Mentoring is well-known for its positive impact on diversity and inclusion across a wide variety of organizational contexts. Despite these demonstrated advantages, efforts to develop diverse leaders either through access to informal mentoring relationships or via formal mentoring programs are often complex, expensive, and frequently produce mixed results. We examine the unique impact of peer mentoring to support and develop African American leaders using a formalized program approach. Our findings show that peer mentoring is effective in providing a safe environment for the necessary work of identity to take place among African American leaders. This identity work takes the form of holding behaviors such as enabling perspectives, empathic acknowledgement and containment that are critical for the development, support and validation of diverse leaders. Our findings clearly show the benefit of external identity peer mentors for providing support and validation for African American leaders that can be absent within traditional hierarchical mentoring. By examining the outcomes of an actual leadership development program over time, we provide recommendations on how to enhance diverse leadership development by recognizing and cultivating the positive impact of identity-based peer mentoring. Mentoring is well-known for its positive impact on diversity and inclusion across a wide variety of organizational contexts . It is cIt is the case that effective leadership development programs, especially for diverse leaders, can face unique challenges in cultivating positive relationships and often fail to value or include different forms of leadership identities ,7. DiverMentoring has traditionally been defined as a series of relationships that are dynamic, reciprocal and have a dual impact on both the mentor and the mentee . Based oMentoring has gained increasing attention as a powerful tool to enable the careers of individuals as they advance through the ranks in organizations ,4,15,16.Within this work, the various challenges that diverse leaders face when mentors are different in terms of gender, race, and/or other demographic factors are also well documented ,28. For Hostile or unsupportive environments not only yield barriers for feelings of psychological safety and inclusion, they also signal  a lack of credibility for diverse mentees as future leaders and executives . The keyWhile diverse mentoring relationships  within a single organization may have many advantages that are critical for individual and organizational outcomes, the reality is that these relationships are complex, more likely to produce conflict, and may not meet all of the needs for women and people of color within organizations ,27. ThisThough mentors have traditionally been viewed as senior individuals, Kram and Isabella  providedWhile peer mentors provide career and psychosocial support in a manner comparable to that of traditional senior mentors, important conceptual differences exist between these types of mentorships, often related to the horizontal or vertical nature of the relationship. An important distinction to note for our purposes relates to reciprocity. Traditional mentoring relationships are frequently conceptualized and measured as unidirectional , whereas peer mentorships are described as either uniquely mutual or reciprocal ,36. BothGiven the changing nature of organizations in terms of being more networked and flat, peer or lateral mentoring is more readily available within the environment and provides critical career and social support ,40. PeerClearly there is an important contribution to be made by examining peer mentoring as a source of support and as a tool for developing diverse leaders. First, this examination responds to a number of scholars who call for greater attention to the diverse types of mentoring and their impact on key career and organizational outcomes ,15. SecoSocial learning theory emphasizes the importance of observing and modeling the behaviors, attitudes, and emotional reactions of others in learning and personal development. For instance, one study by showed that employees’ performance of organizational citizenship behaviors was related to the frequency and consistency of organizational citizenship behavior by other peer employees in their workgroup . Ibarra Social information processing also plays a key role in shaping perceptions, attitudes, and behaviors in organizations. The core argument of social information processing theory is that because organizations are complex and ambiguous environments, perceptions are influenced by the social context in which they are formed. This belief is especially relevant for leadership development because leadership competencies are complex, dynamic and contextual in nature ,51. ThisSimilarly, Coleman, Katz, and Menzel found that doctors’ decisions to prescribe a new drug were similar to the decisions of professional associates with whom they had talked about the drug . Mark anWe agree that social information processing can occur through peer mentoring relationships because peers share information and knowledge related to the completion of their work through such ties . For exaSocial influence among peers can also occur when individuals draw comparisons between themselves and other individuals in order to better understand ambiguous situations . TraditiSocial comparison is prevalent in organizations because, in many cases, evaluations regarding individual job performance within the organization are subjective. Social comparison can result in similar perceptions between individuals when one identifies with his or her peers. However, such social comparison may also lead to negative identity when there are status differences as is the case for traditional hierarchical mentoring, particularly when power and aspects of diversity or identity intersect . In thesThus, peer relationships are often used for social comparison because they develop between individuals with similar attributes or aspirations ,65. For Overall, these theoretical perspectives  provide the basis for our argument that peer relationships serve as an important source of social influence, identity development and social support. Prior evidence suggests that individuals make social comparisons with their peers, identify with individuals they consider models or exemplars of what they would like to become themselves and exchange knowledge and information with peers they see as knowledgeable . IndividWork by Ibarra and Petriglieri  specificFor example, Illeris outlines identity work through the lens of transformative learning as the stripping away of multiple layers of self-identity . This trThe importance of identity work is supported by Heaphy and Dutton who argue that individuals are active participants in their own development as they engage in the co-creation of who they are as individuals and especially as leaders . This meDespite the substantial research on the importance of mentoring for leadership development, there is still a need for more examples of how to facilitate, support and integrate identity work within formal efforts to effectively develop diverse leaders. Some research points to leadership development initiatives that shift away from traditional hierarchical, power-focused, and competitive views of leadership toward collaborative, inter-connected, and values-based approaches as one effective evidence-based model . Recent This insight suggests that paying attention to and developing efforts to support the necessary identity work is critical. Clearly this perspective will require a shift from leadership development-based approaches that tend to focus on reinforcing traditional organizational norms and definitions of leadership and toward more relational, inclusive, and collaborative definitions of what it means to be a leader . This tyHolding environments  have traMentoring as development networks have been described as holding environments especially for leaders . These dEmpathetic acknowledgement involves individuals such as mentors, colleagues and peers who recognize, confirm and validate the lived experiences, conflicts and challenges encountered by an individual or leader. Within the context of identity work, empathetic acknowledgement often involves the confirmation of issues such as unconscious bias, sexism, racism and various forms of discrimination that are validated as legitimate experiences from a point of mutual understanding and empathy. Enabling perspective is where others help the individual to make sense out of conflicting or contradictory information by engaging in self-reflection and insights shared by peers, mentors, and other developmental relationships. Containment involves the accessibility of others and often involves showing compassion, warmth and accepting others’ feelings without judgement or condemnation. The holding behavior of containment is focused on creating a safe environment for others to express emotion and serves as a non-judgmental sounding board and involves high levels of interpersonal trust. In contrast, enabling perspective focuses on mutual sensemaking where the end results in what is often referred to as negotiated interpretation of sharing identity-related experiences or challenges. Empathic acknowledgement can also involve creating safe spaces for shared experiences. However, this phenomenon places a greater emphasis on validation and identification that helps to restore regard and respect given challenging situations and experiences.These holding behaviors are like psychosocial functions of mentoring relationships as documented previously by Kram’s original work and later research by Higgins and Kram ,10. In cThus, we explore peer mentoring among African American managers within a formalized program to support identity-based leadership development and cultivate mutually beneficial holding environments. We examine a formal diverse leadership pipeline program that provides facilitated workshops along with peer mentoring circles over a 1-year period. Using qualitative interview and focus group data, we explore the role that containment, empathic acknowledgement and enabling perspective experiences serve as impactful tools for identity-based mentoring and leadership development among African Americans. Our objective is to provide an evidence-based approach for the use of holding environments via peer mentoring in order to provide support for the range of identity-specific challenges that diverse leaders uniquely face within traditional organizations. Our goal is to demonstrate that the use of facilitated peer mentoring with an explicit focus on identity-related work and the cultivation of holding environments can increase the overall effectiveness of formal mentoring efforts specifically for developing African American leaders.Our research setting was an executive education leadership development program aimed at strengthening the pipeline of African American leaders. Individuals were recruited from a variety of different organizations, industries and geographic regions within the United States. University faculty designed the program and facilitated all of the workshops which covered key topics including leadership competency, mentoring, organizational politics, racism and bias (both unconscious and deliberate). A key feature of the executive education program was to enable each participant to establish personalized developmental goals necessary for their advancement and overall well-being in the next phases of their career.Participants were engaged in several day-long interactive workshops that included fundamentals of effective mentoring, peer mentoring and building a diverse development network. Each program session was facilitated by University-based research faculty members and included both current research and best practices along with cases and interactive exercises to help engage participants. A total of 41 African American mid-level managers participated in this year long program from companies within the private, public and non-profit sectors. Participants were recruited based on having achieved some level of current leadership responsibility within their respective organizations. All participants were based in the U.S. but worked in both local and global companies.Participants were placed into peer mentoring “circles,” which each consisted of 3–4 program participants. Each peer circle participated in workshop exercises and discussions during the program to help with the initial phases of relationship cultivation. Each peer circle was also assigned one of the faculty members as a “coach” to answer questions, provide check-ins to ensure the circles were staying connected and serve as a resource for questions, concerns and emerging needs throughout the program’s duration. Attention was paid to ensure that each peer circle had a balance of diversity in terms of industry, gender, geographic region and professional expertise. All members were classified as “mid-career” in terms of overall tenure within their respective organizations. In addition, participants from the same organizations were deliberately not put into the same peer mentoring circles. Between program sessions, each faculty member did “check-ins” virtual meetings with the participants, for both ongoing evaluation and data collection.Two key points of data collection took place during the formal program. The first assessment took place three months after the initial program session where overview materials were presented, peer circles were formed and a discussion of the benefits and responsibilities of mentoring relationships were outlined. Each faculty coach conducted an hour-long telephone interview with each participant within their assigned peer mentoring circles. This initial point of data collection was to gather early-stage mentoring information including contact frequency and to solicit feedback from each participant. Several interview questions about their mentoring experiences were included and adopted from existing mentoring questions developed and validated by Ragins & McFarlin  and ThomAt the completion of the formal program, faculty coaches conducted in-person focus group interviews with each mentoring circle. A set of questions were developed that addressed participants’ overall evaluation of the mentoring experience. Participants were also asked about the content of their interactions, sharing information, and reflections on the value of their peer-to-peer interactions. These discussions used open-ended questions which allowed for a content examination of the development of “holding behaviors” within the peer mentoring circles without prompting or pre-disposing responses to various questions.Our primary measures focus on the presence and frequency of holding behaviors. We examined participants’ open-ended responses and coded mentions of behaviors or actions that illustrated the presence of holding behaviors as outlined by Kahn :Containment—creating a safe, reliable interactions that includes opportunities for others to express emotions, feelings and reactions. Key behaviors include: accessibility, attention, compassion and acceptance.Empathic Acknowledgement—creating empathic context that affirms the other’s sense of being knowledgeable, worthwhile and valued. Key behaviors include: empathy, validation and acknowledgement of others self-exploration and questioning.Enabling Perspective—creating a context in which others can discover a sense of competence, positive sense of self and creating a space for rational thought and action. Key behaviors include: self-reflection, sensemaking, task focusing and negotiated interpretation.Our analysis focused primarily on qualitative data that was collected during both mid-program check-in interviews and focus groups at the end of the formal program. Thirty-seven of 41 participants were interviewed during the program mid-point which yielded a follow-up rate of 90%. This initial interview phase was to confirm that the mentoring relationships formed during were taking place and involved some level of meaningful interactions. A vast majority of participants reported having “occasional or frequent” (78%) interaction. Mentoring connections typically occurred via telephone (43%) or some combination of telephone and email (49%). Consistent with previous research, these early interactions tended to focus on “getting to know one another” with discussions centering on work-related activities and on the developmental action plans required as part of the formal program (56%). The overall level of satisfaction within this initial phase was quite high. A majority of participants (81%) reported that their relationship was either “effective” or “very effective” when asked to verbally respond based on a 5-point Likert-type scale. Using responses to questions that were adapted from previous mentoring scales, we examined the presence of either career or psychosocial mentoring functions during the early phase of these peer relationships. Among the most frequent types of mentoring functions reported were: role modeling (85%); acceptance and confirmation (75%); exposure and visibility (82%), counseling (52%) and friendship (27%).Interestingly, some participants reported also talking about personal issues such as family–work balance and managing workplace conflicts, but these types of activities occurred less frequently among participants during this early stage of relationship initiation. During this early phase, a number of participants mentioned the importance of allocating time to cultivate their mentoring relationships and the value of having access to African American peers as part of the program, particularly when their organization lacked racial diversity among its leadership. While some discussion surrounding issues of race and how to be effective as a person of color in the organization was reported during our initial follow-up, the frequency of this type of sharing was limited during this initial phase of the peer mentoring program.Peer Mentoring Interviews. Overall perceptions of the mentoring experience among our African American leaders were quite positive and remained positive across the year-long program. During the last session of the program, we conducted face-to-face group interviews to discuss the mentoring experience and gain a better understanding of the value that participants received from interacting within their peer circles. Interestingly, we witnessed a change in the type of information that was shared between members of the mentoring circles as they continued to cultivate their relationships. Circle members reported most frequently talking about their own personal experiences related to career or life success and satisfaction relative to the previous interviews. Later discussions seemed to shift toward more specific leadership and personal development concerns . It is reasonable to conclude that these “tough” issues emerged as interpersonal trust was developed overtime during the program. There was also more information about the shared experiences surrounding racial identity related to their roles as leaders within the organization which was less prominent during the initial phase of the mentoring relationship.Peer Mentoring Holding Behaviors. One of key aspects of our analyses was to examine the presence and type of holding behaviors within these peer mentoring relationships. We categorized participant responses based on the definitions of holding behaviors and found evidence of all three types as described by Kahn  but in vPeer conversations that helped to make sense out of the experiences that were being faced which were similar because of issues of race and leadership. These conversations provided an “objective sounding board” because peers who were external to their organizations were often cited as most valuable. These conversations also included opportunities for peers to provide “greater focus” to one another in terms of what could be changed or controlled about their current circumstances. Participants shared that these conversations where sensemaking took place often lead to useful strategies for how to manage challenges, tasks and their overall careers. This finding is similar to what Kahn  calls, “Next most frequently reported within these peer relationships at the end of the year-long program were holding behaviors within the category of “empathic acknowledgment.” These peer experiences focused on affirming ones’ sense of self, providing validation for others’ skills, knowledge or abilities and the expression of empathy where there is a shared identification of experiences around issues of race, gender, and what does it means to be a “leader.” Participants mentioned that these peer relationships were valued because of the “safe haven” that provided “confidentiality” and “someone you can safely share ideas with.” The presence of empathy, affect, understanding and psychological safety were key aspects of these leaders’ experiences of holding behaviors from peer mentors of color see . Issues We received fewer responses that would be examples of containment according to Kahn . This diDuring our group discussions, we also provided an opportunity for participants to share challenges and issues they faced within these peer mentoring circles. The comments from participants reflected an interesting paradox about the benefits versus limitations of external peer mentoring. While enabling perspective, empathic acknowledgement and containment were clearly benefits of these relationships, peer mentors external to the organizations have some limitations. For example, participants frequently shared that while external peers provide a “safe haven,” the limitation is that they “may not appreciate some nuances of the organization” or “lack knowledge of key players” within their company. Issues such as “not knowing the structure” or “not knowing everything about their industry” were frequently mentioned. Lacking internal knowledge of the organizational dynamics was cited as a particular limitation when dealing with political issues or dynamics that are specific to a particular industry .A second limitation that participants shared about having external peer mentors related to the notion of mentors as a source of protection within their specific organizations. Several participants shared that while valuable, these formal peer mentoring relationships provided “limited protection” because they did “not have knowledge of the political scheme and key players” or ties inside of the same organizations. One participant shared that although supportive, their peer mentor “does not have their ear to the ground to help you navigate” as another example of the lack of protection. This finding is interesting as previous work tends to restrict issues of protection to being provided exclusively by more senior sponsors rather than lateral peer mentors. However, these African American leaders frequently mentioned that peers provided protection and a buffer within the environment, but that support is specific to internal rather than external peer mentors. The notion of “lack of protection” was frequently mentioned as a challenge and issue of concern among these African American leaders.Based on our research findings, we contend that engaging in identity-based sensemaking for emerging African American leaders is essential for supporting their development especially as they build competence and confidence throughout their careers. This identity-based sensemaking is foundational for diverse leaders as they navigate unique challenges while often lacking access to informal networks and sources of information which allow them to effectively “make sense” of their experiences within the organization. Furthermore, the feedback and advice they may receive from non-Black mentors often comes from vastly different experiences within their organization . FeedbacPeer mentoring was clearly seen as a positive resource that provides enabling perspectives which create a context for people to engage in sensemaking, self-reflection, and negotiated interpretation . Our proAs we observed from participants’ feedback, having protected conversations to better understand their role as a leader within the context of race, gender and other social identities provides a critical developmental experience to help them understand and navigate their organizational and career journeys. Thus, these enabling perspective holding behaviors provided by peers are a vital resource for developing clarity in both thought and action as a leader. An enabling perspective may also be a resource that is uniquely provided by peers external to the individual’s organization. Our participants frequently mentioned that peer circles provided an unbiased perspective or sounding board that was not biased or clouded by the dynamics within their own organizations. Clearly this is a critical aspect of the sensemaking process that external peers can provide and one that is important to the unique experience of being an African American leader.One of the key features of our formalized program is the longitudinal nature of the peers mentoring effort. Clearly having peer mentoring over this year-long period allowed for the development of holding behaviors that Kahn  labeled We observed less frequent examples among our peer mentors of containment, which takes place when peers “remain in other’s vicinity, allowing time and space for uninterrupted contact and connections” p. 269) . This ab69 . ThisThis research confirms the importance of peer mentoring, identity work and holding environments for the development of diverse leaders. Over this year-long program, we witnessed important “identity work” being done within these peer mentoring circles. Perspectives based on social categorization argue for identity work as a process of integration of identities . It is cThis acknowledgement suggests that identity work must be part of the explicit content of mentoring diverse leaders that provides a “safe place,” helps to support sensemaking and provides validation in order to overcome biases and traditional obstacles. This approach may be useful for other types of mentoring where issues of trust and psychological safety are important such as leadership programs that are devoted to role modeling of ethical behavior . If ment


In vivo experiments showed that circ_0021205 inhibition reduced tumorigenesis in mice. In addition, mechanisms investigation demonstrated that circ_0021205 exerts its oncogenic function by sponging miR-204-5p to regulate the expression of RAB22A. Overall, this study revealed that circ_0021205 might serve as a potential diagnostic biomarker or therapeutic target for CCA.Cholangiocarcinomas (CCA) are biliary tract tumors that are often challenging to diagnosis and treatment. Accumulated evidence reveals that circular RNAs (circRNAs) are involved in multiple cancer progression. However, the function of circRNAs in cholangiocarcinoma remains largely unclear. In this study, we found that circ_0021205 expression was up-regulated in CCA and positively correlated with tumor size and TNM stage. To further explore the role of circ_0021205 in CCA, cell functional assays were performed. The results showed that circ_0021205 promoted the proliferation, migration, and invasion of CCA cells.  Cholangiocarcinoma (CCA) is a highly malignant tumor found in the epithelial cells lining the bile duct, which is categorized according to anatomical location as intrahepatic, perihilar, and distal cholangiocarcinoma . In the Circular RNAs (circRNAs) are produced by reverse splicing, which is characterized by covalently closed continuous loops and canonical splicing sites . Due to MicroRNAs (miRNAs) are non-coding RNAs which play roles in the negative regulation of gene expression at the post-transcriptional level . In the Cholangiocarcinomas tissues and adjacent tissues were obtained from Lishui Hospital of Zhejiang University. All patients gave written informed consent before this study start. This study was approved by the ethics committee of Lishui Hospital of Zhejiang University. The clinicopathological characteristics of 27 CCA patients were as shown below:All cell lines were purchased from China Center for Type Culture Collection (CCTCC). Normal biliary cell line HIBEC and CCA cell lines  were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium , CCA cell lines QBC939, RBE, and HuCCT1 were cultured in Dulbecco’s Modified Eagle’s Medium . All medium were supplemented with 10% fetal bovine serum , 100 U/mL penicillin and 100 μg/mL streptomycin.Total RNA (10 μg) from HuCCT1 and KMBC cells were incubated with 5 U/μg RNase R  for 15 min at 37 °C. Subsequently, the treated RNA was reverse transcribed and the level of circ_0021205 was examined by quantitative real time PCR (qRT-PCR) assay. Experiments were repeated for three times.The si-circ_0021205-1 (GTCTAATTCCTGTGGTGAAGA), si-circ_0021205-2 (ATTGTCTAATTCCTGTGGTGA), miR-204-5p mimic (5′-UUCCCUUUGUCAUCCUAUGCCU-3′), and miR-204-5p inhibitor (5′-AGGCAUAGGAUGACAAAGGGAA-3′) were purchased from Thermo Fisher Scientific (United States). The oe-circ_0021205 pasmids were constructed using pLO5-ciR vectors . All cell transfection were performed using Lipofectamine 3000 .CT–ΔΔ algorithm. All primers used in this study were listed in Total RNA of CCA tissues and cell lines was extracted using TRIzol reagent , then the concentration and purity of RNA was measured by NanoDrop 2000 . The qRT-PCR was performed using SYBR Green qPCR Mix . For circRNA and mRNA, GAPDH was used as endogenous reference. For miRNA, U6 was used as endogenous reference. All qRT-PCR reactions were run on the ABI 7500 Fast Real-Time PCR System . The relative expression of RNAs was calculated with 2RAB22A-WT, or RAB22A-MuT sequences were co-transfected with miR-204-5p mimics or NC-mimics into HuCCT1 and KMBC cells. After 24 h of incubation, cells were lysed by 1 × PLB, then the luciferase activities were measured using the Dual-Luciferase Reporter Assay System . Experiments were repeated for three times.The pGL3 Luciferase Reporter Vectors  containing circ_0021205-WT, circ_0021205-MuT, 3 HuCCT1 and KMBC cells were seeded into 96-well plates and cultured for 2–4 h. Then added 10 μL CCK-8 reagents  to each well. After 2 h of incubation, the optional density (OD) at 450 nm was measured by the Multiskan FC with Incubator . Experiments were repeated for three times.3 × 102 HuCCT1 and KMBC cells in logarithmic growth phase were seeded into 12-well plates, and cultured for 2 weeks. Then cells were fixed using 4% paraformaldehyde for 15 min and stained by crystal violet staining solution for 15 min. Slowly washed the plates with running water and air-dried. Counted the number of colonies in the plates. Experiments were repeated for three times.2 × 10For migration assay, the transwell chambers  were paved without matrigel mix. For invasion assay, the upper transwell chambers were paved with matrigel mix. HuCCT1 and KMBC cells were suspended in medium without FBS and seeded into the upper transwell chambers. And medium supplemented with 10% FBS was infused into the bottom chambers as cell chemo attractant. After 24 h of incubation, the upper chamber was fixed with 4% paraformaldehyde and then stained by crystal violet staining solution for 15 min. The cell numbers were counted in five different fields under the microscope. Experiments were repeated for three times.Cells were lysed using RIPA buffer containing protease inhibitor . 30 μg total protein extracted from CCA cells was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked by 5% skimmed milk and incubated with the primary antibodies  overnight at 4°C. And incubated with the HRP-conjugated secondary antibodies  at room temperature for 1 h. Subsequently, the protein bands were visualized using the Clarity Western ECL Substrate . The primary antibodies used in this study were as shown below: .7 HuCCT1 cells stably transfected with si-circ_0021205 or si-NC were subcutaneously injected into the mice, respectively. After the injection, we measured the tumor and calculated the tumor volume every 4 days. 20 days after injection, the mice were euthanized, then tumors were excised and measured. We then used the tumor tissues for qOCR, WB, and IHC analysis.All male BALB/c nude mice were purchased from XX. 1 × 10t test and One-way ANOVA followed by Dunnett’s multiple comparisons test were used to analyze the difference between groups. P < 0.05 was considered statistically significant. Each experiment was triplicated.GraphPad Prism 8.0  and SPSS 22.0  were used in this study. Student’s To investigate the correlation between circ_0021205 expression and CCA development, we collected 27 pairs of CCA tissues and adjacent tissues, and detected the circ_0021205 expression using qRT-PCR. As shown in in vitro.To explore the role of circ_0021205 in CCA, the expression of circ_0021205 was down-regulated in HuCCT1 cells using si-circ_0021205-1 and si-circ_0021205-2  and up-r1, and detected their levels bind to specific circ_0021205 probe in HuCCT1 . Tumor volumes were recorded every 4 days (in vivo.To further explore the role of circ_0021205 y 4 days  and the y 4 days . Then thy 4 days . We theny 4 days . Next, wy 4 days . Six-monIn recent years, emerging evidence illustrates that many circRNAs function as tumor suppressors or promoters in several cancers, such as hepatocellular carcinoma, lung cancer, gastric cancer, and cholangiocarcinoma . So far,In this study, we found that circ_0021205 was significantly up-regulated in CCA tissues and cells compare with the controls and associated with tumor size and TNM stage. Subsequently, the functional study demonstrated that circ_0021205 promoted the proliferation, metastasis, and invasion of CCA cells. Currently, accumulating studies reported that circRNAs could serve as miRNA sponges to regulate the expression of downstream genes. The “circRNA-miRNA-mRNA” axis has been reported to be involved in the development of multiple cancers. For example, circCCDC9 suppresses the progression of gastric cancer by regulating CAV1 via miR-6792-3p . Circ-ZKIn our study, RIP experiment and liciferase report assay indicated that circ_0021205 could act as the sponge of miR-204-5p and RAB22A is the target gene of miR-204-5p. The rescue experiments demonstrated that circ_0021205 exerts carcinogenic effects in CCA by target RAB22A via miR-204-5p. RAB22A, a member of the RAB family of small GTPases, is known to be involved in several immune functions, and plays important roles in the endocytic recycling and the formation of T cell conjugates . Also, tIn conclusion, this study demonstrates that circ_0021205 promotes CCA progression through miR-204-5p/RAB22A axis, which may provide a potential biomarker for CCA diagnosis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Ethics Committee of Lishui Hospital of Zhejiang University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee of Lishui Hospital of Zhejiang University.JT and WC designed this study. LZ, SF, DZ, CK, and YY performed these experiments. RQ, ZZ, and CL analyzed the data. XL and JJ wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Large attention has been posed to the so-called Ca2+microdomains, dynamic Ca2+ changes spatially restricted to fine astrocytic processes including perisynaptic astrocytic processes (PAPs). With presynaptic terminals and postsynaptic neuronal membranes, PAPs compose the tripartite synapse. The distinct spatial-temporal features and functional roles of astrocyte microdomain Ca2+ activity remain poorly defined. However, thanks to the development of genetically encoded Ca2+ indicators (GECIs), advanced microscopy techniques, and innovative analytical approaches, Ca2+ transients in astrocyte microdomains were recently studied in unprecedented detail. These events have been observed to occur much more frequently (∼50–100-fold) and dynamically than somatic Ca2+ elevations with mechanisms that likely involve both IP3-dependent and -independent pathways. Further progress aimed to clarify the complex, dynamic machinery responsible for astrocytic Ca2+ activity at microdomains is a crucial step in our understanding of the astrocyte role in brain function and may also reveal astrocytes as novel therapeutic targets for different brain diseases. Here, we review the most recent studies that improve our mechanistic understanding of the essential features of astrocyte Ca2+ microdomains.The glial cells astrocytes have long been recognized as important neuron-supporting elements in brain development, homeostasis, and metabolism. After the discovery that the reciprocal communication between astrocytes and neurons is a fundamental mechanism in the modulation of neuronal synaptic communication, over the last two decades astrocytes became a hot topic in neuroscience research. Crucial to their functional interactions with neurons are the cytosolic Ca Despite these limitations, several recent studies revealed that the intracellular Ca2+ activity occurring in ultra-small processes , where “the most important calcium transients occur,” is crucial for the dynamic cooperation with neurons , where the majority of localized Ca2+ transients occur and may or may not propagate to nearby shafts. A strict correlation was reported between node and spine size, and also between Ca2+ transients and spine size , which could differ in terms of Ca2+ sources and handling from nodes.The nanoscopic size of these structures is an obstacle for functional imaging of Cae needed . A recenrocesses . In linerocesses , Arizonorocesses , in contine size , suggest2+-sensitive membrane-permeable chemical dyes. This approach is, however, inadequate for revealing Ca2+ changes at fine PAPs. The only available, though invasive, tool to study Ca2+ signaling at PAPs was the microinjection of chemical dyes into individual astrocytes through glass pipettes  into the cytosol can be initiated by stimulation of metabotropic Gq G-protein-coupled receptors (GPCRs) through activation of PLC, production of IP3, and activation of IP3 receptors (IP3Rs) on ER membranes , which is known to be enriched in astrocytes, partially reduces the number or amplitude (∼60–70% of reduction) of spontaneous Ca2+ microdomains  mice they observed spontaneous Ca2+ elevations with cytosolic GCaMP6f and spontaneous ER Ca2+ release with G-CEPIA1er, in IP3R2–/– mice they could detect spontaneous cytosolic Ca2+ elevations without decreases in ER Ca2+ levels. In contrast, in fine processes of IP3R2–/– mice, Gq receptor activation evoked a significant Ca2+ release from ER, although attenuated with respect to WT. Interestingly, this IP3R2-independent Ca2+ release induced low cytosolic Ca2+ elevations but robust Ca2+ transients in mitochondria . The opening of mPTP is facilitated by the production of reactive oxygen species owing to a rise in the metabolic demand after increased neuronal activity, suggesting a direct link between metabolic rate and Ca2+ microdomains . Na+-dependent neurotransmitter transporters localized in PAPs, such as the GABA transporter GAT3 . During SOCE, depletion of ER Ca2+ stores activates the Ca2+ sensor stromal interaction molecules (STIMs) to translocate to the junctional ER-plasmatic membranes, where STIMs interact with and activate the store-operated Ca2+-release-activated Ca2+ channels (CRAC channels) formed by Orai proteins. Recently, 2+ transients evoked by thrombin in PAPs from Orai1–/– mice were substantially attenuated, suggesting that Ca2+ microdomains rely on coordinated Ca2+ release from intracellular stores and transmembrane Ca2+ influxes from the extracellular space.Although Ca2+ microdomain essential features is lacking. Key questions remain unsolved. For example, which proteins are involved in Ca2+ influxes shaping microdomains? Astrocytes are mechanosensitive cells and the high motility of astrocyte processes  containing Ca2+ fluctuations (2+ fluctuations, waves involving principal processes, and sparkly signals at microdomains, providing as principal output, the raw fluorescence data along time from each region. Traces obtained through GECIquant can be further processed to obtain additional features, such as the amplitude and frequency of the Ca2+ events for each ROI, as performed in Many experimental and analytical approaches have been developed to properly dissect the features of Caritories  albeit wtuations . The sof2+ microdomains. Time-series images are processed to remove background noise and binarized using an amplitude threshold. Microdomains are selected based on additional criteria of amplitude and duration. Besides producing a spatial map of active regions, the software provides information about the number, frequency, amplitude, and time course of events.In 2017, CaSCaDe software was developed, overcoming the need for two scripts . CaSCaDeWeber’s laboratory worked to develop a processing suite aimed at simplifying the analysis workflows. This is the main purpose of CHIPS, a MATLAB based open-source toolbox that gathers different algorithms and provides the user with options for a number of analysis types. CHIPS offers helpful pre-processing functions, such as motion correction and denoising. Different algorithms can then be applied to analyze data and plot the resulting outputs .2+ variations may become larger than the defined ROI, leading the signal to invade other territories, finally resulting in signal fragmentation among adjacent ROIs. Conversely, larger ROIs may fail to faithfully capture the signal amplitude if Ca2+ increases only in a portion of the ROI.All the analytical procedures described so far rely on the concept of ROIs applied to microdomains, defined by spatial coordinates and measurable along different time series. However, constraining microdomains in defined boundaries may result in signal detection inaccuracy. Regions involved in CaThe turning point in this concern was represented by AQuA software , which r2+ indicator used and on the acquisition signal to noise ratio, with optimized but user-adjustable parameters. AQuA can work also on a single cell level and isolate events in astrocytic processes, but without distinction between principal branches and thin processes. The identification of events is achieved through different image processing steps, including thresholding and smoothing, and the application of a number of algorithms that aim to define the temporal limits and spatial propagation features of individual events. The user proceeds step-by-step and can inspect traces and spatial boundaries of the events along time, as training which is recommended to achieve the best parameters to apply to the specific experimental datasets. AQuA outputs a broad and rich collection of features for detected events, such as area, amplitude, duration, frequency, and propagation features, revealing itself as a valuable tool for astrocytic Ca2+ analysis.AQuA software does not include pre-processing functions but offers suggestions about pre-processing steps to be considered. A valuable feature is the possibility to choose between different data type pre-sets, depending on the Ca2+ activity recorded through 3D imaging. This approach captures the entire extension of individual astrocytes, adding a significant step of complexity to the comprehension of Ca2+ signal dynamics (While the approaches described so far refer to 2D imaging, it is worth mentioning that a study from Volterra’s group focused on the analysis of astrocytic Cadynamics .All in all, the development of different analytical approaches confirms the important role acquired by astrocytes in the studies of brain function. In the near future, analytical and experimental advances will enable us to unveil new types of modulation exerted by astrocytes.2+ activities at microdomains. In this restricted and elusive subcellular region, an unexpected complexity has emerged. Here, the stage of Ca2+ activity has been populated by new actors but many questions remain unanswered. For example, how these players interact? How Ca2+ events propagate from fine to major processes? What is the role of spontaneous microdomain Ca2+ activity in brain functions? The use of different types of preparations, Ca2+ detection techniques, and analytical tools in different brain regions has provided invaluable results that, however, cannot always be easily compared. New shared techniques and analytical tools are thus needed. Elucidating all the mechanisms of neuron-astrocyte interplay at tripartite synapses is crucial to understanding brain functions and dysfunctions (Over the last decade, the combined use of innovative tools has provided compelling evidence that astrocyte dynamic interactions with neurons have fundamental roles in brain function. The different players involved in this interplay are beginning to be elucidated, particularly regarding Caunctions , and wil2+ events with MZ. VH created the figure. AC wrote the chapter on the locus of Ca2+ with GL. MZ wrote chapter on the Analysis of Ca2+ events with AL. GC contributed to the manuscript. MG-G wrote the chapter on the genesis of Ca2+ events. GL wrote the abstract, introduction, the chapter on the site of Ca2+ events with AC, and the discussion with the help of all other authors. All authors contributed to the article and approved the submitted version.AL wrote the introduction with GL and the chapter on the Analysis of CaThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AML cell lines exhibited distinct glycan fingerprints differing in relevant glycan traits correlating with their cellular phenotype as classified by the FAB system. By implementing transcriptomics data, specific glycosyltransferases and hematopoietic transcription factors were identified, which are candidate drivers of the glycan phenotype of these cells. In conclusion, we report the varying expression of glycan structures across a high number of AML cell lines, including those associated with poor prognosis, identified underlying glycosyltransferases and transcription factors, and provide insights into the regulation of the AML glycan repertoire.Acute myeloid leukemia (AML) is characterized by a dysregulated expansion of poorly differentiated myeloid cells. Although patients are usually treated effectively by chemotherapy, a high rate of relapsed or refractory disease poses a major hurdle in its treatment. Recently, several studies have proposed implications of protein glycosylation in the pathobiology of AML including chemoresistance. Accordingly, associations have been found between specific glycan epitopes and the outcome of the disease. To advance this poorly studied field, we performed an exploratory glycomics study characterizing 21 widely used AML cell lines. Exploiting the benefits of porous graphitized carbon chromatography coupled to tandem mass spectrometry (PGC nano-LC-MS NPM1 or the PML-RARA fusion gene. However, if the AML subtype is not specified otherwise, morphological and phenotypic criteria similar to the FAB classification are still considered.Acute myeloid leukemia (AML) is a genetically heterogeneous disease characterized by clonal expansion of irregularly differentiated cells of the myeloid lineage termed blasts . It is tDespite the ongoing advances in cancer therapy and the general understanding of AML biology, the predominant treatment regimens remained largely unchanged for the past decades . For thoN-glycans (linked to Asn residues) and O-glycans (linked to Ser or Thr residues), as reviewed by Pang et al. [In the last decade, it has been increasingly recognized that AML blasts that reside in the bone marrow create a hostile pro-tumoral microenvironment, which may be responsible for the dysregulation of normal hematopoiesis as well as chemoresistance, immune evasion, and eventually, relapse of the disease ,11,12. Tg et al. .x/a) antigen expression in AML and its direct interaction with E-selectin in the bone marrow niche is intriguing. Reported implications of this interplay include altered homing of leukemic cells as well as regulation of their cellular proliferation and quiescence, respectively [x/a and E-selectin is a major determinant of chemoresistance in AML [Especially, the role of the sialyl Lewis x/a , more than 90 distinct N- and O-glycan structures could be structurally characterized and relatively quantified. We report a comprehensive library of glycans present in common AML cell lines and identify the associated antigens, e.g., T antigen, sLex/a, and α-2,8 sialylation, as a valuable tool for future research. Based on a principal component analysis (PCA), we identified a strong association between the glycomic fingerprint of AML cells and their phenotypic and cytochemical characteristics as classified by the FAB system. In addition, we linked acquired glycomics information to the available transcriptomics data to identify the involved glycosyltransferases (GSTs) and, eventually, gathered evidence for the upstream involvement of key hematopoietic transcription factors (TFs) in AML protein glycosylation.While substantial evidence has been gathered that points towards an essential role of protein glycosylation in AML, e.g., in niche formation and chemoresistance, an exploratory glycomics study identifying and characterizing relevant glycan structures has not been conducted to date. Moreover, associations of AML classes as specified by FAB or WHO and their glycomic fingerprint were hitherto not investigated. In turn, this may provide potential benefits to the further stratification of the disease. Therefore, we set out to thoroughly characterize the strategy  and stat2. Cell lines KG-1, KG-1a, HL-60, PLB985, NB-4, ML-1, OCI-AML2, OCI-AML3, EOL-1, MOLM-13, MOLM-14, MV4-11, THP-1, U937, HEL, HEL 92.1.7, TF-1, and M-07e were cultured in media with 10% FBS , whereas Kasumi-1 and ME-1 were grown in media with 20% FBS and AML193 with 5% FBS. Media for TF-1 and M-07e additionally contained 20 ng·mL−1 granulocyte-macrophage colony-stimulating factor . Cells were washed thoroughly with phosphate-buffered saline before conducting the glycomics analysis.AML cell lines were obtained from the Department of Hematology , Department of Immunopathology—Sanquin Research , and the Department of Biosciences . An overview of used cell lines is listed in N- and O-glycans were analyzed based on polyvinylidene difluoride  membrane-based glycan release workflow using a 96-well plate format, as previously described [−1, guanidine hydrochloride (Thermo Fisher Scientific) to 5.8 mol·L−1, and incubation at 60 °C for 30 min. Subsequently, proteins were washed with water before applying PNGase F  overnight at 37 °C. In this step, 10 ng maltoheptaose DP7  was included as a spiked internal standard to monitor sample preparation and PGC nano-LC-MS2 performance. Released N-glycans were collected, incubated in approximately 6 mmol·L−1 ammonium acetate  for 1.0 h at room temperature, and dried by vacuum centrifugation. N-glycans were reduced by resuspension in 50 mmol·L−1 potassium hydroxide  supplemented with 1 mol·L−1 sodium borohydride (Sigma-Aldrich) at 60 °C for 3.0 h. After the removal of N-glycans from the immobilized proteins, O-glycans were released by reductive β-elimination in a 50 mmol·L−1 KOH solution supplemented with 500 mmol·L−1 NaBH4 at 55 °C for 16.0 h. Likewise, 5 ng maltopentaose DP5 (Elicityl) was introduced in this step as an internal standard for O-glycans. Next, both N- and O-glycans were desalted on a strong cation exchange resin  self-packed into 96-well filter plates  followed by removal of boric acid by co-evaporation with methanol in a vacuum centrifuge. Eventually, released glycans were purified on carbograph material  that was also self-packed into 96-well filter plates. Purified released glycans were dried employing a vacuum centrifuge and resuspended in 10 µL of H2O prior to analysis.escribed . BrieflyN- and O-glycans with α-2,3 neuraminidase S and α-2,3,6,8,9 neuraminidase A , respectively, and subsequent PGC nano-LC-MS2 analysis.Sialic acid linkages were determined by enzymatic digestion of purified N- and O-glycan alditols was conducted on an Ultimate 3000 nano-HPLC (Thermo Fisher Scientific) equipped with a self-packed trap column  and a self-packed separation column  for N-glycans. Separation of O-glycans was conducted on a 0.10 mm i.d. column. In all of the cases, Hypercarb™ KAPPA material (Thermo Fisher Scientific) was used for column packing. Separation was conducted at a flow rate of 600 nL·min−1 and a column oven temperature of 45 °C. For N-glycan analysis, 5.0 µL of sample were injected, whereas for O-glycan analysis, 4.0 µL of sample were injected. The nano-LC system was hyphenated to an amaZon ETD speed ion trap mass spectrometer via a CaptiveSpray ESI source . Isopropanol was used as dopant solvent. For further details, see [Chromatographic separation of released ils, see .1 and MS2 information, general glycobiological knowledge on N- and O-glycan biosynthesis in humans [2 fragment assignments is listed in N-glycans) and 2 (O-glycans). For relative quantification of glycan abundances, the data were converted into the mzml format and quantified employing Skyline 20.2.0.343, taking advantage of the small molecule interface [Glycans were identified based on retention time, acquired MSn humans , as welln humans  and estan humans ,38. If an humans . Compilenterface . GlycansMissing values were imputed by the minimum positive number (0.01) before employing statistical evaluation of the data. PCA was conducted in SIMCA 13.0.3.0. . All of the other data evaluation steps were performed and visualized using the programming language “R”. Moreover, rCCA was conducted employing the mixOmics package . Cell liN- and O-glycans present in AML cell lines appertaining to a broad range of classes, we made use of an established sample preparation protocol relying on PVDF-assisted glycan release in a 96-well format [N- and O-glycans were released from the immobilized proteome, purified, and subjected to in-depth structural characterization and relative quantification by means of PGC nano-LC-MS2. Glycan structures were assigned and quantified as detailed in the Materials and Methods section. Identified glycan structures and their assigned MS2 spectra are supplied as N-glycans) and 2 (O-glycans). Glycans were abbreviated according to their monosaccharide composition: Hexose (H), N-acetylhexosamine (N), fucose (F), N-acetylneuraminic acid (S), phosphorylation (P), and sulfation (Su). Glycan isomers are labeled by letters as suffix, e.g., H5N4S2a and H5N4S2b starting with isomers of earlier retention time. Quantitative information on glycan fractional abundances is summarized in the N- and O-glycans, taking advantage of the well-studied retentive behavior in PGC chromatography [2 analysis. Some sialic acid linkages of more complex multisialylated glycans could be additionally assigned based on matching retention times with well-studied glycans of fetuin that was measured alongside AML glycans [To jointly characterize l format . Sequenttography , and con glycans . HoweverN-glycosylation of AML was assessed in 21 cell lines and revealed a cumulative number of 68 glycans that passed the quality criteria for being considered in a qualitative and quantitative manner. The distribution of these glycans in the respective cell lines is visualized in N-glycan profiles of individual AML cell lines showed a high diversity of structures covering all four main glycan types, namely oligomannose, paucimannose, hybrid, and complex type glycans  obtained for the phenotypically distinct AML cell lines MOLM-13 and TF-1 are depicted in N-glycans were measured simultaneously. As evident from the left panels, in both depicted cell lines the oligomannose structure comprising nine mannoses (H9N2) was the predominant glycan. However, the levels of phosphorylation (7.3%) and paucimannosidics (17.7%) are elevated in MOLM-13 compared to TF-1 . Of note, MOLM-13 cells express three structural isomers of truncated hybrid type glycans  with a cumulative fractional abundance of 2.2% that are absent in TF-1. Concerning complex type glycans (right panels in x/a antigens was clearly upregulated in MOLM-13 cells (4.5%) compared to TF-1 (0.3%). Despite the high abundance of bisected glycans  and TF-1  are shown in x/a and sLex/a antigens were prominent in MOLM-13 (3.9%). H antigen was absent in MOLM-13, yet detected in TF-1 with a fractional abundance of 0.7%. Interestingly, the pronounced expression of α-2,8 sialylation was detected in TF-1 at a fractional abundance of 5.4%.In general, all of the AML cell lines showed high expression of core 1 structures, i.e., sialyl-3/6T antigen and disialyl-T antigen ranging from 26.8% in HL-60 to 82.3% in PLB-985. Additionally, core 2 glycans were identified at a fractional abundance from 17.5% in PLB-985 to 72.0% in HL-60. Core 3 and core 4 structures were not detected. Of note, diverging levels of H antigen expression, fucosylation, sulfation, and extension by LacNAc repeats were found. Mostly α-2,3 sialylation of terminal galactose residues was found next to lower levels of α-2,6 sialylation of the core N-acetylgalactosamine  and extension of sialic acids by α-2,8 sialylation. To illustrate these differences, exemplary EICCs of 2 95% with the exception of MV4-11, which seemed to differ pronouncedly in its glycomic phenotype.To assess whether the glycomic fingerprints of cell lines show associations with their AML class or a specific recurring mutation, we performed unsupervised PCA. Exemplarily, MOLM-14 was included in three biological replicates in the PCA (N- and O-glycomics separately to see if either one displayed pronounced grouping (data not shown). As both independent PCAs showed clustering of cell lines based upon their FAB classification, we continued to evaluate N- and O-glycomics in a combined manner , extension by LacNAc repeats (KG-1: 56.2% and KG-1a: 21.7%), and sLex/a expression (KG-1: 5.0% and KG-1a: 0.4%). Concerning N-glycans, the main drivers were found to be paucimannosidics (KG-1: 10.4% and KG-1a: 4.2%), core fucosylation (KG-1: 18.6% and KG-1a: 33.5%), and (s)Lex/a (KG-1: 0.5% and KG-1a: 4.4%).What stands out in the PCA are the FAB groups M4 (acute myelomonocytic leukemia), M5, and M6 comprising most of the investigated cell lines, which show an apparent separation in the first and second principal component a. AML ceN-glycans (red) and O-glycans (blue), as specified earlier (x/a (O-glycans) expression. On the other hand, M4 and especially M5 cell lines exhibit elevated levels of paucimannosidics, (s)Lex/a (N-glycans), and hybrid type glycans.To gain better insights into what drives the discrimination of different FAB classes, we included—next to individual glycans—glycan types and derived traits from  earlier . On the x/a antigens were detected on both N- and O-glycans throughout all of the FAB classes. Yet, expression was highly variable on O-glycans ranging from 1.1% in M6 to a surprising 12.3% in the M7 cell line M-07e. (s)Lex/a epitope expression on N-glycans varied less with the lowest expression in M3 (0.8%) and the highest in M7 (2.8%). H antigen was absent or of low abundance in most of the FAB classes.As it was indicated from the PCA model that glycomic signatures associate with the phenotypic FAB class, relevant glycan epitopes and their variation across FAB classes were further explored . ImportaO-glycans. In addition to O-glycan features, the M3 type showed unique abundances of N-glycan related features comprising the highest levels of phosphorylation (12.1%) and bisection (4.2%), but lowest levels of paucimannose (4.2%), core fucosylation (16.2%), and (s)Lex/a (0.8%). In the context of sialylation, the highest levels of α-2,6 sialylation of the core GalNAc of O-glycans was found in the M6 subtype (40.0%), which is additionally reflected by the highest abundance of core 1 glycans (62.2%) within all of the FAB classes. Interestingly, α-2,8 sialylation of O-glycans could solely be detected on rather differentiated cell lines (M4 to M7), but was absent from cell lines belonging to the M2 and M3 class. N-glycan associated derived traits such as antennarity and sialylation  were of highest abundance in the M6 subtype  compared to the lowest abundance that was found in M4 cell lines . On top of the evidence presented in Solely, the M3 type showed pronounced H antigen expression with 1.2% fractional abundance on x/a, and H antigen. As GSTs may be rather specific for their substrates, we examined N- and O-glycans separately.Derived glycan traits were further examined by Pearson correlation analysis with transcriptomic data of GSTs obtained from public datasets . A significant correlation was determined for α-2,6 sialylation of core GalNAc in O-glycans: ST6GALNAC1 (r = 0.48) and ST6GALNAC5 (r = 0.52) expression correlated well with this glycosylation trait. α-2,8 Sialylation of terminal sialic acids was associated highly significantly with the expression values of ST8SIA6 (r = 0.96).For α-2,3 sialylation, we detected rather weak correlations in both x/a abundance (N-glycans) was obtained for FUT7 (r = 0.38), although it did not pass the significance threshold of p ≤ 0.05. (s)Lex/a abundance (O-glycans) significantly correlated with FUT9 (r = 0.51) and additionally showed a positive correlation with FUT7 (r = 0.41). Both FUT6 (r = −0.54) and FUT1 (r = −0.54) showed a significant negative correlation with this trait in O-glycans. Lastly, H antigen expression correlated positively with FUT1, yet not significantly.The highest positive correlation of fucosyltransferase expression with (s)LeTAL1, GATA1-3, SPI1, CEPBA, and CBFA2T3. Correlation of N- and O-glycan features with these TFs was assessed by regularized canonical correlation analysis Lex/a, as well as T antigen. On the contrary, TAL1, GATA 1, and GATA2 correlated negatively with these features. In particular, (s)Lex/a on N-glycans correlated highly with most of the hematopoietic TFs investigated: TAL1 (r = −0.42), GATA1 (r = −0.44), GATA2 (r = −0.44), GATA3 (r = −0.46), CBFA2T3 (r = −0.22), CEBPA (r = 0.40), and SPI1 (r = 0.32). A strikingly high correlation was also observed for α-2,8 sialylation on O-glycans. SPI1 and CEBPA correlated highly negatively with this glycomic signature , whereas the other TFs showed highly positive correlation (r = 0.91 for TAL1 and r = 0.89 for GATA1). Additionally, the total levels of sialylation including different linkages on both N-and O-glycans as well as associated traits, e.g., antennarity and complex type glycans correlated positively with GATA2 and CBFA2T3. On the contrary, paucimannose, hybrid, phosphorylation, and α-2,3 sialylation on O-glycans showed rather weak associations with hematopoietic TFs.To determine responsible factors further upstream of the presented glycomics data, we investigated amongst other potential candidates hematopoietic TFs. Based on current literature suggesting their involvement in AML pathobiology ,48,49,50is rCCA, . Based oST8SIA6 with several TFs, e.g., r = 0.69 for TAL1 and r = −0.59 for CEBPA, which were also matching well to the downstream-reported glycan signatures. In addition, FUT7 showed pronounced correlation values ranging from r = −0.50 for TAL1 to r = 0.43 for CEBPA. Potential downstream products, i.e., abundances of (s)Lex/a epitopes on N- and O-glycans followed the trends observed for FUT7 transcript levels. FUT9 mainly showed the same associations as observed for its isoform FUT7, however, associations were less distinct. Although oligomannose and complex type glycans showed a moderate correlation with hematopoietic TFs, key mannosidases MAN1A1 and MAN2A1 showed only weak correlations with the TFs investigated. Additionally, the GSTs MGAT5 and MGAT4A that are responsible for branching of N-glycans showed correlation values of r = 0.42 and r = 0.43 for GATA1, and r = 0.26 and r = 0.27 for GATA2, respectively, which was clearly reflected by the acquired glycomics data. The extent of α-2,3 sialylation on N-glycans followed the same association patterns. In addition, TAL1 and GATA1 showed pronounced associations with ST6GALNAC5 with correlation values of r = 0.75 and r = 0.77, respectively. Out of the three GlcNAc transferases responsible for core 2 synthesis, only GCNT3 showed a major association in the rCCA (r = 0.44 for GATA1). Intriguingly, the expression of ST6GAL1 was highly correlated with most of the TFs, however, the expression values were not well reflected by the glycomics data. In N- and O-glycomics, corresponding GSTs, and correlated hematopoietic TFs for the glycomic-wise distinct subtypes M5 and M6.Next, GST expression was included to assess correlations with said hematopoietic TFs  and to vN- and O-glycome of 21 widely used AML cell lines that cover most of the genetic and phenotypical diversity encountered in AML. Exploiting the major advantages of the PGC nano-LC-MS2 platform, namely its excellent separation power for glycan isomers and the in-depth structural characterization provided by fragmentation in negative ion mode, we assessed a plethora of glycan species and obtained quantitative information.In this study, we explored the N-glycome of AML cell lines. As illustrated in N-glycan types  could be identified in all of the cell lines, albeit in drastically varying abundances. Oligomannose type structures were of high abundance in all of the AML cell lines (average abundance of 54.4%). The high prevalence of this glycan type was previously observed for a small number of AML cell lines [N-glycome was the strong expression of paucimannosidics  [ST8SIA4 and suggested N-glycans as the dominant effectors [ST8SIA6 in chemo-sensitive AML cell lines.Contrary to the high complexity across different cell lines observed for ructures . In tota45-01A1) . In thisffectors . NotablyN- and O-glycan data were combined and evaluated by means of PCA Lex/a antigen was abundantly expressed in all of the investigated AML cell lines. However, the two isomers (s)Lex and (s)Lea, which are characterized by the α-1,4 linkage and the α-1,3 linkage of the fucose, respectively, cannot be easily discriminated. Therefore, we performed correlation analyses to see whether their expression correlates with FUT3 ((s)Lea) or FUT4-7, 9 ((s)Lex), as depicted in FUT3 and (s)Lex/a epitopes present on N- and O-glycans, we assume that the reported glycans mainly carry the (s)Lex isomer. This is further supported by the positive correlation of (s)Lex/a expression with FUT7 in the N-glycan derived trait and with FUT7, as well as FUT9 in the O-glycan derived trait.The sLerognosis ,61. As dx expression on AML cells was to date only studied by specific monoclonal antibodies and subsequent flow cytometric analysis [x was expressed throughout all of the AML cell lines investigated albeit in varying abundance Lex epitopes were present on both N-glycans as well as O-glycans further complicating the picture as both types of protein glycosylation may be responsible for its reported implications.To our knowledge, sLeanalysis ,23,62. Ibundance . In partx  in AML is intriguing: The expression of sLex appears to be a feature of the low differentiation status of AML blasts. Upon the induced differentiation of the AML cell line THP-1, the expression of sLex antigens on its surface was lost [x epitope, however, was reported to be largely unaltered. Furthermore, a recent report by Barbier et al. could demonstrate that AML blasts induce an upregulation of E-selectin expression in cells of their microenvironment [x with these receptors, pro-survival pathways, i.e., AKT/NF-κB, were activated leading to enhanced chemoresistance of these leukemic blasts. Disruption of said interaction in the AML niche by the glycomimetic drug Uproleselan or genetic deletion of E-selectin in a murine model reportedly abolished this effect. In addition, they could show that disruption of E-selectin binding reduced the proportion of quiescent AML blasts significantly. Additionally, high expression of sLex associated GST FUT7 and ST3GAL4 has already been linked to a dismal prognosis in AML patients [x is expressed in a wide variety of different AML subtypes as specified by FAB or WHO classification, though to a different extent. Therefore, targeting chemoresistance by interruption of the sLex—E-selectin axis may prove an efficient mode of action in fighting AML and is currently investigated in clinical trials [x epitope expression may shape the pathology of AML in a profound manner.The role of sLewas lost . The Lexironment . Upon inpatients . Our datl trials . Taking TAL1, GATA1-3, SPI1, CEBPA, and CBFA2T3 were found to significantly associate with several glycan features as well as upstream GST expression, as assessed by rCCA  [TAL1, GATA1-2, and CBFA2T3, a set required for the maintenance of both HSC and leukemic stem cells (LSCs) [SPI1/CEBPA. Moreover, this set of TFs was found to be substantially enriched in M6 and M7 cell lines in our dataset  . The lows (LSCs) ,48,49 we dataset . GATA3 sFUT7 expression correlated highly negatively with SPI1/CEBPA and concomitantly correlated negatively with the observed (s)Lex/a expression in AML cell lines. TAL1, GATA1-3, and CBFA2T3 showed opposing correlations with FUT7, again supported by the downstream abundance of glycan (s)Lex/a antigens. In addition, ST8SIA6 correlated highly positively with TAL1 and negatively with CEBPA/SPI1, reflected concurrently by the abundance of this epitope in the glycomics data.To corroborate our findings, we included rCCA of said TFs with the expression of relevant GSTs that relate to these altered glycan traits. As anticipated from the analysis of downstream glycan traits, GST expression showed a high correlation with hematopoietic TFs. For instance, N-glycans, i.e., antennarity, complex type, total sialylation, and α-2,3 sialylation, were grouped closely in the hierarchical clustering and correlated positively with GATA2 and CBFA2T3. Concomitantly, we detected positive associations with the expression of branching enzymes MGAT5 and MGAT4A. These traits associated with invasiveness and metastasis in many cancers [GATA2 as well as GATA3 expression after induced hypomethylation by hypoxia or hypomethylating agents in ovarian and breast cancer [GATA2-3 to elevated levels of MGAT5 and ST3GAL4 and reported downstream changes on the glycan level. While we could substantiate the findings for GATA2 in our study, we did not observe clear associations of GATA3 with extensive branching of N-glycans in the case of AML. Considering these findings, hematopoietic TFs may substantially regulate the differentiation status of AML blasts alongside their cytochemical characteristics as classified by FAB and, thus, may substantially shape their glycomic phenotype.Furthermore, traits linked to increased sialylation and branching of  cancers  have beet cancer ,66. NotaN- and O-glycan biosynthetic pathways including corresponding GSTs and hematopoietic TFs were compiled  were described that carry the (s)Lex/a antigen, which has profound implications in chemoresistance, metastasis, and immunomodulation in AML, and is currently of high interest with several clinical trials registered. In this regard, striking differences in the expression levels of these cancer-associated antigens across different FAB subtypes could be reported. By integrating our glycomics data with transcriptomics data from public repositories, we could propose the involvement of specific GSTs in the expression of certain glycan epitopes. Eventually, we provide evidence for the upstream involvement of hematopoietic TFs in the glycosylation machinery that are both found severely dysregulated in AML.Although a lot of evidence for the involvement of aberrant protein glycosylation in AML has been gathered, a global exploratory study on the


Therefore, the current review aimed to fill the gaps between elevated Ca2+ levels and the pathogenesis of AD. Specifically, we mainly focus on the molecular mechanisms by which Ca2+ affects the neuronal networks of neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, and autophagy. Furthermore, the roles of Ca2+ transporters located in the cell membrane, endoplasmic reticulum (ER), mitochondria and lysosome in mediating the effects of Ca2+ on activating neuronal networks that ultimately contribute to the development and progression of AD are discussed. Finally, the drug candidates derived from herbs used as food or seasoning in Chinese daily life are summarized to provide a theoretical basis for improving the clinical treatment of AD.Alzheimer’s disease (AD) is a neurodegenerative disease that is characterized by the production and deposition of β-amyloid protein (Aβ) and hyperphosphorylated tau, leading to the formation of β-amyloid plaques (APs) and neurofibrillary tangles (NFTs). Although calcium ions (Ca As an antagonist of NMDAR, a Ca2+ transporter on the surface of the nerve cell membrane, memantine significantly inhibits Ca2+ influx and was the first Food and Drug Administration (FDA)-approved drug for the treatment of moderate to severe AD in patients  is a hub protein that interacts with phosphorylated tau, Aβ, and γ-secretase, and it contributes to their toxic effects on triggering cell death and potentially leading to the dementia that is a characteristic of AD -1β and tumor necrosis factor α (TNF-α) via calcineurin (CaN) in glial cells -activated microglia stimulates the production of proinflammatory cytokines, such as TNF-α and IL-6  by bepridil, nitrendipine or nimodipine attenuates neuroinflammation by deactivating astrocytes and microglial cells in LPS-stimulated or artificial cerebrospinal fluid (aCSF)-injected (i.c.v.) rats and astrocytes from the CA1 region of the hippocampus  induce cell apoptosis through Ca2+ release-activated Ca2+  increases the production of cytokines in SOD12+ balance via their transporters. For example, TNF-α, IL-1β, and IFNγ increase the influx of Ca2+ into microglial cells, which indicates crosstalk between Ca2+ and neuroinflammatory factors in cultured hippocampal neurons  neurons  in IL-1β-stimulated OSCC cells  via the JAK/CaN pathways  mRNAs and proteins  and downregulating mitofusin 1/2 (Mfn1/2) and dynamin-like GTPase (OPA-1) in primary cultures of mouse cerebral cortical neurons  into neurons in adult rats  mice  and simultaneously induces neurogenesis during early cortical column development . CaSR activation in vivo induces the homing of granule cell precursors during differentiation, mainly through CaSR interactions with integrin complexes , S47445, a novel AMPAR-positive allosteric modulator (AMPA-PAM), exerted significant neurogenic effects on the proliferation, survival and maturation of new hippocampal neurons  into neural cells is markedly inhibited by regulating the levels of the distal C-terminus (DCT) upon treatment with nimodipine and knock down of Cav1.2 expression  and require connexin hemichannels, P2Y1 ATP receptors, and intracellular InsP3-mediated Ca2+ release, suggesting critical roles for radial glial signaling mechanisms in cortical neuronal production  through TRPC1-deactivating Ca2+-dependent mechanisms -induced autophagy flux in neonatal rat ventricular myocytes (NRVMs) and HeLa cells , Aconitum, Cablin, dandelion and Astragalus, plantule of Nelumbo nucifera, Salvia miltiorrhiza, Radix Salvia miltiorrhiza, Stephania tetrandra, Cnidium monnieri, and Carthamus tinctorius L., respectively, inhibit Ca2+ influx by deactivating Ca2+ transporters on the cell membrane, such as L-type Ca2+ channels, VDCC, G protein-coupled receptors, TRPCs, and TRPVs in different animal and cell models , derived from herbs, such as Cephalotaxus fortunei, magnolia tree, Polygonum cuspidatum, and Ginkgo biloba, respectively, modulate Ca2+ homeostasis by regulating the activities of transporters located in the ER through mechanism partially dependent on SOCE or mitochondria (Matsubara et al., 2+ transporters to restore Ca2+ concentrations (Although several FDA-approved chemical drugs are currently available for treating AD, the identification of new compounds targeting Ca animals . For exatrations .2+ concentrations are increased in the cytosol of neuronal cells via transportation from the extracellular space and intracellular stores through transporter-dependent mechanisms. Ca2+ accumulation in neuronal cells induces the production and deposition of Aβ and hyperphosphorylated tau in APs and NFTs, leading to impaired learning ability in patients with AD. Moreover, transporters in the cell membrane, endoplasmic reticulum, mitochondria, and lysosomal membranes are critical for mediating the effects of Ca2+ on neuroinflammation, neuronal injury, neurogenesis, neurotoxicity, neuroprotection, autophagy, and synaptic plasticity, which contribute to the cognitive decline associated with AD (2+ transporters. Moreover, Ca2+ transporters are progressively becoming new therapeutic targets for treating AD.During the development and progression of AD, Ca with AD . Based oP-PG and L-LC contributed to conceptualizing and drafting the manuscript. YY contributed to summarizing the data presented in The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The second most common childhood leukemia, acute myeloid leukemia (AML), is a heterogeneous disease with a poor prognosis. In order to improve outcomes, efforts to understand the genomic/transcriptomic landscape of AML have been performed. However, there is a significant gap in our understanding of leukemic cell proteomics. In comparison to the proteome, the transcriptome alone cannot adequately represent the biological functions within cells and it is often not a target for immediate drug development. In the current study, we investigated cytogenetic differences at the proteomic level and sought potential predictive biomarkers and druggable proteins.THY1 (CD90), NEBL, CTSF, COL2A1, CAT, MGLL (MAGL), MACROH2A2, CLIP2 (isoform 1 and 2), ANPEP (CD13), MMP14, and AK5.Acute Myeloid Leukemia (AML) is a heterogeneous disease with several recurrent cytogenetic abnormalities. Despite genomics and transcriptomics profiling efforts to understand AML’s heterogeneity, studies focused on the proteomic profiles associated with pediatric AML cytogenetic features remain limited. Furthermore, the majority of biological functions within cells are operated by proteins  and most drugs target the proteome rather than the genome or transcriptome, thus, highlighting the significance of studying proteomics. Here, we present our results from a pilot study investigating global proteomic profiles of leukemic cells obtained at diagnosis from 16 pediatric AML patients using a robust TMT-LC/LC-MS/MS platform. The proteome profiles were compared among patients with or without core binding factor (CBF) translocation indicated by a t or inv(16) cytogenetic abnormality, minimal residual disease status at the end of the first cycle of chemotherapy (MRD1), and in vitro chemosensitivity of leukemic cells to cytarabine (Ara-C LC50). Our results established proteomic differences between CBF and non-CBF AML subtypes, providing insights to AML subtypes physiology, and identified potential druggable proteome targets such as  RUNX1/RUNX1T1 and CBFβ/MYH11 fusions, respectively, account for approximately 30% of pediatric AML  as described previously [Gene expression profiling of bone marrow leukemic blasts at diagnosis was obtained using the GeneChipeviously . The MASeviously .Differential protein expression analysis was analyzed in R studio software  with Bioconductor packages. Normalized proteomic data for each patient sample were grouped for categorical analysis in three comparisons: (1) CBF status (CBF vs. non-CBF AML); (2) MRD1 status (positive vs. negative); and (3) Ara-C LC50 status . Herein, differential protein expression for each comparison was performed using rank-sum analysis for unpaired cases via the “RankProd 2.0” package, a non-parametric method for identifying differentially expressed omics datasets . A thresIntegrative analysis among three differential comparisons  were executed in order to identify proteins with significant prognoses and/or potential drug targets.https://cytoscape.org/ accessed on 13 May 2021, version 3.8.2,) was employed to visualize the PPI network relationship between significant DEPs with exclusion of singleton interactions [To investigate the biological functions and processes which are different between CBF and non-CBF AML, we utilized various publicly accessible databases. First, STRING database version 11.0 was utilized to construct an entire PPI network of significant DEPs , includiractions .Subsequently, we used ClueGO version 2.5.7, a Cytoscape plug-in, to visualize the non-redundant biological terms utilizing three signature databases  [GSEA was achieved using pre-ranked scores from proteins, with Log2FC indicating the direction of enrichment and -values) . The def-values)  was usedp-value < 0.05) at a global level and on significant DEPs of three differential comparisons. For genes that have multiple protein–transcript combinations , only protein–transcript pairs with the lowest p-value for that gene were selected in the final correlation estimation.Within-gene pairwise correlations of observed transcripts and proteins expression were conducted for the 15 samples with paired gene expression profiles. The Spearman’s rank correlation coefficient was calculated between proteome and transcriptome with a matched gene symbol among 15 samples. A correlation estimate percentage was then computed for negative correlation, positive correlation, and significant correlation . Following additional quality control, a total of 9800 annotated proteins per sample were included for downstream analysis.TPPP3, CRIP2, SLC9A3R2, and VAMP5 in CBF AML, and upregulated proteins MGLL, MYT1L, CAT, TESC, CLIP2, and CS in non-CBF AML.Comparing proteomic profiles of CBF to non-CBF AML resulted in 117 significant DEPs  with 51 positive and 66 negative Log2FC as shown in the volcano plot A. Fifty-p-value = 1.33 × 10−15) (The STRING database was used in conjunction with Cytoscape in order to construct a protein–protein interaction (PPI) network on 117 significant DEPs in order to identify significant PPIs. STRING analysis resulted in 112 nodes with more interactions than expected with at least one direct or indirect association (115 connected edges vs. 49 expected edges with an average node degree of 2.05 and PPI enrichment × 10−15) A. ClueGO× 10−15) B. ClueGO× 10−15) C.−5 and grouped KEGG term network FDR = 1.8 × 10−9)  . InteresAdditionally, the top gene-sets enriched in CBF AML by GSEA using the GO , Reactome, and KEGG databases resulted in common protein functions particularly linked to oxidative phosphorylation, RNA synthesis/processing, and multiple pathways with metabolic processes. On the other hand, gene-sets enriched in non-CBF AML revealed common functions in the regulation of transport, membrane trafficking, cellular protein catabolic process, and myeloid leukocyte mediated immunity . The fulp-value < 0.05, |Log2FC| > 1), with 17 upregulated and 48 downregulated proteins in MRD1-positive AML , MACROH2A2, NEBL, MGLL (MAGL), HLA-DPA1, HLA-DPB1, CAT, TTC41P, ICA1, TBC1D8B, GOLGA8K, THY1 (CD90), HOXC10, HOXD10, and SNRPE.Since most proteins are druggable targets, we were interested in investigating the proteomic profile between MRD1-positive and negative patients, as well as proteomic feature profile between LC50-high vs. low. One limitation to be noted is that only 1/6 of the CBF-AML was MRD1-positive and 7/10 non-CBF AML cases were MRD1-positive. Thus, we expect some overlap in the proteomic profiles between MRD1 status and CBF status evaluations. Comparing the proteomic profile of MRD1-positive and negative status among 16 patients revealed 65 significant DEPs  with three upregulated and 21 downregulated proteins in high Ara-C LC50 group . Comparison of proteomic profiles between leukemic cells from patients with high vs. low Ara-C LC50 level by median value (0.29 ng/μL) identified 24 significant DEPs  were upregulated in the non-CBF and overexpressed in the MRD1-positive group. Moreover, IGSF6, RBPMS, HLA-B, and PRG3 are also in concordance with downregulation in both MRD1 and Ara-C LC50 differential comparisons.Given the superior prognostic nature of CBF compared to non-CBF AML subtypes, we observed the concordance of dysregulated direction between CBF status with MRD1-positive and high Ara-C LC50 level. The common significant DEPs of three comparisons included p-value for all protein–transcript pairs, only 13.4% (n = 942/7039) demonstrated a significant correlation  (p < 0.05) in only 9.3% (12/129) of the mRNA-protein pairs.  A in leukn pairs. B. Among CRIP2. Overexpression of CRIP2 in CBF AML was previously reported to be highly overexpressed in leukemic stem cells (LSC) and is associated with chemotherapy resistance in AML in response to EZH2 inactivation [CRIP2 promotes oxidative phosphorylation and regresses glycolysis, which stabilizes metabolic features characteristic for AML stem cells [SLC9A3R2 in CBF AML has previously been shown to be upregulated at relapse [TESC also shown to be highly expressed in FLT3-ITD(+) AML mediating sorafenib resistance [CLIP2 (both isoform 1 and 2) in non-CBF was reported for poor progression in upregulating at the transcriptome level in large AML multi-studies [CLIP2 was also shown to be upregulated in an inferior clinical endpoint with MRD1-positive.Despite the growing interest in investigating proteins as functional molecules in cells, no global proteomic studies of leukemic cells in children with AML studies are currently available. In this study, we reported the proteomic profiles of pediatric bone marrow-derived AML cells at diagnosis in three differential comparison strategies: (1) core binding factor , inv(16)) AML vs. non-CBF AML; (2) MRD1-positive vs. MRD1-negative status; and (3) in vitro chemosensitivity of Ara-C LC50-high vs. low level. Within the first comparison, we identified a total of 117 proteins that were dysregulated between CBF AML and non-CBF AML subtypes. Interestingly, several significant DEPs have shown to be of relevance to AML at the transcriptome level such as tivation . Moreove;21, inv, while non-CBF AML has protein features of later progenitor with colony-forming unit, megakaryocyte (CFU-MK) and megakaryocyte.Using pathway analysis to compare CBF and non-CBF AML proteomic profiles revealed involvement in hematopoietic cell lineage development, platelet activation/signaling/aggregation, and endothelial cell proliferation. Between CBF and non-CBF AML, the KEGG pathway within Pathview showed distinct hematopoietic stem cell (HSC) profiles with unique biomarkers. Thus, this validated the observed biological functions of feration . In lineCAT, MGLL (MAGL), MACROH2A2, and CLIP2 (both isoform 1 and 2). Of these, CAT represents catalase, a key antioxidant enzyme which has been implicated in resistance to antileukemic agents such as doxorubicin [MGLL  represents monoglyceride lipase, which has been shown to be overexpressed in aggressive cancers. Given its involvement in oncogenic lipid signaling, MGLL represents an exciting pharmacological target and there are ongoing efforts to identify the inhibitors of MAGL [MACROH2A2,  is a fusion partner of MECOM in AML [CAT, MGLL (MAGL), MACROH2A2, and CLIP2 (both isoform1 and isoform2) in non-CBF AML were also significantly upregulated in MRD1-positive samples, indicating possible noteworthy prognostic factors and druggable targets that would require further evaluation. Other significant DEPs among MRD1-positive and the Ara-C LC50-high level that have been shown to be relevant to AML were identified in this study. The overexpression of THY1  in MRD1-positive was reported to be preferentially expressed on blast cells of high-risk AML, and CD90+ AML has been associated with shorter patient survival [THY1 protein could carry potential prognostic significance in pediatric AML. Remarkably, the overexpression of NEBL in MRD1-positive was shown to be involved in MLL rearrangement, which is the most frequently identified genetic aberration in early infancy AML [CTSF was shown to have a function in azurophil granules and was found to be overexpressed in acute promyelocytic leukemia (APL) FLT3-ITD [GATM was reported to be associated with increased reactive oxygen species within mitochondria, and found to enriched in chemo-resistant leukemic cells to Ara-C in vitro [COL2A1 was studied to be highly correlated to granulocyte–monocyte progenitor (GMP), which is closely associated with AML [The proteins that were overexpressed in non-CBF and in MRD1-positive included orubicin , and MGL of MAGL ,43,44,45M in AML . Collectsurvival . Hence, ancy AML . Among tFLT3-ITD . GATM wawith AML . TogetheANPEP , MMP14, and AK5. ANPEP (CD13) as a potential target in AML is being investigated in a bispecific, and split CAR-T cells targeting CD13 and TIM3 as CD13 was shown to be overexpressed in AML cells [MMP14 is in the matrix metalloproteinase family and is involved in the breakdown of the extracellular matrix in normal physiological processes. MMP inhibitor (MMPI) is also being investigated in the context of AML, as MMPI reduces AML growth, prevents stem cell loss, and improves chemotherapy effectiveness [AK5 is a member of adenylate kinase family that was previous shown to be highly expressed in leukemia stem cells (LSCs) compared to human hematopoietic stem cells (HSCs), indicating potential therapeutic targets for quiescent and chemotherapy-resistant human LSCs [ANPEP (CD13), MMP14, and AK5 are overexpressed in non-CBF compared to CBF AML and could be interesting drug targets. However, they are underexpressed in both MRD1-positive and Ara-C LC50-high level, suggesting further studies are needed to investigate this phenomenon.Integrative analysis of significant DEPs among three comparisons with cytogenetics such as CBF, MRD1, and Ara-C LC50 clinical endpoints, revealed 100% concordance between cytogenetic features and clinical endpoints in our results. This suggested that the difference in prognosis between AML subtypes based on CBF status could also be explained at the proteome level. The three proteins that were significantly identified among three differential strategies included ML cells . MMP14 itiveness . Next, Aman LSCs . OverallFinally, the significant correlation between the proteome and transcriptome within 15 samples was lower than expected, implying that changes in the transcriptome only resulted in minor or transient changes in the proteome of leukemic cells. However, this could be due to the small sample size, a limitation in our pilot study.In conclusion, we report the first global untargeted proteomic analysis of leukemic cells in pediatric AML, leading to the validation of biological function between CBF and non-CBF AML subtypes and new insights into AML pathobiology and potential druggable targets. This pilot study also demonstrates the high feasibility and importance of global proteomic analysis in pediatric AML. To further support these results, we are currently expanding our proteomic research in a larger cohort.