, and high-strength entailed 1222 N and above. Notably, the classification devised in this study is closely aligned with the widely accepted and internationally recognized ALA specification. This strong consistency with global standards reinforces the reliability and applicability of the classification system developed in this research. In conclusion, this study enhances understanding of wooden chair strength performance and offers practical insights that lead to higher-quality products and improved consumer satisfaction. Its recommendations can potentially drive positive change within the industry and benefit manufacturers and consumers. Furniture could be described as the most important product in human life in every area of everyday life, directly affecting the level of comfort of the individual and society, responding to social and cultural needs . A totalTürkiye’s share in world furniture exports in 2016 was 1.1 percent; in 2022 it was 2.1 percent . This shThe furniture industry is one of the oldest and most developing sectors in Türkiye. In recent years, facilities that produce world-class products have been established in the sector, and a position has been reached in which it sells products throughout the country and the world through dealership organizations. The sector improves its products and increases its diversity every year. The sector aims to reach 25 billion dollars of production and 10 billion dollars of exports in 2023 and to be among the world’s top 10 and Europe’s top 5 largest furniture manufacturers .In Türkiye, furniture manufacturing companies rarely use furniture testing techniques. In fact, although there are test standards compatible with European standards to determine the performance of all types of furniture , the appConsidering the reflection of the standards in the furniture industry of Türkiye, according to the International standard code list (ICS), “Wood Technology” is classified as 79 and “Furniture” is classified as 97.140. Furniture standards are prepared by furniture technical committee (CEN/TC 207) and international furniture technical committee ISO/TC 136) and are grouped as home furniture, office, outdoor, and educational furniture .,30.10,30In the tests, chairs were loaded using the cyclic stepped increasing loading method at a specific rate and speed for each performance test. After completing this phase, the load amount was increased once again within a preset range to repeat the first step. These procedures were carried out repeatedly until the acceptable design load values were obtained or the furniture displayed failure, such as openings or breaks, etc. The speed was 20 cycle per minute, and there were 25,000 cycles every step. By comparing this performance value to acceptable design load values defined in the standards for light, medium, and heavy usage, required optimizations can be made. This performance value was then tested for durability. In this approach to testing, “light service” represented the use of private and domestic spaces, “medium service” represented the use of office places which are not very intensive, and “heavy service” represented the use of institutional places such as hospitals, schools, libraries, and restaurants ,30.This test method includes pulling the chair seat frame system from the front to the back while sustaining this loading until the chair permanently deforms, the joints begin to open or and members break, etc. This test aims to establish the doubling strength of the side frame joints. According to the failure modes of this test, the appropriateness of the application of glue, its quantity, and the accuracy of the joint constructions used in the side frame joints could be evaluated. This kind of loading simulates the process of leaning back when seated in a chair .In the tests, front-to-back loading was performed at 20 cycles per minute a,b. The To prevent the test chair from sliding backward, support pieces were positioned on the back of the chair’s back legs, as indicated in the Using this test method, the chair seat frame system is forced from the back to the front, and this loading is applied until the chair permanently deforms, the joints open, and the members break. Similar to the front-to-back loading test, the aim of this test is to assess the joint strength in the side frames of chair. It is a test that helps to determine whether to use glue, how much glue to use, and whether the joints on the side frame are constructed in accordance with method are all appropriate when the loading direction is changed ,10,30.Tests were carried out with back-to-front loading of 20 cycles per minute a,b. TestTo stop the test chair from sliding forward, support pieces were positioned on the front bottoms of the front legs, as seen in the The chair seat frame system is driven laterally during this test procedure, and the loading continues until the chair permanently deforms, joints open, and elements fail. The major goal of this test method is to evaluate how well the chair performs when subjected to sidethrust coercive forces. Such loadings occur when doing things like lying down, bending the chair sideways for any reason, or pushing the chair laterally by leaning against the armrest, especially when speaking to someone who is lying on their side. This test’s objective is to evaluate the joint strength holding the side frames together ,10,30.In the tests, sidethrust test was carried out at 20 cycles per minute a,b. The To stop the test chair from sliding sideways, support pieces were positioned on the bottom part of the chair, as seen in the The strength values for each loading in this investigation were categorized. A classification study was carried out for the sample group chosen to represent Türkiye, consisting of 21 types and 5 replications from each type, for a total of 105 chairs’ performance values tested for each groups of loading type. In the scope of the study, a total of 315 chairs were tested and evaluated.According to this method, the mean of the data obtained for each loading direction was distributed as a normal distribution, and 34% of the data that contain a standard deviation (SD) and fall below the mean value were deemed to be “weak strength,” while 34% of the data that contain a SD and fall above the mean value were deemed to be “medium strength”. A group of 14% was considered “inadequate”, and 14% was considered “high strength”, since they were out of the target range. In this classification; “inadequate” refers to the chairs that are not suitable for domestic use, “weak strength” refers to the chairs that may be suitable for domestic use if improvements are made in some aspects, “medium strength” refers to the chairs that are suitable for domestic use, and “high strength” refers to chairs that are for use more intensive than domestic use .As expected at this point, the data for each loading group exhibit a regular distribution .It was confirmed from the results of the “Single Sample K-S (Kolmogorov–Smirnov) test” that the sample data of the front-to-back, back-to-front, and sidethrust loading groups were consistent with the defined population normal probability distribution .According to the results of Single Sample K-S (Kolmogorov–Smirnov) test, it can be said that the data obtained for each loading show “normal distribution”. Accordingly, after this stage, the classification stage was identified for each loading direction.The physical and mechanical properties of the wood used in the production of chairs are given with the coefficients of variation in In the classification of front-to-back load capacity test results, the results of the statistics according to the method that the data are considered to be normal distribution are given in According to For the medium-strength group of chair models, the lower limit was calculated, using these data, to be 1450 N; the mean value for the classification and the upper limit was determined to be 1968 N, which was above the standard deviation. In another words, chair types which were within the 1450 to 1968 N range for front-to-back load capacity values were considered to be in the “medium strength” category and considered suitable for domestic usage. For inadequate-strength chair models, the 932 N value, which is less than the mean value, was the absolute minimum. As a result, chair models with front-to-back load capacity values between 932 and 1449 N are regarded as “weak strength”, and require improvements to increase the strength. The highest load capacity limit for medium-strength chairs is 1968 N, and chair types that perform above this (high-strength) can be considered suitable for usage, as well as for heavier services like in libraries and restaurants. The classification of the chair models is given in The evaluation of each chair types for the classification performed for front-to-back load capacity is presented in As seen in Accordingly, the group mean was calculated as 895.9 N and the SD was 271.2. The COV of the group is 30.2%. The high COV demonstrates the inconsistency of the cyclic back-to-front load capacities of chairs.These data were used to identify the lower limit value for the medium-strength of chairs as 896 N, the mean value for the categorization, and the maximum limit as 1167 N, which was above the SD. In another words, “medium strength” groups of chair types with load capacities within the range of 896 to 1167 N for back-to-front load capacity values were allowed and assessed as suitable for domestic usage. The lower limit for weak-strength chairs was the 625 N value, which was less than the mean value. As a result, chair models with back-to-front performance values between 625 and 895 N are regarded as “weak strength” groupings, and were thought to require modifications for strength development. It was simple to consider chair models that perform over the maximum limit of medium-strength chairs, 1167 N, to be ideal models for domestic usage, as well as heavy services like libraries and restaurants.The evaluation of each chair types for the classification performed for back-to-front load capacity is presented in In accordance with this, 12 chairs out of the 21 that were evaluated, or 57%, fell below the medium strength and, while being made for domestic usage, were deemed incapable of meeting the usage requirements. According to the classification results, 4 models  were defined as inadequate-strength, 8  as weak-strength, 3  as medium-strength, and 6  as high-strength. It may be noted that the 6 high-strength chairs are substantially stronger than required for domestic usage, while the 12 models still in the medium-strength category need serious strength improvement optimizations. In this group, there were only three models that were acceptable for domestic usage under back-to-front loading.As a result, a group mean of 935.1 N and a SD of 285.7 were determined. The group’s COV is 30.5%. The high COV demonstrates the inconsistency of the cyclic sidethrust load capacities of chairs.These data were used to identify the lower limit for the medium-strength chairs to be 935 N, the mean value for the classification, and the upper limit to be 1221 N, which was above the SD. Accordingly, chair types were recognized as “medium strength” groups and assessed as suitable for domestic usage if their sidethrust load capacity values ranged from 935 to 1221 N. The lower limit for weak-strength chair models was 649 N, which was less than the mean value. As a result, chair models with sidethrust load capacities between 649 and 934 N were determined to be “weak strength” categories and in need of improvements for strength development. Chair types that have strength above 1221 N, which is the upper limit of medium-strength chairs, could be considered as models suitable for heavy usage, such as in libraries and restaurants.The classification results for the sidethrust load capacity of the chair models are presented in The evaluation of each chair types for the classification performed for sidethrust load capacity is presented in According to ▪ Use of defective materials;▪ Designs with inappropriate cross-section geometry and dimensions;▪ Constructional and application errors at the joining points;▪ Dimensional and formal non-conformity of corner support elements;▪ Insufficiency and inappropriateness of demountable fasteners;▪ Not using side, front, and back intermediate rails in chair design.The deformation characteristics observed as a result of the performance tests and the deficiencies generally observed as a result of the strength values obtained can be summarized below:According to the results of the performance tests, the critical points in the furniture frame systems are joints. In other words, the strength of the joints represents the strength of the whole system. Accordingly, it may be possible to obtain stronger furniture systems with strong joints. Factors such as suitable tenon dimensions and choosing the appropriate adhesive type and applying it in sufficient quantity and appropriately were found to be effective in the joining of the glued chair models tested in the study. In RTA chair models, factors such as the number of fasteners, the diameter of the fasteners, and the effective length of the fasteners were effective in the strength of the joints. In general, the RTA chair models tested in this study performed poorer than the glued chair models.In the experiments, chairs without stretchers performed much worse than chairs with stretchers. It has been observed that the stretcher in the side frame affects the usage performances positively in front-to-back and back-to-front loading models. It has been observed that the stretchers, which are not widely used but located in the front and back frames of a few models, make significant contributions to the sidethrust loading performance. In the study, it is thought that the lack of stretchers, especially in RTA chairs, causes these chair models to show poor performance.The overall evaluation of the tested chair models is shown in Considering the general evaluation of the performance of the chair models tested within the scope of the study; it is understood that six chair models  were successful in all loading direction, six chair models  did not pass any test , and the remaining nine chair models  needed strength-improving optimizations for the loading in some directions.According to The chairs used in this study were procured as models from Turkish chair manufacturing companies, and their cyclic load capacities in various loading directions were identified and categorized according to their strength. For this reason, the manufacturers effective in the place where chair production was centered provided the chair models which were included in this study.The creation of an important database with a significant number of performance test results for each chair evaluated was the study’s most noteworthy finding. For front-to-back loads, chairs performed between 845 and 2802 N, for back-to-front loads, between 445 and 1352 N, and for sidethrust loads, between 489 and 1423 N. According to the test results, there appear to be significant differences in the strengths of the various chair models. While some models are inadequate for domestic use, some models have been found to have unnecessarily excessive strength values. This situation clearly revealed inconsistency among the manufacturing companies. It is thought that this inconsistent quality (strength) level is due to the lack of R&D culture in the manufacturing companies, not allocating enough budget to R&D, not knowing the performance test methods, and not applying product engineering.By using a variety of statistical techniques, the cyclic load capacity values acquired from the chair models are categorized according to strength. For chair models manufactured in Türkiye, as a result, acceptable design loads have been attained. For the front-to-back cyclic load capacities of chairs, it was determined that those between 932 and 1449 N are considered to be weak-strength, those between 1450 and 1968 N are considered to be medium-strength (suitable for domestic use), and those between 1968 N and above are considered to be high-strength . In the case of back-to-front cyclic load capacities, it was determined that those between 625 and 895 N are considered to be weak-strength, those between 896 and 1167 N are considered to be medium-strength, and those between 1168 N and above are considered to be high-strength. The cyclic load capacity values for sidethrust loads are as follows: weak-strength is defined as 649–934 N, medium-strength is defined as 935–1221 N, and high-strength is defined as 1222 N and above. When the classification made in this study was compared with the acceptable design loads determined in the ALA (American Library Association) specification, which is used to evaluate chair performance in many countries of the USA and Europe, it was understood that there was no significant difference between the acceptable design loads. In front-to-back loading, the acceptable design load given in the ALA for domestic usage chairs is 1335 N, while the value found for domestic chairs in the classification in this study is 1450 N. While the recommended design load in ALA for back-to-front loading is 1001 N for domestic use, it was obtained as 896 N in the classification of this study. In case of sidethrust loading, while the recommended design load in ALA for domestic use is 890 N, the recommended value for domestic use in this study is 935 N. Accordingly, the classification developed in this study was found to be highly consistent with the values in a valid specification accepted around the world.It is understood from the literature that the relationships between the dimensions of the furniture and the user’s anthropometric data are important in terms of ergonomics, strength, and functionality, and that the body weight of the users has a direct impact on the strength, especially in furniture used in public spaces ,14,15. APrior to mass production, manufacturers should design and optimize their chair designs in terms of strength and failure modes; thus, it is critical to convert the values from this classification study into a national standard. The standardization, transmission, and display of such data to companies working for them would help them produce furniture of higher caliber, improving the quality of life for consumers in the process. Additionally, economic benefits will thereby be experienced by producers.


Clostridioides difficile (C. difficile) infections represent a major health risk and a high cost to health-care systems. C. difficile causes pathogenic activity in the colon through the production of two toxins, A and B, which provoke cell death. Several studies conducted on the mechanisms by which these two toxins induce various types of cell death in the intestinal mucosa cells and in the immune system cells failed to identify a common basic event. Since calcium is an important regulator of cellular physiology, here we propose a new point of view in which the alteration in physiological intracellular calcium levels by the two toxins may be the key common event responsible for the activation of cell death. Further exploration of this issue with in vivo studies could be important for possible pharmacological interventions aimed at antagonizing the Ca2+ homeostasis alterations that could tone down the C. difficile pathogenicity until specific antibiotic therapy has brought the infection under control.Clostridioides difficile (C. difficile), responsible for 15–25% of gastrointestinal infections, causes health problems mainly due to the toxic activity of toxins A and B (Tcds). These are responsible for its clinical manifestations, including diarrhea, pseudomembranous colitis, toxic megacolon and death, with a mortality of 5–30% in primary infection, that increase following relapses. Studies on Tcd-induced cell death have highlighted a key role of caspases, calpains, and cathepsins, with involvement of mitochondria and reactive oxygen species (ROS) in a complex signaling pathway network. The complex response in the execution of various types of cell death  depends on the amount of Tcd, cell types, and Tcd receptors involved, and could have as initial/precocious event the alterations in calcium homeostasis. The entities, peculiarities and cell types involved in these alterations will decide the signaling pathways activated and cell death type. Calcium homeostasis alterations can be caused by calcium influx through calcium channel activation, transient intracellular calcium oscillations, and leakage of calcium from intracellular stores. These increases in cytoplasmic calcium have important effects on all calcium-regulated molecules, which may play a direct role in several cell death types and/or activate other cell death effectors, such as caspases, calpains, ROS and proapoptotic Bcl-2 family members. Furthermore, some support for the possible role of the calcium homeostasis alteration in Tcd-induced cell death originates from the similarity with cytotoxic effects that cause pore-forming toxins, based mainly on calcium influx through plasma membrane pores. Clostridioides difficile (C. difficile) [C. difficile infection (CDI) range from mild transient diarrhea to pseudomembranous colitis and toxic megacolon [C. difficile is mainly due to two exotoxins, toxin A (TcdA) and toxin B (TcdB), with TcdB being about 1000 times more potent than TcdA on cytotoxic activity. A minor pathogenic contribution is also due to the binary toxin [fficile) ,2 is a Gfficile) ,4,5. Theegacolon ,7. The pry toxin ,10,11,12C. difficile strains [C. difficile in the anthropized environment, and the increasing ability to colonize humans [At present, CDIs have become a serious health problem because they are not only frequent in subjects undergoing antibiotic therapy or in those hospitalized/institutionalized but also because the incidence is increasing in the community, in both elderly and young subjects, in those with severe illnesses e.g., inflammatory bowel disease (IBD) and also as a consequence of increased feeding tube use and other surgical or therapeutic practices ,15,16,17 strains ,19, the e humans .C. difficile is transmitted person-to-person via the fecal–oral route by spores, and hospitals and community health-care settings may become an important source of infection [C. difficile spores can be found almost ubiquitously in hospitals and nursing home environment, health-care workers’ hands, and several medical devices [C. difficile and hospital outbreaks [nfection ,7,21. Vi devices ,7,20,21. devices ,23. NursC. difficile colonization [C. difficile bacteriophages (phages) [So far, attempts to develop non-antibiotic therapies against CDI based on antibodies ,25, vaccnization , C. diff(phages) ,36 and i(phages)  have giv(phages) ,39,40,41C. difficile toxins (Tcds).Consequently, it is necessary to gain further knowledge of the basic mechanisms underlying the pathogenic activity of TcdA and TcdB to find in the network of activated signaling pathways molecular targets to antagonize in order to inhibit the pathogenic activity of 2+) homeostasis followed by the inhibition of Rho-GTPases. This first phase, still poorly understood, is perhaps decisive in the induction of cell death and for the type of cell death and could be of crucial importance to understand the manner of antagonizing the key pathogenic events of C. difficile.As we discuss below, within the pathogenic mechanism of CDI, there is an early phase of about 40 min duration, characterized by the alteration in calcium . Furthermore, we compare the effects of pore-forming toxins (PFTs) and Tcds, because an additional indirect enforcement of the possible role of the alteration in Ca2+ homeostasis in cell death induced by Tcds comes from the similarity with the effects that PFTs cause during induction of cell death, based mainly on the direct Ca2+ influx by plasma membrane pore formation.In this review, we discuss in depth several significant aspects that indicate, albeit indirectly, that in the mechanism of CDI pathogenicity, there is an early phase characterized by the alteration in Ca2+ with all the consequences due to the profound alteration in intracytoplasmic Ca2+ homeostasis, while the pathogenic activity of Tcds until now has been characterized only as the activation of Rho-GTPase-dependent and Rho-GTPase-independent mechanisms after internalization of the Tcd (see below) [The comparison between PFTs and Tcds takes into account the fact that PFTs form a stable pore in the membrane. This pore is the main responsible for the influx of Cae below) ,10,11,122+ homeostasis alteration could be a key event in the mechanism of pathogenicity of C. difficile is discussed and highlighted. Knowledge of this aspect could thus be of crucial importance to understand an alternative manner to antagonize the pathogenicity of C. difficile.Overall, for the first time, the possibility that CaC. difficile have reached the colon, if the right conditions are present, among which the most relevant is the alteration in the microbiota, they enter the vegetative phase with the dissolution of the spore wall and the production of TcdA and TcdB and, in some strains, also of binary toxin [C. difficile penetrates more deeply into the mucosa [Once the spores of ry toxin ,7,42,43.ry toxin ,10,11,12e mucosa ,9,10.C. difficile may contribute to pathogenicity [Although other molecular components of genicity ,45, the genicity ,11,12,46genicity .2+ as an early key event in the process of cell death.However, little emphasis has been placed on the possible key role of alteration in homeostasis of intracellular CaC. perfrigens and C. novi [TcdA and TcdB belong to the large clostridial toxins family, which also includes the toxins produced by  C. novi . TcdB is C. novi ,10,11,49 C. novi ,9,10,11. C. novi . TcdA co C. novi ,9,10,11.Tcds present both a analogous glucosyltransferase enzymatic activity and a similar structural composition with multi-modular domains, referred as the ABCD model  ,9,10,11.The receptors described for TcdA are two proteins expressed in the cell plasma membrane ,9,51. ThThe receptors described for TcdB number three, and are proteins expressed in cell cytoplasmic membrane ,9,51. ThTcdA and TcdB, after binding to the cell plasma membrane receptors for Tcd, prompt its uptake by different pathways. Uptake of TcdB is mediated by clathrin, while that of TcdA is mediated by PACSIN2/syndapin-II ,59,60. FOf interest, TcdA and TcdB may also induce cell death (cytotoxic effect) independently of autoprocessing or glucosyltransferase activities of the Tcds, which occurs mainly by necrosis, but can also occur by apoptosis, pyknosis, or pyroptosis (see below) ,63,64,65Tcds are able to cause death in several types of cells: colonocytes , enterocNotably, Fettucciari et al. showed that EGCs that survive the TcdB cytotoxic effects go into a senescence state as a survival response to a stressor stimulus prompted by TcdB ,80.Although glucosylation-independent necrosis has been observed in vitro, the relevance of this mechanism during CDI has remained an outstanding question. Recently, Peritore-Galve et al. showed that in a mouse model of CDI, epithelial damage occurs through a glucosylation-independent manner not mediated by immune cell influx. In particular, the authors showed that the glucosylation activity of Tcds causes severe edema and inflammation and recruitment of immune cells. Glucosylation activity of both Tcd was necessary to produce weight loss and severe watery diarrhea, while it was not necessary to cause epithelial damage and pseudomembrane formation. Moreover, glucosylation-independent epithelial damage did not require/elicit recruitment of immune cells .2+ homeostasis [Overall, the cytotoxic activity of Tcds, which are multifunctional proteins with intrinsically disordered regions , is due eostasis ,11,37,82The most evident manifestations of the inactivation by glucosylation of the Rho-GTPase members Rac1, Cdc42 and Rho by Tcds are the cytopathic effects (cell rounding) due to derangements of the actin cytoskeleton with cell cycle arrest and cytotoxic effects that cause cell death ,12,46,61The cytotoxic activity induced by TcdB is manifested in all cell types, while this is not the case for TcdA, probably due to the greater cytotoxic potency of TcdB and the different types of membrane receptors targeted by the two Tcds ,9,10,46.Furthermore, the two Tcds show differences in effects at low and high concentrations. In fact, when TcdB is present at low concentrations (picomolar concentrations) and binds to membrane receptors CSPG4 and FZD, it induces mainly apoptosis in a glucosylation-dependent way, while when TcdB is present at higher concentrations (the nanomolar concentrations) and binds to CPSG4, FZD and PVL3 receptors, it induces mainly necrosis in a glucosylation- and autoprocessing-independent way ,56,57,63Studies on the mechanisms by which TcdA ,85,86,87Effector caspase activation by Tcds is mediated through an extrinsic death pathway dependent on receptors that encompasses caspase-8, or through an intrinsic death pathway dependent on mitochondria that include caspase-9 . TcdA anTcds induce apoptosis also, causing alteration in mitochondrial functionality and a low/moderate increase in reactive oxygen species (ROS) by the ROS/JNK/caspase-3 axis ,103.2+, with the subsequent involvement of caspases and cathepsins [However, it has also been demonstrated that Tcds activated pathways of cell death mediated by calpains, likely due to an early increase in intracellular Cathepsins ,86.TcdB at high concentrations induces cytotoxicity, mainly by necrosis, characterized by early alteration in plasma membrane permeability, ATP depletion, cellular leakage, and condensation of chromatin without activation of caspases ,104,105.TcdB also induces cytotoxicity by pyknotic cell death, characterized by ballooning of the nuclear envelope and condensation of chromatin, both glucosyltransferase domain-dependent and -independent, and ROS-independent ,65,104.Furthermore, TcdB induces a form of cell death called pyroptosis, characterized by a strong inflammation and pore formation mediated by gasdermin-D, which is the substrate of proinflammatory caspases implicated in inflammasome generation ,107,108.2+ mobilization, due to Ca2+ release from both the endoplasmic reticulum and damaged mitochondria. This release plays a critical role in the apoptotic–pyroptosis cascade, which can be mediated by caspase activation triggered by inflammasome activation [2+-mediated calpain activation and subsequent activation of caspases and cathepsins [In this context, the initial key event of assembly and activation of the NLRP3 inflammasome seems to be a rapid Cativation  and by tthepsins ,86,100.2+ flux is activated and/or whether there is mobilization of intracellular Ca2+ stores, events necessary for calpain activation.While the activation of calpains and their primary involvement in the apoptotic cascade induced by TcdB in EGCs has been well documented in vitro , there a2+ in cell death induced by TcdA and TcdB, suggesting possible mechanisms of the increase in intracellular Ca2+ induced by Tcds.However, as discussed in the following paragraphs, several studies seem to provide support for a primary role of Ca2+ homeostasis [2+ can increase due to an influx from the extracellular environment, release from intracellular stores such as the endoplasmic reticulum and lysosomes, or by both mechanisms [2+ induced by bacterial toxins by means of Ca2+ sequestration mechanisms in the endoplasmic reticulum and mitochondria and by extrusion via selective Ca2+ pumps [Most bacterial toxins, either by binding membrane receptors or causing pores on the plasma membrane/membrane of intracellular organelles such as lysosomes and endosomes, can alter cytoplasmic Caeostasis ,115,116.chanisms ,114,115.2+ pumps ,114,115.At the same time, the cells try to repair the damaged plasma membrane by endocytosis or shear and removal of the damaged portion of the membrane (shedding of microvesicles) ,119,120.2+ in cell death and more generally its effects on the mitochondria, lysosomes, and Ca2+-sensitive molecules have scarcely been investigated. In contrast, several studies are available on the family of PFTs [As previously stated, Tcds induce rapid cell cycle arrest, and depending on the Tcd concentration, Tcd receptors, and cell type, can cause death by apoptosis or necrosis ,46,59,63 of PFTs ,115,116,2+ in the toxic activity of Tcds. In fact, it has been demonstrated that when TcdB binds to membrane FZD receptors, which affect cytoplasmic Ca2+ levels, inhibition of calpains strongly inhibits TcdB-induced apoptosis [2+ homeostasis. Additionally, a possible key role of Ca2+ in the activity of Tcds is suggested by several previous observations and studies that in the light of current knowledge can now be better understood and contextualized.Recent studies seem to suggest an important role of Capoptosis ,55,56,58poptosis ,122,123,2+ may be involved in the toxic activity of TcdA and TcdB comes from the studies of Giugliano et al. (1984) through the use of Ca2+ antagonists. In fact, C. difficile filtrates, which cause both the morphological changes and cytotoxicity in Vero cells (monkey kidney), are inhibited when lanthanum chloride  or chlorpromazine hydrochloride  are added to the filtrates. This inhibition is dose-dependent, and when LaCl3 and CRP are added together, the protective effect is greater [3 acts as a Ca2+ antagonist, while CRP, among other properties, also binds calmodulin. Therefore, these results suggest that Ca2+-dependent events are fundamental for the activity of Tcds and that they appear to depend from Ca2+ activation of calmodulin [One of the first demonstrations that Ca greater . LaCl3 almodulin .2+ channel blockers verapamil and LaCl3 protect against the cytopathic effect of TcdB when added before, concomitantly, and up to 30 min after TcdB [2+ and calmodulin activation are required for the toxic activity and internalization process of TcdB. In fact, the functionality of Ca2+ channels is absolutely necessary for TcdB to exert its toxicity [Subsequently, in a more organic study, it was demonstrated in MRC-5 human lung fibroblast cells that the Cater TcdB . Furtherter TcdB . Thus, Ctoxicity .2+ in its toxic activity. In fact, in intestinal epithelial cells, IEC-6, the early alterations in cytoskeleton surface blebbing and nuclear fragmentation (BERN) are not inhibited by Ca2+ channel blockers, inhibitor of Ca2+-activated catabolic enzyme, calmodulin antagonists, or Ca2+ ionophores [2+ homeostasis. A further interesting phenomenon induced by TcdA is the strong formation of surface blebbing in about 50% of the cells [2+ plays no role in TcdA activity on IEC-6 [2+ plays a role inTcdA activity on human granulocytes [However, TcdA appears to be different from TcdB regarding the role of Canophores . Thus, The cells . These ron IEC-6 , are conulocytes ,128,129.2+ by TcdA and TcdB in cytotoxic and cytopathic activity can vary according not only to cell type but also to Tcd dose. Indeed, TcdA in human granulocytes induces a rapid and transient increase in cytosolic Ca2+ that begins within 1–3 s, reaches a peak at 30 s, and returns to baseline values after about 2 min. This transient increase is not due to the Ca2+ influx, but to the mobilization of intracellular Ca2+ from intracellular Ca2+ reserves, partly mediated by guanine nucleotide-binding protein G(i) [However, it is clear that the involvement of Caein G(i) ,129.2+ but only for TcdB internalization. However, it is difficult to exclude that during the internalization process, an influx of Ca2+ or its exit from intracellular reserves does not occur. That Ca2+ may, however, contribute to the toxic activity of TcdB is suggested by the demonstration that the direct introduction of Ca2+ ions into the cell via the Ca2+ ionophore, A23187, enhances the morphological changes induced by TcdB. A further interesting aspect of these studies is represented by the cells that survive TcdB, which increase the cytoplasmic ramifications much more than the controls after 26 days of culture, and further by the appearance of flat epithelioid astrocytes with showy bundles of filaments suggesting the transition to the state of senescence [In primary cultures of astroglia, TcdB induces morphological changes which require extracellular Canescence .2+ in the toxic activity of TcdA is confirmed in a model in which TcdA is given to Chinese hamster ovary (CHO) cells and human colonic tumors cells, T84 [2+ channel blockers or reducing agents, leading to the conclusion that the cytotoxicity of TcdA is not a Ca2+-dependent activity [2+ could depend on the type of receptor involved and the cell type. Thus, some receptors once bound by TcdA have the ability to internalize TcdA and induce toxicity by altering Ca2+ homeostasis, while others internalize TcdA without altering Ca2+ homeostasis [The variability in the role of Calls, T84 ,132. In activity . This coeostasis .2+ due to influx of extracellular Ca2+ [2+ increase follows a bimodal pattern that characterizes two cell populations [2+ concentration (2.5 s) and fast Ca2+ influx followed by a sustained steady state; and (2) 75% of cells characterized by a slow increase in cytosolic Ca2+ concentration, always followed by a steady state. In fast Ca2+ influx, Ca2+ initially localizes in discrete regions close to the membrane, where it reaches higher concentrations, then there is a high and homogeneous diffusion of Ca2+ in the cytosol [2+, the latter diffuses into the cytosol [2+ is exclusively due to its extracellular influx, subsequently the Ca2+ released from intracellular stores contributes to the increase in Ca2+ intracellular concentration [2+ for the toxic activity of TcdB occurs with the sequestration of intracellular Ca2+ by 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and Ca2+ clamping, which abolishes the cytotoxicity [Another study demonstrated that the cytotoxic and cytopathic activity of TcdB in human fibroblast cells, NIH-3T3, is due to a sustained increase in intracellular Calar Ca2+ . The celulations : (1) 25% cytosol . Even in cytosol . While tntration . Furthertoxicity . The samtoxicity .2+-independent mechanisms, such as cathepsin activation, may contribute to TcdB toxicity [2+ can activate other Ca2+-dependent protease such as calpains, which can contribute to cytotoxicity [2+ homeostasis, especially if induced from the outside [However, there are other important considerations concerning this phenomenon: (1) Catoxicity ; (2) sustoxicity  and to m outside .2+ and the mobilization of intracellular Ca2+, with the activation of calmodulin. In fact, the association of ethylenediaminetetraacetic acid plus BAPTA completely abrogates TcdA-induced IL-8 secretion, while calpain inhibition abrogates it by about 80% [In human monocytes, TcdA induces an upregulation in the secretion of interleukin-8 (IL-8), one of the cytokines responsible for the inflammatory response that accompanies CDI. In particular, TcdA induces IL-8 upregulation by an influx of Cabout 80% . Furtherbout 80% ,135,136.2+ is required for binding of the cell wall binding domain of TcdA to plasma membrane of CHO cells [2+. Conversely, TcdA at high concentrations has a toxicity independent from Ca2+. Thus, Ca2+ plays a role in TcdA binding to the target cell and renders this more sensitive to TcdA cytotoxicity [Extracellular CaHO cells . In additoxicity .2+ that precedes the final stage of apoptosis [2+ caused by an influx, which is indispensable for altering mitochondrial functionality and the subsequent release of cytochrome C [Most PFTs, in addition to creating pores in cell membranes, creates them in the mitochondrial membranes ,114,115.poptosis . Actuallchrome C .2+. Furthermore, it is possible that the Ca2+ increase induced by TcdB blocks the mitochondrial ATP-sensitive potassium (K+)-channel, thus contributing to apoptosis [TcdB added in vitro to isolated mitochondria causes their swell and releases cytochrome C only in the presence of Capoptosis .2+ also has a very important role in the necrotic response of Hela cells to relatively high concentrations of TcdB (≥100 pM) due to aberrant production of ROS following the assembly of NADPH oxidase (NOX). These high levels of ROS cause mitochondrial damage, lipid peroxidation, and protein oxidation. This phenomenon depends on the activity of L-type Ca2+ channels, because most of their inhibitors block the cytotoxic activity of TcdB [Ca of TcdB ,64.2+ response mechanism to TcdB is characterized by two phases, with an initial peak of Ca2+ increase in 10–15 min, which returns to baseline levels after a further 20 min . This signal is quite small, but it is followed by a second increase at 40–45 min that then persists over time. The increase in intracellular Ca2+ is due to the influx of extracellular Ca2+ [2+ through L-type Ca2+ channels that leads to the rapid activation of PKC with consequent aberrant activation of NOX, which causes the production of a high level of ROS and necrosis as a final event [The Calar Ca2+ ,139. Furlar Ca2+ . Thus, Tal event ,82.2+, also dose-dependent. This increase is due to the upregulation of NOX4, which is mainly responsible for alterations in Ca2+ homeostasis and subsequent endoplasmic reticulum stress. Thus, NOX4 activated by TcdB acting on Ca2+ homeostasis is mainly responsible for cytotoxicity and apoptosis [In another experimental model, at 12 h, TcdB induced a strong increase in intracellular Capoptosis .2+. In fact, in the synaptosomes, where the alpha toxin creates pores allowing the influx of extracellular Ca2+ with the consequent stimulation of phospholipase D, TcdB inhibits the activation of phospholipase D by blocking Rho-GTPase. These results indicate that the Ca2+ influx induced directly by TcdB can be subsequently stopped by TcdB itself through inhibition of Rho-GTPase, which also causes inhibition of phospholipase D, thus preventing the formation of choline and phosphatidic acid [2+ due to extracellular influx and/or mobilization of intracellular Ca2+ stores starts. However, the strongest inhibitory effect is on Ca2+ influx [2+ also depends on the cell type. A relationship between TcdB-induced increase in Ca2 signaling and the phospholipase family is suggested in a macrophage cell model, RAW 264.7, where the phenomenon is independent of Rho-GTPase [Other studies highlight the complexity of the interaction between TcdB and Cadic acid ,141. The+ influx . Of couro-GTPase ,143.2+ alterations.The cytotoxicity of Tcds depends on the cell type because they express different receptors; however, Tcds have the ability to cause cell death in all, further underlining a possible common mechanism of action that could be mediated by Ca2+ homeostasis occur before significant Rho-GTPase inhibition. However, it is possible that after the inhibition of Rho-GTPases, a further alteration in Ca2+ homeostasis occurs, contributing to a stronger activation of Ca2+-dependent molecules, including calpains. This makes the cell death process, which is induced by different molecular mechanisms, irreversible and explains the frequent observation that the inhibition of a single death pathway, for example, that mediated by caspases, does not completely prevent cell death.It has been shown that alterations in CaC. difficile and its Tcds induce activation of pannexin 1 channels, which favor cell death via release of ATP, activation of purinergic receptors and increased intracellular Ca2+ flow, causing the activation of initiator and effector caspases and leading to apoptosis [Loureiro et al.  demonstrpoptosis .2+ mobilization, and PKC activation [Kim et al. demonstrated that TcdA induced colonocytes apoptosis by the release of prostaglandin E2 which by stimulating EP1 receptors induce PLC activation, Cativation .C. difficile by nutrients, including Ca2+, that play a key role in sporulation and germination of C. difficile in the gut [2+ in vitro [2+ uptake or serum Ca2+ levels in CDI in patients or murine models.Loureiro et al.  analyzed the gut , reportiin vitro  and that2+ suggest that in Tcd activity, there is an early phase of about 30–40 min in which Ca2+ homeostasis is altered, with effects that depend on Tcd concentration, cell type and Tcd receptors involved. The increase in intracellular Ca2+ concentration, both distributed throughout the cell and with localized effects on substructures such as vacuoles and vesicular compartments, may be responsible for events crucial for cell survival, such as alteration in mitochondrial polarization, activation of calpains, and alterations in endosome stability, by which the cathepsins are kept under control. There is also the ability of calpains to activate caspases. Then, a series of Ca2+-dependent events fundamental for cell survival takes place, before the occurrence of other events, after about 40 min, resulting from the inhibition by Tcds of Rho-GTPase. These latter are involved in cell cycle arrest and the activation of signaling pathways, which add to the effects already caused by the alteration in Ca2+ homeostasis.Overall, the studies on the relationship between Tcds and Ca2+ homeostasis and the inhibition of the Rho-GTPase family. This in order to be more capable of counteracting the various types of survival pathways in each cell type, including immune cells, the latter with different Tcd susceptibility due to their functional state of activation.All the data above suggest that in their interaction with numerous types of tissue, nervous cells and immune cells, Tcds utilize two important pathways overlapping each other over time, represented by the alteration in Ca2+ has yet to be investigated.It is important to remember that Tcds have a further strategy for neutralizing cells that have resisted toxic activity, i.e., inducing a state of senescence , in whicUnfortunately, there have been no in vivo studies on this issue.2+ that alters its cellular homeostasis, with important consequences on cell fate [2+ homeostasis [2+ homeostasis could have a central role in activating cell death signaling in addition to that derived from Rho-GTPase inhibition [Large clostridial toxins (LCTs) are all PFTs, i.e., toxins that cause pores in the plasma membrane through which occurs an influx of Caell fate ,116,138.eostasis ,138. Althibition ,116.2+ homeostasis  by inositol triphosphate (IP3). This latter is generated through direct or indirect stimulation of G proteins at the plasma membrane level with the subsequent activation of phospholipase A2, favoring the generation of IP3. This in turn stimulates its receptor on the endoplasmic reticulum and final release of Ca2+ [The activation by PFTs of these channels, able to selective release Ca of Ca2+ ,153,154.There are no data available on the possible interaction of Tcds with G proteins; however, TcdA and TcdB are capable of activating phospholipase A2 and therefore of generating IP3 ,156.2+ release from lysosomes through two-pore channels following plasma membrane stimulation of CD38. This activates the pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), responsible for Ca2+ release [2+ release can contribute to PFT activity [It has been suggested that PFTs might induce Ca release . Damage activity .2+ homeostasis.While there are no data on the activity of Tcds on the CD38-NAADP lysosomal pathway, Tcds are able to act on lysosomes, altering their membrane functionality with release of cathepsins ,123,157,2+-dependent cytoplasmic proteins, if activated, are very sensitive sensors of alterations in Ca2+ homeostasis, and thus of the presence of pores, alterations in the Ca2+ channels of the endoplasmic reticulum, and/or alterations in the Ca2+ channels in lysosomes. In fact, PFTs activate calpains, calmodulin, calcineurin, PKC, and phospholipase [Caholipase ,161,162.2+ in the activity of Tcds, TcdB and to a lesser extent TcdA are able to activate these Ca2+-dependent cytoplasmic proteins. For instance, calpains are activated by TcdB in EGCs [As further confirmation of the role of Ca in EGCs . TcdB is in EGCs , while T in EGCs ,156. Bot in EGCs ,165.2+ homeostasis caused by PFTs activate cell death pathways [2+ levels reached, the capacity of Ca2+ extrusion mechanisms, and the cell type and receptor involved are the elements that decide the prevailing type of cell death.The profound alterations in Capathways ,115,116 2+ are the activation of calpains and calmodulins with consequent activation of caspases. Cathepsins released from lysosomes after functional alteration in their membrane also participate to the necrosis process, and to these events should be added the effects of release of ROS [The main events characterizing cell death by necrosis induced by PFTs and mediated by Cae of ROS ,113,166.e of ROS ,116,167.Even Tcds in certain situations can induce cell death by necrosis ,93,106. 2+-activated calpains and calmodulins [PFT-induced apoptosis is characterized by a mitochondrial pathway with release of AIF and/or cytochrome C from the mitochondria and by a caspase-dependent pathway mainly triggered by Camodulins ,115,116.Tcds are capable of inducing apoptosis ,46,47,602+ mobilization. In fact, it has been reported that Ca2+ mobilization activates NLRP3 inflammasome [2+ signaling/mobilization, which can be promoted by several cellular changes induced by pathogens or their toxins, such as K+ efflux and phagolysosome alteration, thus being responsible for ATP-mediated mitochondrial Ca2+ damage. In fact, it has been demonstrated that Ca2+ signal inhibitors block ATP-mediated mitochondrial Ca2+ damage by evaluating mitochondrial ROS production, loss of mitochondrial membrane potential, and release of mitochondrial DNA into the cytosol. Since these processes are needed for NLRP3 inflammasome activation, the critical role of Ca2+ signaling/mobilization in NLRP3 activation is to mediate mitochondrial damage [2+ homeostasis and calpain activation, effects induced by both PFTs and Tcds.The signal that starts from the mitochondria is mediated by NLRP3 in PFTs and likely also in Tcds and depends on Caammasome . The latl damage . Howeverl damage ,111,112,C. septicum whose activated signal pathways are very analogous to those induced by TcdB and TcdA. The first decisive event is the alteration in Ca2+ homeostasis, with its increase, rapidly followed by the activation of calpains, which act on the stability of the lysosomal membrane, favoring the release of cathepsins acting on various substrates [+/Ca2+ exchange pumps, resulting in further Ca2+ influx. This causes an alteration in MOMP that leads to an increase in ROS and a decreased production of ATP. Finally, DNA damage occurs and activates PARP, which in turn contributes to ATP depletion and thus to cell death [An organic view of cell death mechanisms induced by the most studied PFTs is provided by aerolysin, an orthologue of the alpha toxin of bstrates ,167,176.ll death ,167,176.ll death ,167,176.2+ homeostasis, followed by calpain activation and release of cathepsin from lysosomes [This picture of cell death is very similar to that caused by Tcds, especially by TcdB, as shown from studies demonstrating that the initial rapid event is disruption of Caysosomes ,123,157.ysosomes ,91,102. ysosomes .2+, the cell responds with different types of mechanisms to lower the cytosolic ion concentration and to avoid cell death [2+ extrusion by plasma membrane pumps, Ca2+-ATPase; (2) sequestration of Ca2+ within the endoplasmic reticulum reserves by sarco/endoplasmic reticulum Ca2+-ATPase; (3) and sequestration of Ca2+ in mitochondria by the mitochondrial Ca2+ uniporter. If the sequestration becomes excessive, it can lead to intoxication of the mitochondrion itself.Under increased cytosolic Call death ,116,138:2+ load may be effective whether the influx of Ca2+ is slowed or stopped, acting on the pores to eliminate them [These events aimed at reducing the cytosolic Caate them ,116,138.There are three main mechanisms of pore clearance, as follows.2+—migrate to the plasma membrane where the pores have formed [2+, contributing to the formation of a strong interaction with the plasma membrane. The latter forms membrane folds that are subsequently expelled, detaching from the membrane, which then rapidly reseals the externalized microarea called “microvesicles (MVs)” [Ectocytosis: this is a process of elimination towards the outside of the membrane areas that contain the pores by formation of microvesicles, and it takes place by two modalities ,115,116.e formed . Here, as (MVs)” ,118,119.s (MVs)” ,178.2+ homeostasis, with an increase in the cytoplasmic concentration creating MVs that have two main purposes [2+ by ectocytosis; the second is to surround the cell with MVs derived from the membrane containing the receptors for the PFTs. Therefore, the PFTs are bound before they can reach the cell membrane. Despite the fact that Tcds have been convincingly shown to alter Ca2+ homeostasis, there are no direct studies on MV formation in response to Tcds. However, an interesting study on platelets demonstrated that a pathway that regulates the formation of MVs is blocked by TcdB [2+ increase, at the same time prevent the cell to form MVs as a protective response. In particular, the induction of a Ca2+ influx in platelets leads to the formation of MVs through the Rac1 and p21-activated kinase 1/2 (PAK1/2) activation pathway with the involvement of the activation of calpains, which in turn activate caspases [2+ influx, the cell’s response should be to generate MVs, but this cannot happen because TcdB inhibits Rac1 [Multivesicular bodies: this is one of the membrane repair mechanisms that provokes a profound alteration in Capurposes ,119. The by TcdB . This cocaspases . By inhiits Rac1 .This aspect is relevant for Tcd pathogenesis, because if the MV response also has a protective role , TcdB coEndocytosis: this is a process by which the portion of the membrane that contains the pore is internalized and sent to the lysosome, where it is degraded ,115,116.2+ sequestration and/or the pore repair process.It is also evident that in the cases of PFTs and Tcds, there is always in a given population of cells a percentage that does not undergo any form of cell death, suggesting that in some of them are active effective mechanisms of intracellular CaIJs are one of the primary targets of PFTs, and the main consequence of their destruction is bacterial transmigration across the tissue barrier and the deepening of the infection ,115,180.2+ plays a central role in this process of IJ destruction and on the consequent redistribution of ZO-1 [2+, there is consequent activation of calmodulin and the release of pro-ADAM10, which can be activated by furin. At this point, activated pro-ADAM10 is transferred to the cytoplasmic membrane, where it causes shedding of cadherin and therefore IJ destruction [2+ can alter the permeability of the barrier is mediated by the activation of the myosin light chain kinase pathway [Ca of ZO-1 ,115,180. of ZO-1 ,180,181.truction ,180,181.truction ,180,181. pathway ,180.IJs are an important target of Tcds because their destruction favors the deepening of the infection in the submucosal colonic layers ,174,175.CDIs represent an important and serious widespread health problem, with an increasing incidence in both Western and Eastern communities. This increase is still under investigation, but some data suggest that it might be associated with the appearance of hypervirulent epidemic isolates of ribotype 027  or with The most important event in CDI is represented by cell death caused by TcdA and TcdB, which is then followed by inflammation with immediate and long-term consequences.2+ homeostasis the early key event for the activation of the various types of cell death.Important results have been obtained in studies concerning the complex activation mechanisms and signaling pathways that lead to various types of cell death, apoptosis, necrosis, pyknosis and pyroptosis. Given that Tcd dose, cell type, Tcd receptors involved and their expression in various cell types could account for the complexity of the various signaling pathways activated, perhaps it is possible to identify in the alteration in intracellular Ca2+ is the key player in the activation of cell death, the alterations in its homeostasis in terms of duration, Ca2+ concentration and localization in the various cellular microenvironments can in turn explain the great complexity of the activated signaling pathways involved in the various types of cell death. In particular, the inactivation of Rho-GTPases by Tcds may block the mechanisms that could attempt to counteract the increased intracellular Ca2+ concentration.Of course, if Ca2+ homeostasis are quite similar to those elicited by PFTs, which base most of their toxic action on the direct influx of Ca2+ through membrane pores to alter intracellular Ca2+ homeostasis. Further studies of the key role of Ca2+ in the pathogenetic mechanisms used by Tcds in CDI are needed and could be useful to explore new therapeutic strategies, focused on these early pathogenetic mechanisms, in order to prevent or block them, and to stop the subsequent cascade of events leading to this important inflammatory state of the gut.Additionally, it is interesting that TcdA and TcdB mechanisms and effects on intracellular Ca


TRIB1, a complement of the COP1 ubiquitin ligase complex, as a functional FOXO downstream gene maintaining an undifferentiated status. TRIB1 is direct target of FOXO3 and the FOXO-binding cis-regulatory element in the TRIB1 promoter, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), played a critical role in differentiation blockade. Thus, we designed a DNA-binding pharmacological inhibitor of the FOXO-FRE-T interface using pyrrole-imidazole polyamides (PIPs) that specifically bind to FRE-T (FRE-PIPs). The FRE-PIPs conjugated to chlorambucil (FRE-chb) inhibited transcription of TRIB1, causing differentiation in various AML cell lines. FRE-chb suppressed the formation of colonies derived from AML cell lines but not from normal counterparts. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. In conclusion, targeting cis-regulatory elements of the FOXO family is a promising therapeutic strategy that induces AML cell differentiation.Differentiation therapy has been proposed as a promising therapeutic strategy for acute myeloid leukemia (AML); thus, the development of more versatile methodologies that are applicable to a wide range of AML subtypes is desired. Although the FOXOs transcription factor represents a promising drug target for differentiation therapy, the efficacy of FOXO inhibitors is limited in vivo. Here, we show that pharmacological inhibition of a common cis-regulatory element of forkhead box O (FOXO) family members successfully induced cell differentiation in various AML cell lines. Through gene expression profiling and differentiation marker-based CRISPR/Cas9 screening, we identified  PML–RARA, which recruits histone deacetylase complexes to a group of genes involved in differentiation. All-trans retinoic acid directly binds to PML–RARA and promotes its degradation, leading to the induction of differentiation and excellent therapeutic outcomes [Differentiation blockade in progenitor cells is a critical pathological feature of acute myeloid leukemia (AML). To date, various driver genes that contribute to differentiation blockade have been identified. Acute promyelocytic leukemia is a subtype of AML characterized by the fusion gene, outcomes . Other ooutcomes , 3. TherKip)), and overexpression of FOXOs induces apoptosis and cell cycle arrest, while suppression of FOXOs promotes tumorigenesis in prostate, gastric, and thymic tumors [Forkhead box O subclass (FOXO) is a group of transcription factors consisting of FOXO1, FOXO3, FOXO4, and FOXO6. FOXOs are involved in various biological processes such as cell proliferation, apoptosis, and resistance to stress. FOXOs are regulated by phosphorylation through protein kinase B (AKT/PKB), which leads to the nuclear exclusion of FOXOs, resulting in a loss of transcriptional activity. Consistent with the oncogenic role of AKT, FOXO target genes include tumor-suppressive genes (BCL2L11(Bim), BBC3(PUMA), CDKN1B, which are synthetic DNA-binding molecules, have gained increasing attention. Because the pairing of imidazole (Im) against pyrrole (Py) (Im/Py) preferentially binds to a G/C base pair, and a Py/Py pair recognizes an A/T or T/A base pair, PIPs that bind to arbitrary sequences could be created by changing the order of Py/Im and Py/Py . PIPs tatribbles pseudokinase 1 (TRIB1), a component of the COP1 ubiquitin ligase complex, as a functional molecule in AS1842856-induced differentiation in AML. The FOXO binding cis-regulatory element in the promoter region of the TRIB1 gene, referred to as the FOXO-responsive element in the TRIB1 promoter (FRE-T), plays a critical role in the blockade of AML differentiation. Pharmacological inhibition of a common cis-regulatory element of FOXOs, including FRE-T, using PIPs conjugated to chlorambucil (FRE-chb), successfully induced differentiation in AML. Administration of FRE-chb inhibited tumor progression in vivo without remarkable adverse effects. These data revealed that targeting cis-regulatory elements of the FOXO family is a potent therapeutic strategy for inducing differentiation in AML.Here, we identified E. coli (NEB). Virus pools were produced by co-transfection of the sgRNA-expressing vectors with VSV-G and psPAX2 plasmids at a 1:1:0.5 ratio into HEK-293T cells using X-tremeGENE HP DNA Transfection Reagent (Roche). Genomic DNA samples were extracted and sgRNA inserts were amplified by PCR, and the resultant PCR products were sequenced by MiSeq (Illumina). The results of next-generation sequencing are shown in Table https://hannonlab.cshl.edu/fastx_toolkit/), and sequence data were aligned against the reference by BWA software [p values were corrected using MAGeCK [In this study, 30 genes were evaluated through CRISPR library screening to identify genes crucial for AML differentiation blockade. The sequences coding for 1004 non-targeting control sgRNAs and sgRNAs targeting 30 genes were obtained from validated sgRNA libraries published previously . The presoftware . Each re6 cells) were suspended in 50 µL of RPMI-1640 prior to being mixed with an additional 50 µL of Matrigel (Corning) and subcutaneously transplanted into 6-week-old female NSG mice. At post-transplantation day 3, the mice were randomly divided into three groups based on body weight and treated with vehicle, Chb-S, or FRE-chb (1.28 mg/kg per intravenous injection every 2 days). Tumor volume was calculated using the formula (long diameter) × (short diameter)2/2. During the experiments, the researchers were aware of the group assignments.NOD.Cg-Prkdc scid Il2rg tm1Wjl/SzJ (NSG) mice were purchased from NINOX Labo Supply Inc. . SKM-1 cells (5 × 10TRIB1 (pLJM1 TRIB1) was generated by subcloning TRIB1 from pCR3HA into pLJM1 [TRIB1 promoter was amplified from the HL-60 genome and cloned into pGL3 basic (pTRIB1wt-Luc). pTRIB1mt-Luc, in which the FOXO consensus sequence was depleted, was developed by site-directed mutagenesis. Expression vectors for sgRNA and shRNA were constructed by subcloning annealed oligonucleotides into lentiGuide-Puro and pLKO.1 puro, respectively. Target sequences of sgRNA and shRNA are listed in Table 1319 pcDNA3 flag FKHRL1 AAA was a gift from William Sellers (Plasmid #10709) . scramblto pLJM1 . The TRIp value of <0.05 was considered to be statistically significant. The statistical correlation of FOXO3 and TRIB1 gene expressions in AML patients’ samples were assessed by the Spearman’s rank correlation coefficient.Data are shown as mean ± SD values of the indicated number of independent experiments. The sample size was predetermined based on pertinent literature. Statistical differences between groups were assessed by two-sided Welch’s t-test. A LYZ) mRNA expression, in all six cell lines  activity, or lysozyme , we examined whether this element is involved in the induction of TRIB1 by FOXO3. Luciferase assay revealed that this element exhibits responsiveness to the constitutively active form of FOXO3 . Moreover, the deletion of FRE-T induced the expression of CD11b and LYZ associated with the inhibition of proliferation  using PIPs conjugated to Chb (FRE-chb). To this end, the polyamide was designed to bind to ACTGTTTA, which includes a significant portion of the FRE-T, by the polyamide recognition rule [To develop a therapeutic methodology that involves the inhibition of FOXO function for the suppression of ion rule  and to bion rule . Furtherion rule .Fig. 4FRTRIB1 expression in AML cells  in TRIB1 promoter by electrophoretic mobility shift assays. The wild-type FRE-chb probe (WT probe) includes FRE-chb targeting sequence in the TRIB1 promoter and mutant type FRE-chb probe (MT probe) is mutated in its target sequence Table . Becausells Fig. . FRE-chbnes Fig. . Moreovenes Fig. . ImportaIB1 Fig. . These dFinally, to assess the antileukemia efficiency of FRE-chb, we evaluated its half-maximal inhibitory concentration (IC50) in six AML cell lines. FRE-chb significantly inhibited the proliferation of leukemia cells at concentrations in the nanomolar range compared with control PIP, Chb-S, which reportedly does not affect proliferation in AML cell lines Fig. . In fourTo investigate the therapeutic window of FRE-chb, we compared the colony formation efficiency between AML cell lines and human hematopoietic stem cells  from cord blood treated with FRE-chb. The sensitivity of AML cell lines to FRE-chb was much higher than that of HSCs, indicating the therapeutic advantage of FRE-chb in AML Fig. . To examTRIB1 expression by FOXO3. Therefore, inhibiting FOXO signaling using PIPs is a promising therapeutic strategy for AML.Although the FOXO family of transcription factors represents a promising target for differentiation therapy in AML, the efficacy of FOXO inhibitors in vivo is limited. In this study, we developed a novel type of FOXO inhibitor targeting cis-regulatory elements of the FOXO family (FRE) using a PIP conjugated to Chb (FRE-chb). FRE-chb induced differentiation in various mutated AML cell lines. Mechanistically, FRE-chb induced differentiation by inhibiting the induction of FOXO3 induces differentiation in human AML cell lines harboring different types of mutations, and FOXO1 overexpression is sufficient to induce differentiation blockade and a preleukemic state in HSPCs. Considering these observations, FOXO1 and FOXO3 could be crucial regulators of differentiation blockade and represent promising drug targets for differentiation therapy.A critical finding of our research was that the FOXO inhibitor (AS18428569) induced differentiation in various AML cell lines. Although Lin et al. showed that AS1842856 inhibits the proliferation of AML1-ETO-expressing AML cell lines, the efficiency of the compound against other mutation statuses of AML and the mechanistic action has not been elucidated . We founTRIB1, a component of the COP1 ubiquitin ligase complex, as a functional molecule involved in compound-induced AML cell differentiation. Substrates of the COP1 ubiquitin ligase complex include transcription factor C/EBPα, whose overexpression induces differentiation in AML. Inactivating mutations of C/EBPα are detected in 10–15% of AML cases and promote leukemia in mice, highlighting the importance of C/EBPα in differentiation blockade [TRIB1 reduced C/EBPα protein levels, resulting in progression of leukemia in murine models [TRIB1 is a crucial oncogene regulating C/EBPα and differentiation status in AML. Importantly, we identified the FOXO-responsive element in the TRIB1 promoter (FRE-T) and found that transcriptional activation of TRIB1 though FRE-T plays a critical role in AML differentiation blockade. These data support that the FOXO–TRIB1 axis is an important pathway maintaining the undifferentiated status of AML.Although FOXO downstream genes have been characterized through RNA-seq and ChIP-seq analyses, the mechanism of differentiation blockade by FOXOs remains unclear because of the uncertainty of their functional target genes , 15. Thrblockade . Furthere models , 43. TheTRIB1 suggests that other FOXO targets also contribute to the maintenance of the undifferentiated state in AML. The FOXO downstream genes include MYC, a crucial molecule of leukemogenesis and differentiation blockade in AML. Smita et al. revealed that FOXOs are important molecules for maintaining MYC expression in breast cancer [MYC, and knockdown of FOXO3 reduces MYC expression in AML cell lines . We clearly demonstrated that FRE-chb efficiently impedes the FOXO–Supplementary Fig. 1-5Table S1-S6supplemental materialChecklistOriginal Data FileConsent for Authorship Change