Diabetes is the ninth leading cause of death, with an estimated 1.5 million deaths worldwide. Type 2 diabetes (T2D) results from the body’s ineffective use of insulin and is largely the result of excess body weight and physical inactivity. T2D increases the risk of cardiovascular diseases, retinopathy, and kidney failure by two-to three-fold. Hyperglycemia, as a hallmark of diabetes, acts as a potent stimulator of inflammatory condition by activating endothelial cells and by dysregulating monocyte activation. G-protein couple receptors (GPCRs) can both exacerbate and promote inflammatory resolution. Genome-wide association studies (GWAS) indicate that GPCRs are differentially regulated in inflammatory and vessel cells from diabetic patients. However, most of these GPCRs are orphan receptors, for which the mechanism of action in diabetes is unknown. Our data indicated that orphan GPCR26 is downregulated in the PBMC isolated from T2D patients. In contrast, GPR26 was initially upregulated in human monocytes and PBMC treated with high glucose (HG) levels and then decreased upon chronic and prolonged HG exposure. GPR26 levels were decreased in T2D patients treated with insulin compared to non-insulin treated patients. Moreover, GPR26 inversely correlated with the BMI and the HbA1c of diabetic compared to non-diabetic patients. Knockdown of GPR26 enhanced monocyte ROS production, MAPK signaling, pro-inflammatory activation, monocyte adhesion to ECs, and enhanced the activity of Caspase 3, a pro-apoptotic molecule. The same mechanisms were activated by HG and exacerbated when GPR26 was knocked down. Hence, our data indicated that GPR26 is initially activated to protect monocytes from HG and is inhibited under chronic hyperglycemic conditions. Diabetes mellitus (DM) is a chronic, metabolic disease characterized by elevated levels of blood glucose (hyperglycaemia). Type 2 diabetes (T2D) is the most common type of diabetes, accounting for approximately 90% of all cases of DM [es of DM . Hypergles of DM .Inflammation plays a crucial role in the pathogenesis of T2D and its complications. Vascular cells, especially immune cells, such as lymphocytes and monocytes, are involved in the development of vascular inflammation, and promote endothelial activation and monocyte adhesion to the endothelium, sustaining diabetic vascular complications ,5. Despiβ-cell dysfunction [Cellular receptors are fine-tuned regulators of cell response to environmental changes via translating extracellular signals into intracellular activation cascades. The failure of a physiological process, such as the receptor-derived physiological processes in monocytes, dysregulates their function and mediates monocyte pro-inflammatory activation . G-protefunction , receivifunction . Since ofunction ,16,22. Tfunction , GPR55 [function ,24, and function . Howeverfunction ,23,24,25function .GWAS studies on single nucleotides polymorphisms and microarrays data from blood or peripheral blood mononuclear cells (PBMC) samples collected from diabetic patients (T1D and T2D)  were used to identify GPCRs differentially expressed and related to diabetes . Among ahttps://apps.who.int/iris/handle/10665/70523, accessed on 1 June 2022). To exclude any intervening condition due to background diseases, all of participants were examined for T1D, liver and kidney dysfunction, auto-immune diseases, current or prior cancers, acute or chronic inflammatory diseases, and any other unrelated disease. In addition, pregnant, smoking individuals and patients who were receiving immunosuppressive or hormone-containing drugs were excluded from the study. The healthy donors showed normal FBS and HbA1C values and did not report a T2D history, autoimmune disease, obesity, or any other chronic disease. This group was matched to diabetic cases based on gender, age, and ethnicity. Human PBMC were obtained from volunteers following an oral and written informed consent. Institutional approval was obtained from the Ethics Committees of Tarbiat Modares University (IR.MODARES.REC.1397.109). The study was conducted in accordance with the ethical guidelines of the Helsinki declaration.Whole blood samples were obtained from 32 diabetic patients and 32 healthy donors referred to the outpatient clinic of Tehran Heart Center, Iran. The diagnosis of T2D was based on WHO criteria, and diabetic patients were selected based on standard criteria (fasting blood glucose (FBS)), N 125 mg/dL (6.9 mmol/L), and HbA1c N 6.5% (47 mmol/mol) . HbA1c was quantified using an enzymatic method according to the manufacturer’s instructions .g for 30 min at room temperature with no brake. Then, the PBMC layer was collected, washed with cold PBS, and centrifuged 10 min at 400× g (4 °C). The supernatant was discarded, and a wash step was repeated with PBS. PBMCs were finally collected and used for downstream analyses.PBMCs were isolated from fresh whole blood samples by density gradient using Ficoll Histopaque . In detail, 2 mL of Ficoll Histopaque per 1 mL of blood were placed in a 50 mL conical centrifuge tube. Fifteen mL of anticoagulated blood was diluted with an equal volume of Phosphate-buffered saline (PBS). The diluted blood was layered over the Ficoll Hypaque solution gently. Gradients were centrifuged at 400× −ΔCt. Primer efficiencies for the tested genes were comparable to those for reference genes. Primer sequences are reported in Total RNA was extracted from PBMCs using TRIzol Reagent  according to the manufacturer’s instructions. One µg of RNA was reverse-transcribed with a high-capacity cDNA reverse transcription kit according to the manufacturer’s instruction . Gene expression was evaluated by qPCR using the GoTaq qPCR master mix . All samples were tested at least in duplicate . The relative gene expression analysis of GPR26 was according to the Livak method . Express2. CTHP-1 were exposed for 24, 48, and 72 h to media containing either 5 mM D-glucose , or 25 mM D-glucose  to simulate a condition closed to the diabetic hyperglycemia. PMBCs were treated similarly for 24 and 48 h. Cell viability in each experiment was >90%, as indicated by trypan blue staining. THP-1 and PBMC total RNA was isolated as described above.PBMCs for cell culture experiments were obtained from additional 12 healthy volunteers after giving oral and written informed consent. Human monocyte cells  were cultured in a Roswell Park Memorial Institute 1640 medium  supplemented with 2 mM L-glutamin, 1% penicillin/streptomycin, and 10% fetal bovine serum (Gibco), and incubated at 37 °C in a humidified atmosphere containing 5% COTHP-1 were chemically transfected , Termofisher Scientific) for 24, 48, and 72 h with LNA single-stranded antisense oligonucleotides  designed for highly effective, strand-specific, knockdown of GPR26 mRNA, or with scrambled controls, alone or in combination with NG or HG. RNA isolation and cDNA synthesis were performed as mentioned before. Efficiency of GPR26 Gapmers was validated by qPCR and western blot. All data belong to at least three biological replicates.Whole proteins were extracted from THP-1 using a RIPA lysis buffer  containing phosphatase and proteinase inhibitor cocktails . The protein content was determined using the Pierce BCA Protein Assay Kit . Twenty µg of total proteins were loaded on a 4–12% precast polyacrylamide gel  and electro-transferred onto polyvinylidene difluoride (PVDF) 0.45 µm membranes according to the manufacturer’s instructions. Proteins were incubated with antibodies indicated in the ® 200 PRO, Tecan, Männedorf, Switzerland). ROS levels were expressed as DCFH-DA relative fluorescence intensity and reported as fold change of the control.To examine the possible role of GPR26 in generation of ROS, its amount was measured using the OxiSelect Intracellular ROS Assay Kit  based on the manufacturer’s instructions. Briefly, THP-1 cells were transfected with GPR26 GapmeRs and exposed to NG or HG as indicated before. Then, cells were incubated with 2′, 7′-Dichlorodihydrofluorescin diacetate (DCFH-DA) during the last 30 min of treatment. At the end of the incubation, cells were washed three times with PBS to remove any DCFH-DA excess. Fluorescence was analyzed at 485 nm excitation/530 nm emission using a fluorescent plate reader . Luminescence is proportional to the amount of caspase activity present. Luminescence was measured in a plate luminometer . Six technical replicates per condition were performed for each of the 3 biologically independent experiments.THP-1 cells were cultured and transfected with GPR26 GapmeRs as mentioned above. To determine the activity of caspase-3, cells were incubated at 37 °C with a luminogenic caspase-3 substrate containing the tetrapeptide sequence DEVD  for 45 min as previously described [THP-1 were transfected with GPR26 GapmeRs for 24, 48 and 72 h. At the end of the experiment, cells were labeled with 5 μM/L × 10escribed , with soTHP-1 monocytes were plated on 8-well chamber slides (Ibidi) and treated as described before. Cells were fixed using the ViewRNA Cell Plus Assay , and incubated with antibodies against GPR26  and β-catenin . Proteins were detected using appropriate fluorescently labeled secondary antibodies feasible for 3D confocal microscopy, such as STAR-488-conjugated anti-rabbit IgG antibody , and Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H+L) . Cells were embedded in Vectashield Antifade Mounting Medium with DAPI  to counterstain nuclei.For detailed GPR26 localization studies, three-dimensional confocal laser scanning microscopy (CLSM) was performed with a Leica SP8 3X microscope equipped with a 63×/1.40 (Leica) oil immersion objective. Optical zoom was used where applicable. A UV laser (405 nm) was used for excitation of DAPI. A tunable white light laser for selective excitation of Star635P and Cy3 fluorochromes was used for the detection of GPR26 and β-catenin, respectively. Time gating of the detected emission signals (0.8–6.0 ns) was used for all STED channels to enhance image resolution. All data were acquired in three dimensions and voxel size was determined according to Nyquist sampling criterion. Image reconstructions were performed using the LAS X software package v.3.0.2 (Leica) and deconvolution was performed combining the Huygens Professional software package v.19.10  using the unsupervised CLSM algorithms.GPR26 quantification and cell localization were performed using the Imaris 8.4.2 software equipped with the imaging processing toolbox MATLAB. Spatial distribution of automatically calculated GPR26 voxels  was defined according to the voxel localization to the cell surface, which corresponded to the membrane surface calculated using the spatial distribution of the β-catenin . A voxel value of 0.5 µm was used as threshold to discriminate membrane versus cytosolic GPR26. Multiple wells per experimental conditions were performed in independent experiments. The analysis of the staining was performed in a blinded manner.t test. Not normally distributed variables were compared by the Mann–Whitney test. Analysis of variance (ANOVA) was used to determine the significant differences between more than two groups. The correlation between the expression levels of GPR26 and the clinical features of patients were assessed by Spearman or Pearson tests based on the normality test result. In all cases, p values < 0.05 were considered significant.Statistical analysis was performed using GraphPad Prism 9.3.1 . Data were initially evaluated for normal distribution with the Shapiro-Wilk test. Comparisons between two groups with normally distributed variables were analyzed by Student’s unpaired The raw data are available in the Diabetes mellitus is a chronic, metabolic disease characterized by elevated blood glucose levels, which leads to additional chronic complications, such as cardiovascular, inflammatory, and kidney diseases [https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death, accessed on 1 June 2022). In Iran, diabetes is the 6th leading cause of death, with a death rate of 27.47 per 100.000 of population . Genome-wide association studies (GWAS) provided evidence that occurrence of certain single nucleotide polymorphisms (SNPs) in GPCRs are associated with an increased risk for diabetes and cardiovascular complications [diseases . Diabeteications . Hence, ications   in diabetic patients  and healthy donors (r = −0.35 p = 0.04)  . Taken tWe analyzed the expression of GPR26 in human cultured monocytes treated with different doses of glucose to simulate a hyperglycemic condition and to study GPR26 functional role. Accordingly, THP-1 cells were treated with 5 mmol/L  or 25 mmol/L  of glucose for 24, 48, and 72 h. Interestingly, treatment of THP-1 with HG increased the expression of GPR26 a. The prTo corroborate the data, human PBMC were isolated from healthy donors and treated with NG or HG for 24 and 48 h. Accordingly, we found that GPR26 expression was increased only at 24 but not at 48 h of HG exposure b. AccordNext, we analyzed GPR26 cellular localization and relative levels to test whether HG affected GPR26 exposure on the monocyte membrane surface. THP-1 were treated with NG or HG for 24, 48, and 72 h and stained for GPR26 and β-catenin as membrane marker. Immunofluorescence analysis 3D z-scan confocal images confirmed that HG increased whole GPR26 levels at 24, 48 but not at 72 h c. The nuTaken together, these data suggested that GPR26 might be activated to counteract the deleterious effects promoted by HG, which in turn impaired GPR26-related protective effect by inhibiting its membrane localization on the monocytes from T2D patients.To study the functional role of GPR26, THP-1 cells cultured in NG or HG were transfected for 24, 48, and 72 h with 50 nM of LNA single-stranded antisense oligonucleotides to knockdown GPR26, or with scrambled controls. GPR26 knockdown was confirmed by the analysis of GPR26 at mRNA and protein levels at all time points a. TransfAccumulating evidence indicates that high levels of glucose promote dysfunctional monocyte activation, characterized by an increased inflammatory and apoptotic activity, partly by enhancing the production and accumulation of reactive oxygen species (ROS). To study the role of GPR26 on HG-mediated production of ROS, GPR26 was knocked down in THP-1 cells treated with NG or HG for 24, 48, and 72 h. Accordingly, we confirmed that HG increased ROS levels in HG-induced THP-1 cells compared to NG throughout the entire 72 h c. KnockdHigh levels of glucose can promote the activation of the MAPK signaling pathway, including p38 and ERK1/2, to promote monocytes-endothelial cell adhesion . MoreoveKnockdown of GPR26 increased p38 MAPK activation at all time points b. P38 phTreatment of human monocytes with HG promotes the phosphorylation of p38 MAPK and ERK1/2, leading to NF-κB transactivation and related monocyte inflammatory activation . GPRCs cTo explore whether GPR26 affected NF-κB (p65) activation, THP-1 were transfected with GPR26 GapmeRs alone or in combination with NG or HG incubation . Our resNext, we examined the role of GPR26 on hyperglycemia-mediated monocyte inflammatory activation and adhesion to HAoECs. Accordingly, knockdown of GPR26 increased the adhesion of THP-1 to HAoECs at 48 and 72 h of NG b. HG-indSeveral in vitro studies indicate that HG induces cell apoptosis in monocytes ,49. CaspAutophagy is an important physiological process activated to maintain cellular homeostasis and to adapt to extracellular cues. Autophagy is closely associated with ROS generation and inflammation . Indeed,https://www.who.int/news-room/fact-sheets/detail/the-top-10-causes-of-death, last accessed on 1 June 2022). T2D is one of the major public health diseases diffused in Iran, the Middle East’s second-largest country, due to its high prevalence rate [Diabetes is among the top 10 leading causes of death worldwide . However, data regarding the role of GPR26 in diabetes and inflammatory associated complications are missing. We analyzed the expression of orphan GPR26 in the PBMC isolated from T2D patients and confirmed that GPR26 was downregulated. However, GPR26 expression was initially enhanced in human monocytes (THP-1) and human PBMCs treated with HG, a surrogate of hyperglycemia, whereas it was decreased when cells were exposed to prolonged and chronic HG levels. Two hypotheses were generated to explain these results. First, the group of diabetic patients was composed of insulin and non-insulin treated patients, which might have influenced the expression of GPR26. Second, GPR26 might be initially activated to protect monocytes against HG, whereas prolonged and chronic monocyte exposure to HG might impair GPR26 protective role. Our data indicated that both hypotheses might be reliable. Indeed, when diabetic patients were divided according to their insulin treatment, we found that GPR26 expression was further downregulated in insulin patients compared to non-insulin treated patients. This data might be due to insulin-mediated alleviation of hyperglycemic adverse effects on monocyte activation. In line with this, we noticed that the expression of GPR26 negatively correlated with the BMI and HbA1c percentage in diabetic and non-diabetic subjects. In addition, GPR26 exposure on monocyte membranes was decreased by chronic levels of HG, whereas GPR26 accumulated in the cytoplasm. Hence, these data supported the hypothesis that GPR26 might be activated to protect monocytes against hyperglycemia-mediated detrimental effects.Analysis of GWAS studies from diabetic patients indicated that occurrence of certain SNPs in GPCRs are associated with an increased risk for diabetes and cardiovascular complications . In addiIn the diabetic state, chronic or intermittent hyperglycemia promotes a phenotype named “glucose toxicity”, which is often associated with an imbalance of the cellular redox state, inflammation, and related apoptosis . Indeed,Previous studies showed that elevated ROS production leads to p38 MAPK andERK1/2 activation, which in turn promotes NF-κB-mediated transcription of pro-inflammatory genes and monocyte activation . Our datROS induced -p38 MAPK and ERK1/2 activation results in caspase 3 activation and apoptotic cell death , despiteAutophagy is fundamental to maintain cell homeostasis, removal of ROS, and inflammation . Our resTo the best of our knowledge, this is the first work demonstrating the expression and role of orphan GPR26 in monocytes from diabetic patients. Although additional data are needed to prove the protective role of GPR26 against T2D in vivo, we started to depict possible signaling pathways differentially regulated by GPR26 in THP1, independently or not from HG.Indeed, our study has some limitations. Expression assay of GPR26 in PBMCs of diabetic patients was performed in limited samples, and future studies with a large number of samples are needed to confirm our results. Moreover, analysis of GPR26 expression and function in other cell types related to diabetes and cardiovascular complications, such as endothelial cells that are closely related to immune cell activation in diabetic patients, could provide more information and better clarify the role of GPR26 in T2D. Since the ligands of GPR26 are still unknown, future studies devoted to their identification might offer the basis for modulating the expression and activation of GPR26 in T2D patients.The results obtained in the present study defined a new role for GPR26 in human monocytes and PBMCs in T2D patients. Our data suggested that THP1 might initially activate a mechanism of defense in response to hyperglycemia, which is partly mediated by a positive feedback loop that involves GPR26 activation. The protective effect might be impaired by chronic and intermittent hyperglycemic conditions. Treatment of T2D patients with insulin might counteract HG-mediated pathological activation of monocytes, a phenomenon partly reflected from a reduction in GPR26 expression. Moreover, our data suggested that GPR26 exerts a protective role independently from HG by inhibiting pro-inflammatory monocyte activation, ROS production, and apoptosis.


Efforts to support flagging mentoring programs facing shortages of experienced clinical mentors have had an unexpected and welcome effect. Supplementing traditional mentoring programs with peer-mentoring have not only addressed gaps in practice, structure, support and mentee oversight but have offered mentees charged with peer-mentoring duties the opportunity to take on mentoring roles under senior supervision. This study evaluates the experiences of peer-mentors within a local research mentoring program to better understand and advance this endeavor.Semi-structured interviews and post-interview surveys based on recent reviews on mentoring were employed. Adapting the Systematic Evidence Based Approach, data was analysed using thematic and content analysis. Results were combined using the Jigsaw Perspective to ensure that key elements of the different mentoring stages were identified.The interviews and surveys revealed the following domains: Motivation, Initiation, Practicing, and Mentoring Environment.These findings provide novel insight into a structured framework that may help guide the experiences, training, assessment, and oversight of peer-mentors beyond the auspices of our local program. These general observations will equip host organizations with the direction they need to take in designing and executing peer-mentoring training and assessment programs of their own. Whilst the stages of peer-mentoring need further evaluation and an effective means of assessment and support pivotal, we believe our findings suggest that peer-mentoring may not only help to address the shortfall in mentors but is an invaluable learning experience that prepares and instils key values, beliefs and principles in young would-be mentors. A shortage of experienced clinical mentors , changinAs a result, some programs have supplemented their traditional mentoring approaches with electronic-mentoring (e-mentoring) , peer-me, a research mentoring program at the Divisions of Supportive and Palliative Care (DSPC) and Cancer Education (DCE) at the National Cancer Centre Singapore (NCCS), adopted a Combined novice, e-mentoring and peer-mentoring (CNEP) approach.  program .Combined novice, near-peer, e-mentoring palliative medicine program: A mixed method study in Singapore”.Whilst the PMI has supported more than 100 single-authored, mentee co-authored and/or mentee first-authored publications in peer-reviewed journals, and more than 150 posters at international conferences in palliative medicine, medical ethics, medical education, end-of-life ethics and health services research over the last 12 years, its novice mentoring approach has been especially susceptible to mentor shortages. To attend to these gaps, the PMI adopted the CNEP approach detailed in Krishna et al. ’s study Designed around mentoring reviews –25 and fWhilst the success of the CNEP approach in enhancing the mentoring experience of mentees have been well established, feedback by peer-mentors have revealed rather surprising findings. Successful PMI mentees report taking on the role of mentor under senior supervision as a means of advancing their mentor training and interests. With two peer-mentors becoming mentors in the PMI, these unexpected findings demand closer scrutiny. The notion that peer-mentoring could be employed to train future mentors is especially appealing not only to sustain the culture and structure of the program but to mould their development around the program’s time-honoured values, beliefs and principles. With no studies we are aware of looking into the effects of peer-mentoring as a means of mentor training, we propose an evaluation of the experiences of peer-mentors in the hope that insights provided will not only enhance the PMI experience but facilitate the design and support of similar programs in other settings.how does being a peer-mentor impact a mentee’s development?” and “what factors influence a peer-mentor’s development?”, we adopted a combination of semi-structured individual interviews to capture individual experiences in our peer-mentoring program and post-interview surveys designed using the MEntor Reflection InstrumenT (MERIT) framework  on others." (peer-mentor 3)Practical perspective-taking on this new position required an alignment of expectations and an understanding of their roles and responsibilities. All twelve participants reported being introduced to the mentoring approach and discussing their roles and responsibilities. For nine participants (75%), their concerns required individual discussions with their mentor or senior peer-mentor.“She explained whatever I had to do and said that I could ask her for help whenever I need[ed] to or had any clarifications. So, I think that helped to allay my fears." (peer-mentor 8)A peer-mentor’s role brought with it a deeper appreciation of the mentoring approach and the impact of the mentoring environment upon their experiences. The role of peer-mentor also brought a greater sense of independence  and reaffirmed the peer-mentor’s values and beliefs ."This idea of being generous or paying it forward or giving back to people [was] reinforced during my process as a peer-mentor." (peer-mentor 3)Peer-mentors also reported enhanced leadership , reflective , communication  and active listening  skills as well as being better at nurturing relationships  and role modelling . Peer-mentors also reported being more adaptable, accountable to their mentees and mentors, approachable, collaborative, confident, empathetic, open-minded, humble, meticulous, patient, resilient, self-aware, trustworthy and independent.Participants reported that the mentoring program’s structure and culture influenced their development. Peer-mentors attributed their overall development to the program’s consistent structure , clarity on roles and job scopes  and the presence of online platforms . Similarly, the mentoring program’s supportive culture  and duty to respect each other  helped them mature as mentors. These changes were also facilitated by the mentor’s guidance and support  and their approachable, available and proactive nature  some 3–4 weeks after the initial interviews reflected similar findings Peer-mentors also believed that their mentoring approach was guided by a combination of their previous experiences in the program, the presence of role modelling, regnant values, professional expectations and the mentoring approach and culture.In keeping with the SEBA methodology, the initial results were reviewed by the expert and research teams. This process revealed that, without appropriate information on the structuring and practice of the PMI CNEP program, much of the nuances surrounding the findings would be lost. As a result, the discussion section will include information on the structure, culture, and environment of the PMI’s practice.The data accrued here suggests the presence of mentoring stages. This finding is not surprising given earlier reports of mentee ‘mentoring stages’ , 22 deriAs shown in Enlisting ‘PMI competent’ mentees provide an indication as to whether prospective peer-mentors will find the roles, responsibilities and codes of conduct expected of peer-mentors acceptable. In addition, the personalized invitation and the offer to collaborate helps to build the mentee’s confidence. It provides them with the assurance that their efforts, competencies and potential are recognized and appreciated. It also provides motivated ‘PMI competent’ mentees a chance to build on their skills and knowledge within a working environment and learning structure that they are already aware of and are comfortable with. Aside from the chance to lead a team on a project that they are likely to be interested in with a mentor that they have worked with in the past, the invitation to be a peer-mentor also provides them with a chance to improve practice, culture, and structure of the PMI. The prospective peer-mentor is then given time to consider the offer and discuss their queries with the mentor before accepting the invitation.Stage 2 is based on Domains 1 (Motivation) and 2 (Initiation). It begins with acceptance of the invitation and an agreement of roles, responsibilities, codes of conduct and expectations associated with the peer-mentoring role. This stage can be difficult for new peer-mentors, and they often rely upon their mentors or senior peer-mentors for guidance and support.Drawing on regnant CNEP practice to contextualize the data garnered in Domain 2 (Initiation), this stage sees new peer-mentors build on their ‘cognitive base’ and adopt appropriate time management and self-management strategies to contend with the additional work. The mentors should provide additional resources and training to prepare the peer-mentor for their new role and responsibilities. Timelines, expectations and the approach adopted for the specific mentoring project are also discussed and agreed upon. As highlighted in Domain 2 (Initiation), this stage also represents a key opportunity to agree upon lines of communication and reiterate codes of conduct, expected assessment outcomes, feedback loops, methods used and support mechanisms available. These processes underscore the personalized nature of recruitment and support of a peer-mentor. It also marks the beginning of a new mentoring relationship between the mentor and peer-mentor.Stage 3 sees the establishment of the peer-mentor’s place within the mentoring project. Drawing on Domain 3 (Practicing), this stage sees peer-mentors orientated to their roles and introduced to the mentees. This stage proceeds with mentors and peer-mentors working together to create an open and supportive mentoring environment that allows open discussion and trusting relationships between mentees, peer-mentors and the mentor. These personalized relationships provide them with the opportunity to hone their listening, communication, leadership, empathetic and reflective skills under senior supervision.Stage 4 sees the maturation of peer-mentoring skills, growing confidence, and the development of deeper reflective practice on the part of the peer-mentor. Drawing from current CNEP practice, we see peer-mentors acquire a more nuanced understanding of teamwork, the mentoring process, the relationships with the people around them and the mentoring environment. This stage brings personal and professional development for the peer-mentor. Having previously experienced the mentee’s role, trajectory and evolving needs, it is notable that peer-mentors will likely begin to exhibit desirable mentoring traits . Drawing on Domains 3 (Practice) and 4 (Mentoring Environment), Stage 4 is marked by the peer-mentor’s growing confidence. This is exemplified by greater adaptability, patience, approachability, open-mindedness, empathy, collaborativeness and self-awareness which are key professional characteristics .Domain 4 (Mentoring Environment) also provides a glimpse of the support provided for professional and personal development within the CNEP program. This includes role modelling and guidance by the mentor, and guided reflections, peer-mentoring experiences, interactions and mentee feedback. In addition, Domain 4 (Mentoring Environment) reveals the impact of the mentoring structure in shepherding practice, the effects of the mentoring approach on steering practice and interactions, and the mentoring culture that focuses on ‘paying it forward’ and supporting one another. The post-interview surveys reveal that the peer-mentor’s own reflections and insights personalize the effects of the structure, approach and culture within the mentoring environment.Stage 5 also draws on Domain 4 (Mentoring Environment). It sees peer-mentors using their experiences to help their fellow peer-mentors. The data reveals the organic formation of a support system where more experienced and senior peer-mentors guide and share information with more junior peer-mentors.Drawing on the post-interview surveys, Stage 6 sees the peer-mentoring network begin to influence the wider mentoring program. This is primarily by shaping existing mentoring codes of practice, the program’s culture and expectations, and its hidden curriculum.Stage 7 draws on current CNEP practice and focuses on the more senior peer-mentor taking on the mentor’s role in their own projects. Stages 6 and 7 serve to underline the importance of longitudinal and holistic review of the mentoring relationship and the program as a whole . In doinWe believe this framework will help guide and structure the experiences, training, assessment, and oversight of peer-mentors beyond the auspices of the PMI. These general observations may equip host organizations with the direction they need to take in designing and executing peer-mentoring training and assessment programs of their own.This study is not without its limitations. Participants in this study were represented by 12 peer-mentors in a single centre research-based mentoring program for medical students and junior doctors in Singapore. This setting and the unique approach adopted by the mentoring program may raise some questions as to the applicability of these findings outside the program and in other clinical settings.Additionally, as peer-mentors were interviewed at a single time point, often after completion of a project or in the later stages of their research process, recall bias and the halo effect may surface.Suggestions of peer-mentoring reducing the presence of hierarchy within programs and thus facilitating more open and honest exchange of ideas may also be compromised given the presence of strict levels of seniority in medical school and the practice of medicine. This may lead to the possibility of social desirability bias amongst responders.In forwarding these new insights, this study underscores the critical responsibility of mentors and the host organization in nurturing peer-mentors–particularly evident in their role in recruiting, training, overseeing and enabling a supportive and safe environment for mentees, peer-mentors and mentors alike. Whilst the stages of peer-mentoring need further evaluation and an effective means of assessment and support pivotal, we believe our findings suggest that peer-mentoring may not only help to address the shortfall in mentors but is an invaluable learning experience that prepares and instils key values, beliefs and principles in mentees with greater mentoring aspirations. The impact of these experiences on their professional identity formation ought to be the focus of future study.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


It participates in a wide range of biological functions in the central nervous system (CNS). However, the role of SERCA2b in glutamate-induced excitotoxicity and its mechanism must be elucidated. Herein, we demonstrate that SERCA2b mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22 cells. In this study, SERCA2b mutants accelerated Ca2+ depletion through loss-of-function (reduced pumping capacity) or gain-of-function (acquired leakage), resulting in ER stress. In addition, the occurrence of ER Ca2+ depletion increased mitochondria-associated membrane formation, which led to mitochondrial Ca2+ overload and dysfunction. Moreover, the enhancement of SERCA2b pumping capacity or inhibition of Ca2+ leakage attenuated Ca2+ depletion and impeded excitotoxicity in response to hypo-glutamate stimulation. In conclusion, SERCA2b mutants exacerbate ER Ca2+-depletion-mediated excitotoxicity in glutamate-sensitive HT22 cells. The mechanism of disruption is mainly related to the heterogeneity of SERCA2b mutation sites. Stabilization of SRECA2b function is a critical therapeutic approach against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS.Glutamate-induced excitotoxicity is a pathological basis of many acute/chronic neurodegenerative diseases. Sarco/endoplasmic reticulum Ca Glutamatergic neurotransmission controls many cognitive, motor, sensory, and autonomic activities. In addition, glutamate is critical in maintaining the balance between excitation and inhibition in the CNS . Glutamapoptosis .Impaired glutamate homeostasis has severe neuropathological consequences and is associated with a variety of CNS disorders, such as epilepsy, Alzheimer’s disease (AD), Huntington’s disease, and Parkinson’s disease . Neuropr2+ is an essential cellular signaling regulator that acts as a crucial second messenger in many cellular processes and “enforcers” of glutamate excitotoxicity. The activation of the glutamate receptors leads to an inward flow of extracellular Ca2+ and an increase in intracellular Ca2+ levels ([Ca2+]cyto). The elevated amount of [Ca2+]cyto further triggers the opening of Ca2+-releasing channels in the endoplasmic reticulum (ER), activating inositol 1,4,5-trisphosphate receptors (IP3Rs) and ryanodine receptors (RYRs). This process is known as calcium-induced calcium release (CICR), which is a primary mechanism of calcium signal generation and amplification in the cells . Mitochondrial Ca2+ overload results in increased mitochondrial membrane permeability, imbalance of redox reaction, release of reactive oxygen species (ROS), and release and activation of apoptosis-related factors, all of which are associated with glutamate-induced neuronal death   . ERS canss (ERS) . ER Ca2+2+-ATPases (SERCA2b) pumps are membrane-embedded P-type ATPases pumps that mediate the translocation of [Ca2+]cyto to the ER lumen by hydrolyzing ATP against a concentration gradient  mechanism of ATP2A2 mutations demonstrated in Darier’s disease . The G806S mutant has been published previously , whereas2 and 95% air. Glutamate   was applied to HT22 cells 24 h for subsequent analysis. According to the DNA transfection protocol, the SERCA plasmids were transfected into HT22 cells by jetOPTIMUS . The control group (CON) mentioned in this article refers to the cells transfected with the empty plasmid (pcDNA3.1) only. The cells were analyzed for the glutamate response 24 after transfection.HT22 cells were obtained from Kunming Cell Bank of the Chinese Academy of Sciences , and they were cultured in DMEM medium  supplemented with 10% FBS , 100 U/ml penicillin, and 100 μg/ml streptomycin . Cell culture was performed at 37°C in an incubator filled with 5% COThe pcDNA3.1-SERCA2b-3xFLAG plasmid containing human SERCA2b was purchased from GenScript. Site-directed mutagenesis was performed using the QuickMutation™. After sequencing confirmation of the wildtype SERCA2b, all mutants  studied were induced by site-directed mutagenesis using Serca2b as a template and confirmed by sequencing again. Primers used for site-directed mutagenesis PCR are listed in 3 cells per well. After 12 h, cells in each well were incubated with 10 μl CCK-8 solution  for 2 h at 37°C. The incubation plates were then placed in a microplate reader  to determine the optical density (OD) values at 450 nm. The curves were plotted according to the OD values. Thiazolyl Blue Tetrazolium Bromide assay (MTT): Cells were seeded into 96-well plates at a density of 5 × 103 cells per well. After 12 h, cells in each well were incubated with 10 μl MTT solution  for 4 h at 37°C. After that, 100 µl of Formazan solution  was added to each well, mixed properly, followed by incubation for another 4 h. Then the OD of each well was measured at 570 nm. Lactate dehydrogenase (LDH) release assay was performed using the LDH Cytotoxicity Assay Kit . Briefly, cells were plated in a 96-well plate, and 60 μl of LDH assay working solution was added to each well according to the operation manual, mixed well, and incubated at room temperature (about 25°C) in a dark place for 30 min. Then the OD value of each well was measured at 490 nm. The absorbance after Triton X-100 treatment was regarded as Amax. Cell Counting Kit-8 assay (CCK-8): Cells were seeded into 96-well plates at a density of 5 × 102+ and Mg2+. Cells were scanned and imaged at 5 s intervals under a microplate reader  by successive addition to a final concentration of 1 mM glutamate, 10 mM caffeine, and 5 mM EGTA. For the rescue of [Ca2+]ER experiment, CDN1163  or thapsigargin  was added and incubated for 10 min, followed by glutamate stimulation. Fluo-2 AM Ex/Em = 549/578 nm, Fluo-4 AM Ex/Em = 494/506 nm.Cells were incubated with 5 μM Fluo-4 AM  or Rhod-2 AM  for 20 min. After washing with PBS twice, the buffer was replaced with HBSS without CaCells were fixed with 1% osmic acid for 2 h at 4°C and centrifuged at 200 rpm for 10 min; then, cell pellets were collected. The cell pellets were fixed again by 1% osmic acid and 0.1 M PBS (PH7.4) for 2 h at 20°C, followed by dehydration, infiltration, embedding, sectioning, and staining (2% Uranyl acetate and lead citrate). Finally, they were observed under an electron microscope .Cells were incubated with 10 nM tetramethylrhodamine, ethyl ester (TMRE)  for 30 min at 37°C at 24 h after glutamate stimulation. Then, cells were washed twice with a pre-warmed cell culture medium and observed in a confocal laser microscope . Cells treated with 10 μM carbonyl cyanide-m-chlorophenyl hydrazone  were used as a positive control. Image J software was used for analyzing fluorescence intensity. Ex/Em = 550/575 nm.Cells were incubated with 10 nM 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA)  for 1 h at 37°C, and then centrifuged at 1,000 rpm for 5 min. The cell pellets were observed under a confocal laser microscope . ImageJ software was used for analyzing fluorescence intensity. Ex/Em = 500/525 nm.The cells were rinsed with ice-cold PBS two times before the total protein was extracted. The extraction process took place on ice. After adding RIPA buffer  the homogenate was centrifuged at 12,000 rpm for 10 min at 4°C. For SERCA2 solubility analysis experiments, the addition of 1% Triton X-100 and repeated ultrasonic fragmentation helped lysis, and the supernatant was subjected to denaturation  to enhance lysis. The bicinchoninic acid protein assay kit determined the protein concentration. Equal amounts of protein (20–40 μg) were separated by a 10%–15% SDS-PAGE gel and transferred onto a PVDF membrane. PVDF membrane was blocked with 5% skim milk at room temperature in TBST for 1 h. Membranes were incubated with primary antibodies  was used to detect apoptotic cells according to the manufacturer’s instructions. In brief, samples were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature, incubated with working solution  for 1 h, and shielded from light at 37°C. The nuclei were then stained with the DAPI for 10 min. The specimens were analyzed using a confocal laser microscope  method to determine the relative quantitative level and was expressed as a fold-difference to the relevant control (recognized as 1 ± 0.00).Total RNA was isolated using TRIzol Lysis Reagent (Invitrogen), and cDNA was synthesized by All-in-OneTMFirst-Strand cDNA Synthesis Kit . For qPCR, amplification reactions were performed using All-in-One™qPCR Mix , run on the ABI-Ⅶ Real-Time PCR detection system. The primers were synthesized by Tsingke Company (China); the details of sequences are listed in 2and 3 mM β-mercaptoethanol. The concentration of microsomes was determined by the Lowry method.According to the modified Parsons’s protocol, microsomes (crude ER) were isolated by serial centrifugation . BrieflySERCA2 activity was measured by the inorganic phosphorus method . Briefly, ATP was decomposed into ADP and inorganic phosphorus by ATPase. The reagents were added separately, then mixed according to the protocol, and the reaction was carried out at 37°C for 10 min. After that, 100 ul of the sample was added and mixed for the enzymatic reaction. After centrifugation at 3,500 rpm for 10 min, 150 μl of the supernatant was collected to determine phosphorus. The absorbance was measured at A636 wavelength with 0.02 μmol/ml standard phosphorus solution as a reference. The amount of ATPase to produce 1umol of inorganic phosphorus per mg of microsomes per hour was defined as 1 ATPase activity unit (μmolPi/mg protein/hour).p-value of less than 0.05 was considered statistically significant. Results were expressed as means ± SEM.The student’s t-test was used to determine significant differences between the two groups. One-way analysis of variance was utilized to determine significant differences among multiple groups. A Excitotoxicity plays an essential role in CNS injury. Intracellular calcium overload and mitochondrial dysfunction are the leading causes of high glutamate-induced neuronal death . We test2+, as the process of proteins folding by the molecular chaperones requires high levels of Ca2+. We next measured the levels of [Ca2+]cyto concentration in response to glutamate stimulation and ER Ca2+ storage levels. After 10 min of loading with Fluo-4 AM, cells stimulated with 1-mM glutamate showed a transient increase in calcium fluorescence signal and gradually returned to the resting levels. The magnitude of the glutamate response was significantly lower in the G860S mutant than in the WT group , and voltage-dependent anion channel 1 (VDAC1). After confirming that ER Ca2+ uptake is impaired in SERCA2b mutants, we wondered whether the mitochondrial function is altered and involved in hypo-glutamate-induced excitotoxicity. Following experiments, we discovered an increased proportion of mitochondrial-ER contacts in the mutants compared to WT group cells by transmission electron microscopy , which provides energy for the assembly, folding, and modification of ER and the uptake of Cacroscopy . WB resucroscopy , suggest2+ flux to mitochondria, resulting in mitochondrial Ca2+ overload. We then assessed the kinetics of mitochondrial Ca2+ by specifically labeling mitochondrial Ca2+ with Rhod-2 AM. Subsequently, mitochondrial Ca2+ fluorescence showed a strong response to hypo-glutamate stimulation in the four mutants, followed by a return to the resting levels  was significantly decreased after glutamate stimulation  showed shortened recovery time from the [Ca2+]cyto oscillations compared to the group without CDN1163 intervention  pathogenesis. To confirm this hypothesis, WT and G860S cells were treated with thapsigargin before glutamate stimulation. Thapsigargin is a non-competitive inhibitor of SERCA and does not affect the activity of other ATPases. Calcium signal imaging results showed that thapsigargin reduced the glutamate response time of G860S and partially restored ER Ca2+ storage . In the GM1 gangliosidosis model, GM1 accumulates in the microstructure domain of the MAM, which increases ER Ca2+ depletion and Ca2+ flux to the mitochondria by interacting with phosphorylated IP3R, ultimately leading to mitochondrial Ca2+ overload-mediated apoptosis  exacerbated Capoptosis . In glucinvolved . In neurhic pain . Unlike et of AD .2+ regulatory mechanisms can buffer excitatory stimuli from low glutamate concentrations and maintain intracellular homeostasis. However, for SERCA2 mutant cells, this excitatory stimulus is a devastating signal, mainly attributed to the LOF effect due to abnormal SERCA2 expression or activity. Many studies have confirmed that different SERCA2 mutations lead to LOF effects in pump defects or reduced protein levels that underlie dominant diseases. Gordon-Smith et al. found a significantly higher rate of disrupting ATP2A2 mutations in cases with Darier’s disease accompanied with neuropsychiatric disorders  and the other factor is the increase in leakage (GOF type), which determines the difference in the scheme of restoring ER Ca2+ homeostasis. It is also true that CDN1163 enhances the activity of LOF-type pumps  to inhibit glutamate-induced excitotoxicity, but not the GOF-type mutant (G860S)  respond well to sodium channel blockers (SCBs), whereas patients with LOF (decreased channel availability and membrane excitability) mutations experience exacerbated seizures due to SCB use  . Genetic SCB use . Notablyactivity , suggestrecovery . CDN1163at model . The dysat model .2+. As depicted in 2+ depletion caused by SERCA2b mutations disrupts this balance, leading to the activation of a series of cell-death signaling pathways. Targeted treatments against organelles may be a potential approach to synergistically enhance the neuroprotective function of glutamate receptor inhibitors or glutamate transporters  or GOF (acquired ion leakage), leading to ERS. The occurrence of ER Ca2+ depletion increases MAM formation, contributing to mitochondrial Ca2+ overload and dysfunction. Rescuing the SERCA2 pumping capacity or inhibition of Ca2+ leak attenuates Ca2+ depletion and inhibits excitotoxicity in response to hypo-glutamate stimulation. The stabilization of SRECA2b function is a critical therapeutic target against glutamate-induced excitotoxicity. These data will expand understanding of organelle regulatory networks and facilitate the discovery and creation of drugs against excitatory/inhibitory imbalance in the CNS.The current study demonstrates that SERCA2 mutants exacerbate the excitotoxicity of hypo-glutamate stimulation on HT22. SERCA2 mutants accelerate ER Ca


Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca In addition, BNCD microelectrodes are suitable for measuring electrochemical potentials, showing sensitivity in good agreement with Nernst’s law [Nanocrystalline diamond (NCD) has proven to be an excellent material for the fabrication of microelectrodes and microelectrodes-arrays (MEAs). Although an electrical insulator, diamond can reach quasi-metallic conductivity when doped with boron. Boron-doped nanocrystalline diamond (BNCD) microelectrodes present excellent properties such as very low background current <1 μA/cm μA/cm2, 2+ and Mg2+ can be determined with the aid of BNCD microelectrodes, which allow their application for the investigation of ion-dependent biological processes [Several applications can be found for BNCD electrodes and microelectrodes, including treatment of polluted waters ,4, electrocesses ,2.2+ channels [Annexin A5 (AnxA5) is responsible for many physiological events such as the inhibition of phospholipid-dependent blood clotting ,20, moduchannels ,24,25,26channels . In addichannels ,29. A lechannels ,31,32.2+ channel specifically in the membrane of matrix vesicles (MVs) [2+ concentration, thus hampering mineralization by MVs [2+ accumulation ability [2+ in MVs, all factors that affect the function of these proteins during the mineralization process must be identified, such as their locations and their specific functions within the mineralization process as a whole.Human AnxA5 ~ 35 kDa) was the first to have its three-dimensional structure characterized ,34. It ies (MVs) ,35,36,37 kDa was es (MVs) ,39,40 den by MVs ,42. More ability . To unde2+ transport mediated by AnxA5 when inserted into the lipid bilayer of liposomes (proteoliposome) as MV-mimetic systems. Since the measurements technique using a BNCD microelectrode proved to be highly sensitive, we were able to evaluate the selectivity of the transport mechanism.In this paper, we describe the use of a BNCD microelectrode to track Ca® system. Analytical grade bovine serum albumin (BSA), tris-hydroxymethyl- amino-methane (Tris), sodium dodecylsulfate (SDS), polyoxyethylene-9-lauryl ether (polidocanol), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl- snglycero-3-phospho-L-serine (DPPS), calcium chloride, magnesium chloride, sodium chloride, chloroform and ethylenediaminetetraacetic acid tetrasodium (EDTA) were purchased from Merck KGaA, St. Louis, MO, USA and used without further purification.All aqueous solutions were prepared with ultrapure apyrogenic water from a Millipore DirectQg for 1 h at 4 °C. The pellet containing proteoliposomes was resuspended in an appropriate volume of the same buffer resulting in a 0.6 mg/mL of total lipid. Liposomes and proteoliposomes were characterized by dynamic light scattering (DLS) and zeta (ζ)-potential measurements. Protein concentration incorporated in the lipid bilayer of liposomes was estimated in the presence of 2% (w/v) SDS, and BSA was used as standard [Liposomes constituted by 9:1 DPPC:DPPS (molar ratio), containing up to 1.5 mg/mL of lipids were prepared as previously described . Brieflystandard .v/v) glutaraldehyde  to avoid vesicle deformation and disruption. The mixtures were homogenized and then 5 μL of the sample was dropped onto freshly cleaved mica substrate, left to dry at room temperature and imaged by AFM as described by Bolean et al. [n = 100 vesicles were analyzed.Liposome and proteoliposome samples were prepared with stock buffer and stabilized by adding 1:1  Clean a blank sapphire wafer.(2) Deposit a ~60 nm layer of amorphous silicon (α-Si) for buffering the thermal expansion mismatch between sapphire and diamond and promote the diamond nucleation.(3) Grow a ~500 nm thick film of intrinsic NCD.(4) Grow a ~250 nm film of BNCD.(5) Transfer the pattern of the conducting structures via optical lithography, metal mask protection and reactive ion etching (RIE) in an argon-oxygen atmosphere.(6) Deposit a 1 µm thick passivation layer of silicon nitride by plasma-enhanced CVD (PECVD).4) atmosphere.(7) Transfer the pattern of the microelectrode openings (16 µm in diameter) and bonding pads by optical lithography and RIE in a tetrafluoromethane (CF(8) Dice the wafer for obtaining the individual chips.After fabrication, the chip was assembled on a printed-circuit carrier plate by flip-chip bonding with conducting silver-epoxy, and finally a glass ring was glued over the device for providing a 200 µL volume for the biological environment . A ring-Before the first use, the device was exposed to mild oxygen plasma for 10 min to provide the desired surface termination. This step is of fundamental importance for potentiometric measurements. In fact, oxygen termination is not only responsible for a hydrophilic behavior, but results in a surface band bending and a stable pinning of the surface Fermi level at 1.7 eV above the valence band, thus making it reliably sensitive to variations of the electrochemical potential in the electrolyte .E can be estimated by means of the general Nernst equation by taking into account the activity of all oxidized and reduced species EӨ the potential offset given by the Ag/AgCl quasi-reference electrode, R the universal gas constant, F the Faraday constant and T the absolute temperature. Potential changes are due to concentration changes of certain species, including buffering effects. This means that the contribution of calcium ions to the measured potential is only one among many, and the presence of a buffer can compensate, at least partially, the potential variations due to ion-transport inside the vesicles.In the adopted configuration, the chip is not provided with a calcium-specific ion-selective membrane. This is intentional, in order to measure with the same device, the response of liposomes and proteoliposomes to various ions, thus assessing their selectivity and specificity. Therefore, the microelectrodes sense the cumulative electrochemical potential resulting from all ions present in the electrolyte. This potential After every measurement session, the device was cleaned with a 14% solution of sodium hypochlorite to remove organics and restore a conveniently oxidized surface, then abundantly rinsed with Millipore DirectQ ultra-pure apyrogenic water, and finally dried with a nitrogen gun. No material degradation could be observed by optical inspection, even after several measuring/cleaning cycles. However, we observed from measurement to measurement slightly different baseline potentials after equilibration, which may indicate different characteristics of the surface termination. In fact, it is well known that oxygen can form ketone-bonds, ester bonds or hydroxyl groups when terminating a carbon surface. The random proportions of these three O-termination variants leads to slightly different chemical activity and surface potentials. During the measurements, the front-end electronics was held in a ferromagnetic shielding box, equivalent to a Faraday cage, having a small opening above the device to allow manual pipetting operations. Unfortunately, this simple technique introduced artifacts in the signal recordings, but these did not affect the long-term trends (300 s). Due to the uniformity of the biological sample, the signals detected from the four channels were substantially identical; therefore, only one channel was considered for data analysis. Data presented in the figures are representative of triplicate measurements using different vesicle preparations.The mean diameter and ζ-potential of 9:1 DPPC:DPPS-liposomes (molar ratio) were 110 ± 10 nm and −2.3 ± 0.1 mV, respectively. Proteoliposomes containing 14 µg/mL of AnxA5 had a diameter of 140 ± 15 nm and a ζ-potential −21.8 ± 0.3 mV. These vesicles were also topologically characterized by AFM and tested in vitro as biomimetics of the MV functions ,27,44. ADPPC:DPPS liposomes show spherical and intact (unbroken) vesicles. The presence of negative charges in the DPPS lipid results in a rough surface A, but no2+ inside proteoliposomes was followed by using the BNCD microelectrodes described above, standardized and calibrated using the same method described by Denisenko et al. [2+ in solution were measured. The experiment depicted in 2+ ions. The potential of the microelectrode equilibrated after approximately 100 s, thus reaching a nearly flat baseline. The addition of Tris-HCl buffered liposomes or proteoliposomes into the microelectrode compartment  [As recently described by Lin et al. , Mularskine (PS) .2+ ions from a more to a less concentrated microenvironment [2+ influx into proteoliposomes or MVs are still not well understood.The annexins are the most abundant family of proteins found present in the MV membrane, and the ability of AnxA5 in transporting Caironment ,28,29,422+ influx into fura-2-loaded liposomes in the presence of AnxA5. This protein is able to modulate Ca2+ permeability into liposomes and is not simply due to leakiness of the membrane caused by the presence of protein, once it was shown that the Ca2+ influx was inhibited by the presence of antibodies to AnxA5 [2+ influx ability promoted by AnxA5, AnxA6, and AnxA2 into native MVs isolated from chondrocytes [45Ca2+ influx into 9:1 DPPC:DPPS (molar ratio) liposomes harboring AnxA5. AnxA5-proteoliposomes had the highest affinity for collagens, especially for type II, supportive of a role for AnxA5 during chondrocyte mineralization in joint cartilage [Kirsch et al.  showed bto AnxA5 . Years ldrocytes . Bolean drocytes  also werartilage ,44.AFM enables morphologic analysis of proteoliposomes at the nanoscale without the use of fluorescent dyes, just as it is widely used to image membrane structures by means of fluorescence microscopy. We need a high-resolution imaging technique that works on soft nanosystems with a minimal degree of perturbation of the system, and AFM brings this advantage when compared with other techniques. AFM enables recording of phase images by monitoring the shift between the phase angle of the cantilever oscillations relative to that of the drive signal. Since proteins and lipids have different physical properties, the presence of proteins within lipid membranes can be detected as surface regions with distinct phase angle shifts in AFM phase images, thus enabling the mapping of single proteins as well as protein aggregates on lipid membranes. Thus, darker spots on the surface of the proteoliposomes are domains with lower viscoelasticity when compared with the entire vesicle surface. This suggests that these darker dots are regions with different chemical composition induced by the AnxA5 protein presence in the lipid bilayer of proteoliposomes. Therefore, AFM data suggest the presence of AnxA5 in the proteoliposome, besides showing direct images of the liposomes and proteoliposomes as intact vesicles .It was expected that after AnxA5 incorporation into the bilayer lipid of liposomes, thus forming proteoliposomes, we would obtain proteoliposomes vesicles with bigger diameters when compared with the same vesicles without protein inserted (liposomes) . This be2+/AnxA5 was 50. Since it has been reported that the AnxA5 monomer binds up to 10 Ca2+ ions [2+ in the solution and confirmed that the reduction in the potential was not only due to the binding of Ca2+ to AnxA5, but mainly to the transfer of ions to the lumen of the proteoliposomes, showing the transport function of AnxA5 when incorporated in the vesicles’ membrane bilayer.Under the conditions of the measurements carried out using the BNCD microelectrodes, the molar ratio of Caa2+ ions , this ex2+ transport by AnxA5 into DPPS:DPPC:Chol (2:1:1) liposomes, raising the levels of mineral propagation in odontoblast pre-differentiated cells, leading to the odontogenic differentiation [2+ transport to the inside of vesicles and targeting them to collagen fibers in the extracellular matrix.The lipid DPPS plays a very important role both for improving AnxA5 incorporation into liposomes and for favoring binding of proteoliposomes to collagens . Besidesntiation . Therefo2+ and Mg2+ ions bind to AnxA5, but only Ca2+ is able to support an orientation in the α-helix structures of the protein. While Ca2+ ions are fully accessible to water on the AnxA5 surface, some of the Mg2+ ions are embedded in the protein structure. Indeed, by means of BNCD microelectrodes it was shown here that AnxA5 is able to transport Ca2+ in a selective way when compared with Mg2+.Results described by Fezoua-Boubegtiten et al.  in PS mo2+ and inorganic phosphate (Pi) in MVs can lead to the growth of apatite crystals, which then propagate on the ECM and continue to grow onto collagenous fibers, forming bone tissues. More than any other protein so far studied, AnxA5 accelerates the nucleational activity of the acidic phospholipid-Ca-Pi complexes present in the NC, which triggers de novo calcium phosphate mineral formation in MVs. AnxA5 interacts with Ca2+ and the PS polar head groups present in the MV lipid membrane, and domains are created that apparently exclude Mg2+. Thus, in the presence of PS, AnxA5 shows no affinity for Mg2+, in strong contrast to its high affinity for the Ca2+ ion [2+, they quickly form complexes that co-sediment with the PS liposomes. On the other hand, if Ca2+ is replaced for Mg2+ even at much higher levels, AnxA5 does not bind Mg2+ or co-sediment with the PS liposomes. This behavior is observed because the coordination chemistry of Mg2+ is very different from that of Ca2+ [2+ can form flexible bond lengths and angles with as many as seven different ligands, Mg2+ coordinates with only six ligands in a highly rigid, precise geometry [2+ displays a rigid coordination geometry, which blocks the nucleational activity of PS-CPLX, and consequently prevents ACP from forming hydroxyapatite or octacalcium phosphate crystals.Based on the results obtained, it is possible to re-establish a correlation with the physiological condition during mineralization. Thus, during bone formation, osteoblasts or chondrocytes produce and mineralize the extracellular matrix (ECM). The formation of apatite is initiated in the lumen of MVs and has been described to depend on the presence of a nucleator, the nucleational core (NC), in the lumen of MVs. The components of the NC, and amorphous calcium phosphate (ACP) in association with PS, result in calcium–phosphate–lipid complexes (PS-CPLXs). Further accumulation of CaCa2+ ion ,51. Ther of Ca2+ ,53,54. Wgeometry ,56. Thus2+ ions to be transported by AnxA5, and questions related to the role of AnxA5 in the transport of Ca2+ ions remain. Therefore, this paper describes the high sensitivity of BNCD microelectrodes to track small changes in Ca2+ concentration due to binding to proteoliposomes, which could not be investigated using common Ca2+ selective electrodes. The authors are aware that the reported results are essentially of a qualitative nature; therefore, future work will focus on providing more precise quantitative assessments. However, the results shown here support, with adequate evidence, the hypothesis of selective Ca2+ transport within the proteoliposomes and the possible function of AnxA5 mediating the mineralization initiation process that occurs in MVs.To date, there is no description of the possible ability of Mg


Fluorescein diglycolic acid  was synthesized and used in multivariate determination of Ca2+ and K+. Full-structure computation shows that the complexes of 1 and Ca2+ have comparable energies regardless of additional interaction with lactone moiety. The initial formation of diglycolic-Ca2+ complex followed by macrocyclization is thermodynamically disfavored. A U-shaped pre-organized 1 allows Ca2+ to interact simultaneously with diglycolic and lactone motifs. Both motifs actively participate in Ca2+ recognition and the eleven methylene units in the undecyl arm provides excellent flexibility for reorganization and optimum interaction. Principal component analysis (PCA) of computational molecular properties reveals a simple method in evaluating motifs for cation recognition. Fragment models support full-structure results that negative charge causes significant structural changes, but do not reproduce the full extent of C-O bond breaking observed in the latter. Experimental optical responses show that 1 is selective towards Ca2+ and discriminates against K+ and Mg2+. PCA of emission intensities affords distinct clusters of 0.01, 0.1 and 1 mM Ca2+ and K+, and suggests applicability of this technique for simultaneous determination of cationic plant macronutrients in precision agriculture and a wide variety of other applications.Computational and experimental approaches were adopted to utilize a chromophore diglycolic functionalized fluorescein derivative as a Ca Fluorescein derivatives are one of the most widely used chromophores in the labelling of biomolecules. The hydrophilic nature of fluorescent dyes greatly contributes to its compatibility with biological systems such as in enzyme detection and cell tracking. Fluorescent sensors have successfully been utilized in imaging cell structures and for analyzing biophysical processes: for example, in the detection of Alzheimer’s disease and the activity of certain enzymes, as well as pH and the concentration of certain metal ions ,2,3,4. D2+ and Mg2+, the conventional sensing method often leads to discrepancies between calibration equations and actual sensor characteristics attributed to erroneous or inaccurate sensor measurement . Th. Th6) is3 and enolate 3a were the main sources due to economic reasons. Data from the gas phase are compared with those in methanol medium , top-right (Q2), bottom-right (Q3) and bottom-left (Q4). The first case involves the separation of 3a-Ca2+ from 3a-K+ in a vacuum. The two points (red squares) are very well separated vertically on the right into Q3 and Q2 quadrons, respectively.The scree plot in 3 chromophore (blue triangles), 3-Ca2+ and 3-K+, is also well separated vertically on the left into Q4 and Q1 quadrons, in that order. The results suggest that the chromophore-cation systems afford distinct clusters and variations in the computational molecular properties give rise to good cluster separation. Score plots of PC3 versus PC1 and PC3 versus PC2 have also been analyzed and confirmed most of the extracted information above, albeit with less separations in certain pairs.The second scenario is more important to the practical application since it involves the separation of this pair in the actually used solvent ). The two points (green circles) are even better separated into Q2 and Q1 quadrons, respectively. The pair with enol 1 and fluorescein as fluorophores and with Ca2+ and K+ macronutrients, is provided in 2+.A scree plot from actual experimental data, employing 2+ selectivity with this receptor, (ii) the participation of various types of oxygen atoms, and (iii) the effect of negative charge. This work focuses on two forms of diglycolic moiety as receptor for Ca2+; diglycolic acid 4 and diglycolate anion 4a  the origin of Caanion 4a . In the 6) at the aldehyde terminal and the ether linkage (O3) is 2.74 Å. Cation recognition by 4 or 4a most probably involves three oxygens in the diglycolic receptor. Ca2+, Mg2+ and K+ having Shannon ionic diameter -3-one). Yield 58%. IR : 3525 (OH). 3068 (C-H)Ar. 2850, 2919 (C-H)Aliph. 1712 (C=O)Ester. 1503 (C=C)Ar. 1253 (O-C-O)Ether. 1H-NMR : 8.184 , 7.627 , 7.215 , 6.836 , 6.665 , 6.484 , 6.403 , 3.995 , 3.567 , 1.747 , 1.48 , 1.38 ; 1.23 , 13C-NMR : 184.93 , 165.59 , 164.16 , 159.06 , 154.63 , 134.01 , 132.54 , 131.34 , 130.79 , 130.40 , 129.75 , 117.46 , 114.85 , 114.33 , 105.54 , 100.62 , 69.08 , 65.81 , 32.76 , 32.76 , 32.75 , 32.55 , 29.55 , 29.47 , 29.40 , 29.37 , 25.76 . Elemental analysis; calculated: C, 74.08; H, 6.82; O, 19.10, found: C, 73.40; H, 6.14.7 in 20 mL of freshly distilled acetonitrile (A solution of 0.01 mol of diglycolic anhydride in dry acetonitrile was added dropwise into a gently stirred solution containing 0.01 mol of 4-dimethylaminopyridine (DMAP), 0.01 mol of triethylamine and 0.01 mol of compound onitrile . The mix1 2-oxy)undecyl)oxy)-2-oxoethoxy)acetic acid. Yield 87%. IR : 3525 (OH). 3074 (C-H)Ar. 2926, 2854 (C-H)Aliph. 1634 (C=O)Acid, 1732 (C=O)ester. 1453, 1421 (C=C)Ar. 1264 (O-C-O)Ether. 1H-NMR : 8.186 , 7.819 , 7.692 , 6.882 , 6.686 , 6.614 , 6.399 , 6.23 , 4.20 , 4.17 , 4.023 , 3.857 , 1.725 , 1.319 , 1.339 ; 1.14 , 13C-NMR : 184.33 , 171.54 , 170.20 , 165.82 , 163.90 , 158.82 , 154.13 , 150.47 , 136.12 , 131.05 , 130.57 , 130.48 , 129.82 , 129.11 , 117.14 , 114.69 , 111.26 , 105.01 , 101.29 , 83.23 , 79.59 , 68.43 , 64.62 , 29.54 , 29.44 , 29.40 , 29.14 , 28.91 , 28.79 , 28.54 , 25.98 , 25.83 , Elemental analysis; calculated: C, 67.95; H, 6.19; O, 25.86, found: C, 67.54; H, 6.30.All computations were performed with the G09  system o


Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca Normal stem cells are undifferentiated or partially differentiated cells that are characterized by their ability to self-renew, the process of bringing about indefinitely more cells of the same type, as well as to differentiate in more specialized mature cells. The term “stem cell” was coined by Ernst Haeckel in 1868 to describe the ancestor unicellular organism from which all multicellular organisms were supposed to evolve .Cancer stem cells (CSC), also known as tumor-initiating cells, share features of both cancer and stem cells. These cells constitute a sub-population of tumor-resident malignant cells responsible for recurrence, metastasis formation, and chemoresistance. Experimental evidence indicates that CSC exhibit “stemness” properties, that is, the ability of cells to perpetuate their lineage, to bring about differentiated cells and to interact with their microenvironment to maintain a balance between quiescence, proliferation, and regeneration . AccordiCSC have been reported to derive from “normal” tissue resident stem cells or from differentiated cells undergoing transformation . Further+/CD24−/low) together with expression of ALDH1 is a feature of breast cancer stem cells as compared to non-stem breast cancer cells [CSC show similar surface markers as normal stem cells in a given tissue, but a number of cell surface and intracellular biomarkers are commonly used to identify CSC among differentiated tumor cells and to isolate them. These markers include the clusters of differentiation (CD) CD44, CD24, or CD133, among others, the epithelial cell adhesion molecule (EpCAM) or the intracellular markers aldehyde dehydrogenase-1 (ALDH1) and the Notch, Wnt/β-catenin, Nanog, Sox2 pathways (for a more extensive review of CSC biomarkers in different tumors please see ). CD44 ier cells . Neverther cells . ALDH1 her cells . Intereser cells . The funer cells ; furtherer cells .Different signaling pathways and transcription factors have been reported to play an essential role in the state of cell stemness. Among them, developmental signaling pathways such as Notch, Wnt/β-catenin or Hedgehog play important roles in normal stem cell function. Notch signaling pathway is activated by the interaction of ligands of the DSL family with the receptor protein Notch, a single-pass transmembrane protein. The interaction of the ligand with the Notch extracellular domain (NECD) leads to the cleavage and release of the Notch intracellular domain (NICD) that acts as a transcription factor and interacts with the transcription factors of the CSL family and Mastermind . The CSLThe Hedgehog (Hh) pathway also plays a major role in normal stem cell and CSC biology and tumorigenesis. Hh signaling begins with the interaction of Hh ligands  with Patched-1, and to a lesser extent Patched-2, a twelve-pass transmembrane protein receptor. This process internalizes the Patched receptor and relieves the constitutive repression of the G-protein-coupled receptor Smoothened, which, in turn, leads to the nuclear translocation or the transcription factor Glioma-associated oncogene (Gli) resulting in the transcription of Hh target genes  Figure . The Hh 2+ pathway. The interaction of Wnts with Frizzled, a family of G-protein coupled receptors, leads to the activation of phospholipase C and, thus, IP3 -dependent Ca2+ release from the intracellular stores and subsequent Ca2+ influx across the plasma membrane [The Wnt/β-catenin signaling pathway promotes CSC self-renewal while reducing the differentiation of CSC to proliferating tumor cells . The Wntmembrane . The intmembrane . A recipmembrane .2+ concentration [2+ participates in several pathological conditions, including cancer. Resistance to apoptosis and chemotherapy, high proliferation rate or the ability to migrate and to invade different tissues, have been considered key features in all cancer types for years [2+ [2+ homeostasis has been included as a cancer hallmark.Calcium ion modulates a myriad of physiological processes, such as muscle contraction, secretion or gene transcription, through a sophisticated and well-orchestrated machinery that deftly tunes cytosolic Cantration . In addior years . As stator years ,5. All tears [2+ , thus, i2+ concentration are required to trigger several Ca2+-dependent downstream effectors that modulate cellular pathways, such as calmodulin, which is essential for cell cycle and proliferation [2+ concentration is achieved by the cells either by releasing Ca2+ from intracellular reservoirs, or by the opening of Ca2+-permeable channels in the plasma membrane (PM), which ensures an unlimited source of Ca2+ influx from the extracellular medium. Recent reports have demonstrated that CSC exhibit altered function in those mechanism  membrane, IP3- and ryanodine (Ry)- receptors, are vital for CSC stemness, proliferation, and metastasis in different cancer types, such as glioblastoma [Concerning the Cablastoma , melanomblastoma , and breblastoma .2+ entry, CSC present channels in the plasma membrane that are permeable to Ca2+ and could be gated by a variety of stimuli, such as voltage, second messengers or depletion of intracellular Ca2+ stores. For instance, over-expression of L- and T-type voltage-dependent Ca2+ channels is involved in tumorigenesis, proliferation, migration, and resistance to drugs in ovarian and glioblastoma CSC [2+ channel α2δ1 subunit, which modulates Ca2+ oscillation amplitude and the expression of different genes by keeping transcription factors in the nucleus, has started to be considered a tumoral marker in many cancers, such as lung [2+ entry and have a key role in the CSC physiopathology of different cancers. For instance, TRPC3, which is over-expressed in triple-negative breast cancer cells, is activated by lysophosphatidic acid, promoting the process of self-renewal in CSC [Regarding Catoma CSC ,52,53,54 as lung ,56, brea as lung , and liv as lung  or laryn as lung , with a l in CSC . Enhancel in CSC , glioblal in CSC , or neurl in CSC  and linkl in CSC  in the sl in CSC . Conversl in CSC ,66,67 anl in CSC . This inl in CSC .2+ reuptake, and the proteins involved, such as Ca2+-ATPases, Ca2+ exchangers or mitochondrial uniporter [2+ ATPase (SERCA) presents an important antiapoptotic function in breast CSC, by reducing Ca2+-dependent apoptosis during glucose deprivation. This process is mediated by CaMK2α, which triggers the activation of NF-κB, and, in turn, SERCA over-expression [2+ uniporter and the Na+/Ca2+ exchanger, located in mitochondria and in the plasma membrane, respectively, are highly expressed in glioblastoma CSCs [Caniporter ,71,72 mipression . Anotheroma CSCs ; however2+ influx from the extracellular medium into excitable and non-excitable cells, is physiologically triggered by the activation of phospholipase C (PLC) and the production of IP3, which subsequently leads to the release of Ca2+ from intracellular stores, mainly the ER, resulting in the activation of store-operated calcium channels in the plasma membrane and a rapid increase in cytosolic Ca2+ concentration [2+ homeostasis, which might contribute to tumor development, poor prognosis, and chemotherapeutic drug resistance [Store-Operated Calcium Entry (SOCE), a major mechanism for Cantration ,76. SOCEsistance .2+ concentration in the intracellular stores: (1) as the Ca2+ sensors of intracellular organelles, mediated by EF-hand Ca2+-binding domains in the N-terminus; and (2) as the transmitters of the filling state of intracellular Ca2+ stores to, and the activators of, Ca2+ channels in the plasma membrane. The latter is achieved by direct interaction between different domains within the STIM cytosolic C-region and the store-operated Ca2+ channels  family, STIM1 and STIM2, and their splice variants, possess a single transmembrane domain, with the N-region located either in the ER lumen or the extracellular medium, and a long cytosolic C-region ,79. Bothiewed in ,81,82).2+ Release-Activated Ca2+ (CRAC) channels that exhibit high Ca2+ selectivity and an inwardly rectifying current, termed ICRAC, which its exclusively conducted by members of the Orai family [2+ (SOC) channels, responsible to mediate a non-selective cation current denominated ISOC, formed by both, Orai1 and TRPC1, the first identified member of the canonical Transient Receptor Potential (TRPC) channel subfamily [SOCE could be mediated by two types of channels with different biophysical properties: (1) the Cai family ; and (2)ubfamily ,85.2+ channels, with four transmembrane domains spanning the PM and both, N- and C-terminus, facing the cytoplasm [Drosophila melanogaster Orai1 (dOrai1) presented a hexamer configuration, with the ion pore formed by the first transmembrane domain of the Orai subunits and located in the center of the complex surrounded by the remaining Orai plasma membrane domains [2+ influx, each of them with different biophysical properties that are extensively discussed here [2+ influx forming heteromers, such as the arachidonate-regulated Ca2+ channels (ARC), where three Orai1 and two Orai3 subunits form a pentamer [2+ influx [Orai1 was initially characterized as the main component of CRAC channel during a RNAi screening in 2006, when it was found that the Orai1 R91W mutation was responsible for abrogated CRAC channel function, critical for T-cell activation, in immunodeficient patients . Orai1 aytoplasm . Originaytoplasm ; however domains . The thrsed here ,91. Somesed here . In addipentamer , or inte+ influx .ISOC currents [+, Ca2+, and Cs+ [2+ than Orai1 and allow a massive ion influx from the extracellular medium, required for the maintenance of SOCE and store replenishment [TRPC1 belongs to the TRP channel superfamily, whose members ubiquitously mediate ion fluxes across the whole animal kingdom in a cell type-dependent manner . All TRPcurrents ,100,101. and Cs+ , are lesnishment .2+ stores depletion that will be sensed by STIM proteins  modulating each other, in a reciprocal dependency, in hypoxic hepatocarcinoma cells (HCCs). HIF-1α up-regulates STIM1 transcription, which in turn, induces higher SOCE, activating the CaMKII and P300 pathways, which are required for the accumulation of HIF-1α in HCCs .CDKN1A and G0S2 and the down-regulation of CCNB1 genes [Even less is known about the role of TRPC1 in CSC, since some of the inhibitors used to block SOCE, act over both Orai1 and TRPC1 channels. For instance, treatment with SKF96365, a SOCE inhibitor, impairs CSC proliferation in the glioblastoma stem-like cell line, TG1, triggering these cells to adopt a quiescent state by up-regulation of B1 genes . SimilarB1 genes . Even wh2+ current modulators due to their lower Ca2+ conductivity and greater fast Ca2+-dependent inactivation as compared to Orai1 [2+ influx pathways that are independent of the filling state of intracellular Ca2+ stores [2+ current mediated by a Orai1/3 channel [2+ influx mediated by the physical interaction between Orai1 and secretory pathway Ca2+-ATPase-2 [2+ influx mediated by the physical and functional interaction of Orai1 with the small conductance Ca2+-activated K+ channel 3 [+ channels by Orai1-mediated Ca2+ entry, which in turn leads to plasma membrane hyperpolarization, thus maintaining the driving force for Ca2+ influx and Ca2+ entry through Orai1 channels [As described above, native CRAC channels are hexameric structures comprised by the heteromeric association of Orai1, Orai2, and Orai3. Although all Orai family members can conform the channel, Orai2 and Orai3 also act as Cato Orai1 ,118. Sevto Orai1 ,120, livto Orai1 , and breto Orai1 ,123, Orato Orai1 , breast to Orai1 , oral [1to Orai1 , while Oto Orai1 , as wellto Orai1 , pancreato Orai1 , and proto Orai1  + stores . These p channel ,136,137,ATPase-2 ,139,140,hannel 3 ,142 or whannel 3 ,144. In channels ,145,146.2+ current, promoted a decrease in Sox2 expression, effect that was associated with reduced spheres formation and with the inhibition of their proliferation and self-renewal capacities [The role of Orai family proteins has also been described in the induction of CSC phenotype in a variety of cancers, such as glioblastoma, lung, and OSCC cancer cells. This CSC phenotype includes self-renewal capacity, tumor spheres formation, drug resistance, increased migration ability, and enhanced expression of stemness-related transcription factors and CSC-related markers ,147,148.pacities . Similarpacities . Converspacities .Regarding the role of Orai3 in the CSC phenotype acquisition in cancer cells, it has been demonstrated that Orai3 overexpression is correlated with tumoral aggressiveness and chemoresistance acquisition in breast cancer cells ,147. OraAltogether, the presented data support an essential role of SOCE mediators-STIMs, Orais, and TRPC proteins in the induction of CSC phenotype. However, our current understanding about the role of these proteins in cancer stemness is incomplete since the existing studies do not take in consideration their participation in other pathways, such as the store independent function of STIM1, Orai1, and Orai3, while the possible implications of Orai2 or TRPC1 in the stemness properties of CSC remain unclear.