Mycoplasma pneumonia (MP) infection might be pathogenically closely related to urticaria. This study is a nationwide population-based cohort study from 1997 to 2013, which investigated the association between MP infection and urticaria in Taiwan. A total of 1,175 patients were included for the study group, and 2,350 for the control group. Multivariate Cox regression analysis was performed to estimate the adjusted hazard ratio (aHR) for urticaria. Result showed that 254 patients with new-onset urticaria were involved in the study group and 465 incident cases in the control group. The incidence rates  of urticaria were 37.2 and 32.5 in the study and control groups, respectively. The relative risk is 1.1 (95% CI = 1.0–1.3) indicating no significant correlation between MP and urticaria. The multivariate analysis revealed that the risk of urticaria with MP infection  had no statistically significance difference compared to the control group. However, the risk of urticaria in MP-infected patients aged between 20 and 59 years old was found to have increased  prior to a diagnosis. Urticaria is a vasoactive disorder caused by vasoactive mediators released from activated mast cells which trigger an immune response . The resAlbeit urticaria is reportedly non-fatal, it interrupts daily life, such as sleeping due to itching of skin wheals. Clinically, urticaria is categorized into the immunologic and non-immunologic types. Immunologic urticaria is a hypersensitivity reaction mediated by: 1) IgE; 2) IgG autoantibodies; 3) circulating immune complexes to mast cell–expressing Fc receptors for IgG and IgM; and 4) T-cell activation . Non-immHelicobacter pylori has been reported to be the most bacterial infection associated with urticaria [Mycoplasma pneumoniae-related extrapulmonary diseases including urticarial were demonstrated to have significantly higher total serum IgE levels than those with classical respiratory infections by M. pneumoniae [M. pneumoniae may also play a role in the etiology of acute urticaria in adult. Therefore, this study aims to examine the correlation between MP and urticaria through understanding the underlying mechanism for MP-induced urticaria symptoms.The association between urticatia and infection has been reported more than 100 years , 9. Howerticaria , 15. Timrticaria  describerticaria . Recentleumoniae . Howevereumoniae  demonstrrelative risk of the study group compared to the control group. Under the NHI Program, 98% of Taiwanese benefit from the compulsory, single-payment health care system since 1996. All data derived from the NHIRD are anonymized for epidemiologic research that contains the information of patients’ demographics, health care service data, medication dispensation, International Classification of Diseases, and so forth. Due to the large sample size provided by the NHIRD, previously reported diagnostic information of the Longitudinal Health Insurance Research Database (LHIRD) derived from the NHIRD has been established in 2000. The data used in this study is de-identified, and is derived from the LHIRD from 1997 to 2013. The de-identified, secondary data required no informed consent, and was approved by the Institutional Review Board of Chung Shan Medical University Hospital (CS15134).This was a retrospective study, because the data collected in this study are from an existing record, the National Health Insurance Research Database (NHIRD) from the Taiwan National Health Insurance (NHI) Program, which is managed by National Health Research Institutes (NHRI) , 21. ThiPatients with a first-time diagnosis of MP were identified from the NHIRD by using International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) code 483.0 between January 1, 1997 and December 31, 2013. Because the administrative datasets are always criticized for their poor diagnosis validity, this study defines the MP patients who had emergency department (ED) visit or hospitalization for MP in their medical claims. We excluded 11 patients who were diagnosed with urticaria before 2002; and 839 patients who were diagnosed with MP before urticarial diagnosis. In addition, 1,175 patients with MP were included as exposure individuals. We assigned the ED visit for receiving a diagnosis of MP as the index date. The four controls were age and sex, individually matched with one MP patient, and they were still at risk at the index date. Furthermore, each individual in the study group was propensity score-matched with a non-MP individual. Details (including control group) are being discussed in the following sections. Propensity score matching was performed at a ratio of 1:2 to correct potential confounders including age, gender, and comorbidities. Finally, we included 1,175 patients with MP and 2,350 propensity score matched controls in this study .The identification of patients with urticaria was based on records ascertained by using ICD-9-CM codes 708.x. Patients with urticaria needed to have at least two outpatient visits or one hospital admission to fulfill the case definition. In addition to this, we also find co-morbidities diseases related to urticaria and MP infection, which included rheumatoid arthritis (RA: ICD-9-CM codes 714.0), asthma (ICD-9-CM 493), allergic rhinitis (ICD-9-CM 477), diabetes mellitus (ICD-9-CM 250), zoster , hepatitis B virus infection , hepatitis C infection , gout (ICD-9-CM274). These co-morbidities were considered as the covariates in the multivariate analysis.The absolute standardized difference (aSD) was the indication of difference in study variables between the study and control groups, particularly the population-based and large sample study. The small (<10%) standardized difference agreed with the balance between groups. The Cox proportional hazard regression models were used to calculate the hazard ratios (HR). There were 95% confidence intervals (CI) of associations between pre-existing urticaria and MP infection after adjusting at index date for age, sex, urbanization, co-morbidities or medication usage. The Kaplan-Meier curve was performed for the cumulative probability of urticaria for patients with MP infection. All data analyses were performed using SAS 9.4 , P value less than 0.05 is considered to be statistically significant.The selection of study participants is shown in Result shows that 49.4% and 50.6% of the MP-infected patients were female and male, respectively. After propensity score matching, the difference of baseline characteristics were all small (10%) among the study groups . Table 1p = 0.0913). The adjusted HR (aHR) at 0–12 months, 12–48 months and 48–120 months were 1.272 (95% CI = 0.938–1.724), 1.337 (95% CI = 1.067–1.676) and 1.005 (95% CI = 0.827–1.22), respectively. The aHR for urticaria among subjects with MP was 1.2  after 1:4 age and sex matching  for 3 days. Wu CC et al. found that azithromycin-treated urticaria patients had significantly shorter mean (SD) convalescence period, shorter duration for complete resolution when compared to untreated urticaria patients , 23. Thinfection .Mycoplasma [Urticaria is a benign disease for adults and children since the symptoms are often short-lived, and can be treated with antihistamine. Clinically, patients hospitalized for severe urticaria, or having refractory urticaria are difficult to be treated by antihistamines and any other types of treatments. MP infection has been observed and implicated to play a role in the refractory urticaria. MP infection in adults and children can lead to complications in lungs and other organ (e.g. kidney), as well as skin disorders, such as Stevens-Johnson syndrome and erythema multiforme . Interescoplasma . As a recoplasma . In addicoplasma . The symcoplasma . Taken tThere are several limitations to this study. Firstly, there is the lack of public alertness for MP infection, leading to undiagnosed MP infection and lack of medical service, and thus less people seek urticaria treatment. Secondly, due to the variation in clinical definition for urticaria, potential MP infection can be omitted as a risk factor during diagnosis. Thirdly, severe MP-infected patients who were hospitalized were not included in this study. In addition, the data in this study is limited to Taiwanese population; therefore, other ethnic groups and nations worldwide might not be applied in this finding. However, the data analyzed in this study is pioneer, and is convincing due to the randomized, and large sample size.This 13-year population-based cohort epidemiological study identified an increased risk for urticaria among patients with MP infection. This study provided insight into early awareness of MP infection among urticaria patients and early antibiotic treatment taken as precaution for high risk patients.


This integrative literature review synthesizes the primary research evidence on mentoring female health academics published from 2000 to 2018, to identify the benefits, enablers and barriers to mentoring women. The need for this review is underpinned by the magnitude of change in higher education, the high number of women in health disciplines, limited progress in advancing women’s academic careers, escalating role expectations, faculty shortages and staff turnover. Data were sourced from Scopus, PubMed, EMBASE and Cumulative Index of Nursing and Allied Health Literature. Twenty-seven studies were included. Although effective mentoring facilitates personal and career development, academic craftsmanship, psychosocial support and job satisfaction, it is complicated by organizational factors and personal and relational dynamics. Enablers of mentoring are mentor availability and expertise, supportive relationships, mutuality and responsiveness. Lack of, or inadequate mentoring compromise women’s job satisfaction, career development and academic productivity. Providing female health academics access to experienced, well-connected mentors with common interests who are committed to advancing their career, is an investment in optimizing potential, promoting supportive work environments and increasing productivity and retention. Realizing the institutional potential that mentoring female health academics offers, is contingent on academic leaders valuing mentorship as faculty business and understanding the role that the contemporary academic environment plays in achieving mentoring outcomes. Further empirical and longitudinal research is needed to evaluate effective approaches for mentoring women in the contemporary academic environment. Academic mentorship features prominently in orientation, support of new faculty transitioning to the academic role, faculty development, career advancement, job satisfaction and retention –4. HowevThe roots of mentoring lie in Greek mythology where a mentor was considered a sage and trusted counsellor , 19. TraRecent changes in higher education have spawned alternative forms of mentoring such as collegial, facilitated peer, functional, online and distance mentoring , 20, 21.…that may vary along a continuum from informal/short-term to formal/long-term in which faculty with useful experience, knowledge, skills, and/or wisdom offers advice, information, guidance, support, or opportunity to another faculty member or student for that individual’s professional development (p. 67) .This more flexible conceptualization of mentoring reflects efforts to adapt to the restructured higher education environment which has become a corporatized global knowledge industry , 7, 22. Despite significant attention to advancing women’s careers in academic medicine, only ‘modest’ progress has been achieved , 25. As In a systematic review of mentorship in academic medicine, Sambunjak et al. , 29 highWe adopted an integrative review process based on the five-stage process proposed by Whittemore and Knafl : developAlthough diversity is a key strength of the integrative review process, it renders quality appraisal somewhat problematic and limiting . WhittemOur research questions were: What are the benefits, barriers and enablers of mentoring female health academics, the consequences of a lack of, or inadequate mentoring, and the gaps in knowledge about mentoring women?We conducted a systematic and rigorous search in July 2018 across the electronic databases considered to encompass a wide-ranging multidisciplinary span of research relevant to the healthcare domain: PubMed, EMBASE, CINAHL and Scopus. Boolean connectors AND, OR and NOT were used to combine search terms including mentor*, women, female, higher education, universit* and academi* .Our search criteria incorporated peer-reviewed primary research on the mentorship of female health academics published in English from 2000 to July 2018. Research articles on mentorship where the majority (>90%) were female participants or those reporting gendered findings were also included. We excluded articles published prior to the year 2000 because of the significant changes occurring in higher education that have influenced the need for mentoring and women’s access to mentorship. Reviews, theses, conference proceedings and editorials were excluded, as were studies involving students and clinicians.The database search generated 815 records. After removing duplicates, 372 potential studies were identified. Three authors independently screened the titles and abstracts of prospective papers against the inclusion criteria and identified 57 studies. In the case of disparities, consensus was achieved by examining the full-text and collaborative discussion. Through this systematic process, 34 studies were removed leaving 23 for inclusion. After scanning reference lists of included and review papers, four additional studies were identified, yielding a total of 27 .A summary table was generated synthesizing the data from included studies. Data extracted included author(s), year of publication, country of origin, purpose of study, sample, design and data collection, method(s) of analysis and significant findings germane to the review aims . ResultsAnalysis of the study characteristics presented in career development, personal development, academic craftsmanship, psychosocial support and job satisfaction. The themes associated with barriers to mentoring were personal and relational dynamics and organizational factors. Mentoring was enabled by mentor availability, mentor expertise, supportive relationships, mutuality and responsiveness to shifting needs. The lack of, or inadequate mentoring for female academics led to decreased job satisfaction, limited career development and reduced academic productivity.The key themes reflect the review aims; namely, the benefits, barriers, enablers and outcomes of lack of, or inadequate mentoring for women. Thematic analysis revealed 15 major sub-themes . The theThe majority of the studies reviewed found that mentoring benefited career development by engendering valuable professional growth and fostering women’s careers. Mentorship provided a structured process for career planning and professional development , 48, 55,Mentoring provided women access to successful role models and promoted psychological empowerment and assertiveness, self-efficacy, self-esteem, confidence, job related well-being and problem-solving , 42, 44.The skills deriving from mentoring such as proficiency in academic teaching, research, writing , publicaHaving a mentor fostered psychosocial support by providing encouragement, motivation, confidence, assertiveness, a sense of caring, inspiration and guidance , 52, 59.Mentoring was associated with job satisfaction, tenure and retention , 41, 57 The availability of mentors was key to female faculty having access to mentoring , 35. AvaHaving access to an experienced mentor with expertise in clinical practice, teaching and research facilitated role modelling . Mentor The efficacy of mentorship was enhanced by having a supportive relationship , 40, 59,Repeated references to effective mentoring necessitating a certain rapport and ‘chemistry’ were reinforced by studies demonstrating that matching mentors and mentees based on mutual interest and shared understanding achieved better outcomes for both , 51, 54.Facilitated and collaborative peer mentoring were valued for taking the power out of the mentoring relationship and fostering shared understanding. A benefit of facilitated peer mentoring was that it involved senior academics overseeing peer mentors and required limited institutional resourcing. This form of peer mentoring was valued as a successful long-term strategy to provide women access to colleagues who understood their situation, shared their academic interests and sought to progress their academic skills to achieve career goals , 57.One of the defining needs for female academics in the studies reviewed, was for mentors to be responsive to their shifting needs over time. The need for sameness between mentors and mentees reduced with age and experience . There wAlthough over two thirds of the articles reviewed provide a body of evidence supporting the value of mentoring for female health academics, there were signals that the workplace was not always conducive to mentorship or realising its potential.Personal and relational barriers to mentoring for female faculty included the variable quality of available mentors and incongruent assignment of mentors , 55. TheThe power differential inherent in the hierarchical structure of traditional dyadic mentoring relationships represented an important relational dynamic that sometimes rendered mentees vulnerable to exploitation , 53, 56.Organizational barriers to mentoring female faculty included the lack of mentoring available to women , 51, 53,Eleven of the 27 studies reviewed identified the consequences of a lack of, or inadequate mentoring to be decreased job satisfaction, limited career development and reduced academic productivity.Lack of mentorship increased job stress and psychological disempowerment, limited women’s networking opportunities and detracted from job satisfaction by creating a sense of isolation, discontent and discouragement , 44, 46.Without adequate mentoring, career development, academic productivity and promotion were compromised , 38 and The studies provide evidence indicating links between the lack of or inadequate mentoring and factors that disrupt or compromise academic productivity by limiting effective transition to the academic role, networking, academic craftsmanship and collaboration , 46, 56.More than half of the articles reviewed highlighted specific gender issues. Despite women more often considering mentoring more important than men , and demMany studies reported not having enough senior women to mentor junior women , 46, 53.While Wasserstein, et al.  found noThis review has synthesized the research evidence about mentorship generated in the aftermath of corporatist changes in higher education, with the specific intent to identify the benefits, enablers and barriers to female health academics accessing suitable mentorship, the consequences of the lack of, or inadequate mentorship, and gaps in knowledge. The results, though somewhat limited and the evidence variable and context-bound, reinforce the value of mentoring female health academics as an ideal strategy for improving academic craftsmanship and productivity, promoting women’s career advancement, building female mentoring capacity and promoting retention, issues that resonate with the broader literature , 16, 28.The personal, professional and institutional benefits of mentoring women feature prominently in this review. The accrual of benefits over time are also congruent with the findings of others , 28 and For the ‘craftsman’, the driving motive for performance is mastery  and the Arguably, the notion of academic craftsmanship in the context of mentoring, fits like a hand in a glove, whereby the master academic craftsman, guides the development of a novice or junior craftsman, teaching them their craft somewhat like an apprentice; supporting, coaching, reviewing and refining their mastery of the role and promoting their status in the field . HoweverFactors enabling mentoring of female health academics were the availability of a suitable mentor, mentor expertise, supportive relationships, mutuality and responsiveness to shifting needs. However, similar to our findings, others have found that women have limited access to suitable senior female mentors and that personal and relational factors compromise mentorship between men and women , 4. PartSambunjak, et al.  identifiCollectively, the studies reviewed have reinforced the findings of others that a top-down approach to mentoring, whereby a mentee is assigned a senior mentor without regard for personal factors, may undermine outcomes , 18, 66.The structural barriers that block mentorship for female faculty reflect the higher education environment; overwhelming teaching loads, shortage of mentors and lack of time, institutional support and incentives , 56, 67.This review found the consequences of inadequate mentoring to be isolation, disempowerment, job dissatisfaction, stress and limited career development; factors conducive to burnout and attrition , 11, 69.Despite the need to address gender inequities and career advancement, much of the literature on mentoring in the health sciences is generalised, the level of evidence weak and lacking in gender analysis. The gendered and work similarities between medical, nursing and other health academics suggest mentorship requires attention. Despite the prevalence of women in the health sciences, there were few studies in areas other than medicine that examined mentorship from a gendered perspective. This is an important oversight as female faculty often see themselves as ‘outsiders’ in the context of the academic workplace , 65, andAlthough Athanasiou, et al.  found feDespite various models of mentoring being evaluated, no studies compared the gendered outcomes of different models of mentoring or the use of online mentoring. Most of the studies reviewed utilized cross-sectional, self-report designs with minimal use of validated instruments; factors which limited comparison across studies and precluded a meta-analysis. While six studies examined follow-up, outcomes ranging from 10 months to 6 years, only three studied the longer-term benefits of mentorship over four years or more. The dearth of longitudinal and high-level empirical studies that consider gender in the context of the contemporary higher education environment, continue to limit the evidence available about specific models of mentoring and their outcomes for women.This review was limited to studies of mentoring of women in health faculty, published in English in peer-reviewed journals from 2000 to 2018. Though we undertook a comprehensive search of four databases deemed most likely to elicit mentoring studies in faculties of health, it is possible some relevant studies may have been missed. As this was an integrative review and we sought to include all valuable informational content, we included all studies that met our inclusion criteria, and did not undertake quality ratings. The preponderance of data from academic medicine flags a bias in understanding yet highlights the priority given to mentoring as an investment in recruiting and retaining medical academics. While the lack of conceptual clarity around the notion of mentoring limited our ability to synthesize data, it yielded rich insight into new models and varied possibilities.This integrative review has synthesized what is known about the benefits, enablers and barriers to mentoring in the context of female academics in health faculties from 2000 to 2018, the effects of a lack of, or inadequate mentoring, and gaps in knowledge. In synthesising the evidence, this review provides a compelling case for institutions to invest in mentoring programs as a mechanism to support role transition, empower and retain new faculty and build female mentoring capacity. These results provide evidence that the provision of effective mentoring for female health academics is contingent on the organizational environment, specifically, workplace structures and relationships, and represents a long-term investment that can benefit academics and their mentors. Furthermore, mentorship assists higher education institutions to address faculty shortages, increase retention and productivity, advance female academics’ careers and build female mentoring capacity. The urgent need to address these issues necessitate that new strategies be adopted to capture the enablers and circumvent the personal, relational and organizational impediments to mentoring. This review highlights the need for further research on academic mentorship from a gendered and environmental perspective, particularly large scale empirical studies measuring comparative outcomes of different models of mentoring, and longitudinal studies into the value of mentoring and its potential to advance women’s academic careers.S1 Table(DOCX)Click here for additional data file.


While the majority of patients with acute myeloid leukemia (AML) are above the age of 65 years at diagnosis, the outcome of older AML patients remains disappointing. Even if standard intensive chemotherapy induces morphologic complete remission (CR1), relapses in older AML patients are common leading to poor long-term survival outcomes. Since autologous hematopoietic stem cell transplantation (HCT) offers distinct anti-leukemic effectiveness while avoiding graft-versus-host disease associated with allogeneic transplantation, it represents an option for consolidation treatment in selected older AML patients. However, prospective studies in older AML patients assessing the benefit of autologous HCT compared to chemotherapy consolidation or allogeneic transplantation are lacking. Consequently, clinicians face the dilemma that there is considerable ambiguity on the most appropriate consolidation treatment for older AML patients in CR1. This review highlights the possible role of autologous HCT for consolidation in older AML patients reaching CR1 after induction treatment. Acute myeloid leukemia (AML) is predominantly diagnosed in older patients with a median age of 67 years . The outAutologous HCT has become a therapeutic option for first-line consolidation in younger adults with AML with good and intermediate risk features . It offeA number of retrospective, mostly single-center studies, have reported their experiences with autologous HCT in older AML patients with predominantly promising results, albeit inevitably in a selected patient population ,8,9,10. Noteworthy, the concept of an older AML patient was widely differing among the available reports. Whereas earlier studies identified such patients as being older than 50 years , recent For this review, we retrospectively analyzed all consecutive adult AML patients undergoing autologous HCT in CR1 after two cycles of induction chemotherapy between January 2004 and June 2018 at a single academic institution . Clinical characteristics of these 148 patients are summarized in Only limited data are available assessing differences in tolerance and outcome between younger and older AML patients undergoing autologous HCT consolidation in CR1 after induction treatment. In our single-center cohort introduced above comprising 148 consecutive AML patients consolidated in CR1 with autologous HCT, we observed a median PFS of 30 months and a median OS of 54 months for the entire cohort . SubsequGiven the comparable survival rates in younger and older AML patients in our cohort, these data challenge earlier observations of longer PFS and OS in patients younger than 50 years compared to older patients ,8. Moreop = 0.0166) and OS  compared to patients of this age cohort without any consolidation at all [A prospective comparison between older AML patients in CR1 after induction treatment receiving autologous HCT for consolidation and patients without any consolidation at all is lacking so far. Consequently, recommendations rely on retrospective comparative series with relevant selection bias precluding definite conclusions. In a previous report of AML patients treated at our center, we found that patients ≥65 years with autologous HCT consolidation achieved significantly longer PFS  or chemotherapy consolidation (n = 259) [p = 0.02), with a non-relapse mortality rate of only 4% in the autologous HCT group in this multi-center international trial. The leukemia-free survival (LFS) rates at five years were 38% versus 29%, and the OS rates were 44% versus 41% for the autologous HCT versus chemotherapy consolidation cohorts. Remarkably, 91% of the patients assigned to autologous HCT actually received the transplantation. More recently, the HOVON/SAKK groups [n = 337), chemotherapy (n = 271), or autologous HCT (n = 152). The autologous HCT group had better OS than the chemotherapy group , and the relapse rate was lower (p = 0.003). In summary, these data obtained in patients less than 60 years suggest an advantage for autologous HCT, compared with chemotherapy for consolidation, in terms of relapse rate.Compared to chemotherapy consolidation such as with mitoxantrone and etoposide in the HOVON/SAKK tradition, autologous HCT provides timely hematologic recovery, thereby reducing the probability of infectious or hemorrhagic complications ,9,10,11.n = 259) . The relK groups  comparedHowever, similar prospective data are lacking in AML patients older than 60 years. In the absence of randomized studies, available retrospective data suggest that autologous HCT in selected older AML patients is not increasing TRM. Moreover, older patients appear to benefit from the anti-leukemic effect of autologous HCT similarly to younger AML patients. Finally, one must acknowledge the higher proportion of patients with unfavorable-risk features in the older age cohort, and such patients primarily remain candidates for allogeneic transplantation.Due to the graft-versus-leukemia (GvL) effect, allogeneic HCT provides the most potent anti-leukemic treatment. If severe toxicity and TRM of this procedure would be below 5%, every patient with AML would be a candidate for this strategy. However, high TRM and significant decrease in quality of life due to graft-versus-host disease (GvHD) continue to limit this modality to instances in which the relapse-free benefit by far overcomes the treatment non-relapse toxicity.Autologous HCT does not have the advantage of significant GVL. Consequently, relapse is the leading risk of this treatment. Importantly, however, TRM after autologous HCT is low and there is no GvHD. An increasing number of reports propose that AML patients up to the age of 75 years can safely receive myeloablative chemotherapy supported by autologous or allogeneic transplantation ,13,14,15p < 0.001), higher TRM (p < 0.001), and similar OS (p = 0.19). The EBMT compared, retrospectively, the results of three different HCT approaches in CR1: autologous HCT (n = 1202), allogeneic HCT from matched unrelated donor (MUD) 10/10 (n = 1302), and allogeneic HCT from mismatched unrelated donor (misUD) 9/10 (n = 375) [p = 0.84), and misUD versus autologous HCT (p = 0.49). Similarly, a retrospective study based on the Japanese HCT registry compared autologous HCT (n = 375) with allogeneic HCT from sibling donor [n = 521) versus peripheral blood (n = 380). In the multivariate analysis, the OS of autologous HCT versus allogeneic HCT was not statistically different in both allogeneic HCT groups.In AML patients up to 60 years, the HOVON/SAKK leukemia groups demonstrated  that alln = 375) . The OS ng donor , with thn = 116) were compared with those who underwent haplo-identical allogeneic HCT (n = 50), the autologous cohort showed a higher relapse incidence , much lower NRM , and better OS . In summary, retrospective registry studies indicate that autologous HCT is at least as good as allogeneic HCT from alternative donors in terms of OS, but this issue awaits further clarification in prospective studies.Consistently in these studies, the relapse rate was higher for autologous HCT versus allogeneic HCT, but the TRM was lower. Thus, when an older AML patient considered fit for induction treatment has achieved CR1, the debate will be whether to propose autologous or allogeneic HCT. No prospective trial in older AML patients has dealt with this question, and the available retrospective data are limited. This is even truer in older AML patients with no available donor necessitating the evaluation of alternative donors. The EBMT retrospectively compared the results of autologous HCT and haplo-identical allogeneic HCT in AML patients . When pap = 0.048) and OS (p = 0.058) compared to chemotherapy consolidation, whereas allogeneic HCT was associated with improved leukemia free survival (p = 0.011), but not OS (p = 0.10). A retrospective EBMT cohort study [p = 0.049), whereas it did not differ significantly between autologous HCT and MUD (p = 0.9). The comparison between a subgroup of intermediate risk AML with normal karyotype and wild-type FLT3 and NPM1 indicated again no difference in OS between autologous HCT and MUD (p = 0.88) [There is a consensus that young AML patients with adverse risk features should undergo allogeneic HCT in CR1 whereas favorable risk AML patients in CR1 preferably should have chemotherapy consolidation or, alternatively, autologous HCT. In addition, the SAKK/HOVON leukemia groups  assessedrt study  found si = 0.88) . Finally = 0.88)  summarizFor older AML patients, there is a complete lack of randomized prospective data based on cytogenetic and/or molecular risk groups. All current recommendations rely on reports in young fit AML patients suggesting that there is an unmet need to test these recommendations in older patients. However, the use of biomarkers like minimal residual disease (MRD) may have a different application in older AML patients as compared to young AML; accordingly, the simple translation of one to another is problematic and needs prospective clarification ,2,19.A prerequisite of autologous HCT after confirmed achievement of CR1 is the collection of a sufficient number of autologous stem cells usually obtained following hematologic recovery after the second induction cycle. Differences between younger and older AML patients in the success rates to mobilize CD34+ hematopoietic stem and progenitor cells are widely missing. In a retrospective analysis including 40 patients, Ferrara et al. demonstrated similar CD34+ yield and successful mobilization rates in a limited series of patients above and below 60 years, and they concluded that age does not significantly affect mobilization and collection of peripheral stem cells . HoweverThe predominant use of peripheral blood as the source of autologous stem cells (PBSC) has fueled the interest in predictive parameters associated with the mobilization procedure and the successful engraftment after re-transfusion of PBSC. Compared with stem cells obtained from bone marrow sampling, the use of PBSC is associated with faster recovery of neutrophils and platelets, shorter hospitalization, reduced need of blood transfusions and fewer days of intravenous antibiotics, whereas survival outcomes do not differ ,24,25. IBased on the considerations above, one might conclude that favorable or intermediate risk AML patients, who failed to mobilize a sufficient number of peripheral blood stem cells, might draw similar (or even particular) benefit from autologous HCT consolidation. However, the number of AML patients failing collection of autologous stem cells is poorly studied. We previously reported in a retrospective study of uniformly treated consecutive AML patients in CR1 a 19% rate of mobilization failure in those patients in whom we effectively initiated G-CSF stimulation after two cycles of induction treatment in order to initiate subsequent PBSC collection .Risk factors for poor mobilization in AML patients involve increasing patient age mediating stem cell senescence, loss or dysfunction of the stem cell niche, and altered bone metabolism . Specifi6 CD34+ cells/kg, whereas PBSC mobilization was insufficient in 16 patients (19%). Thus, the mobilization failure (MF) rate appears to be higher in AML as compared to myeloma or lymphoma patients.Collaborative groups or single centers usually aim at collecting hematopoietic CD34+ stem and progenitor cells (PBSC) following hematologic recovery from neutropenia after induction chemotherapy. However, few studies have investigated mobilization failure rates in AML patients in CR1, and the rate of AML patients failing the collection of a sufficient number of CD34+ cells is unknown. We have studied 85 AML patients with morphologic CR achieved after the first cycle of induction treatment (early CR1) . Sixty-np = 0.0074), and normal karyotype patients with mutated NPM1 and wild-type FLT3 were more common in the MF group  [Characteristics of AML patients with MF remain to be elucidated. In our small series of 16 MF patients reported above, these patients had higher numbers of platelets at diagnosis  .A rescue strategy for AML patients with MF is harvesting stem cells from the bone marrow, with all the inconveniences associated with this procedure. The alternative use of the CXCR4 antagonist plerixafor in this condition is problematic given the potent mobilization of residual leukemic stem cells associated with its use in AML patients . Thus, m2 of vinorelbine together with G-CSF as a fully ambulatory mobilization procedure allowing highly predictable collection of a sufficient number of PBSC at day 8 [We previously explored the potential of the cytotoxic compound vinorelbine together with G-CSF as a remobilization procedure in AML patients in first remission who experienced MF after standard induction chemotherapy . In myelat day 8 ,37,38. Iat day 8 . We obseat day 8 ,40. Vinoat day 8 ,42.The combination of busulfan 16 mg/kg administrated over four days given every six hours together with cyclophosphamide 120 mg/kg applied on two consecutive days (BuCy) is the preferred conditioning regimen for autologous HCT in AML patients. However, no prospective randomized trial has ever challenged this regimen, and it is safe to say that optimizing the conditioning regimen before autologous HCT is a largely neglected topic in AML treatment.2) as conditioning regimen (BuMel) [p = 0.003), the LFS (p = 0.004), and the OS (p = 0.0007) of the patients who received BuMel were better than of those who got BuCy, whereas the NRM was similar in both groups (p = 0.66). A recently published subgroup analysis of the EBMT group indicated that the survival advantages associated with BuMel conditioning were limited to poor-risk or FLT3 mutated AML patients whereas the outcome in favorable or intermediate risk AML was comparable [FLT3 both conditioning regimens are valid.Noteworthy, the EBMT retrospectively compared the results of 596 AML patients in CR1 who received the BuCy regimen with 257 patients who had a combination of busulfan with high-dose melphalan (140 mg/m (BuMel) . Remarkamparable . In summIn summary, prospective studies are lacking, which compare different conditioning regimens before autologous HCT in older AML patients. All recommendations for older patients rely on studies conducted in younger patients, and optimizing autologous HCT for older AML patients remains an unfinished topic.FLT3, or, more recently, IDH1 and IDH2. However, maintenance treatment with targeted compounds and/or with hypomethylating agents following autologous HCT may also represent promising strategies to be studied to further improve the outcome of older AML patients after HCT.Whereas there is increasing interest in autologous HCT consolidation in good and intermediate risk older AML patients in CR1, the basis for this option largely relies on retrospective reports, many of them based on single center experiences or selective registry data. These studies typically suggest that selected older AML patients in CR1 similarly tolerate (and benefit from) conditioning treatment and autologous HCT as younger AML patients. However, treatment recommendations for older AML patients concerning autologous HCT consolidation suffer from a lack of data obtained from prospective studies, and such data are needed to provide a rational for proposing autologous HCT consolidation to older AML patients as stated in previous reviews by others ,46,47. I


Ischemic heart diseases (IHD) are the leading cause of death worldwide. Although the principal form of treatment of IHD is myocardial reperfusion, the recovery of coronary blood flow after ischemia can cause severe and fatal cardiac dysfunctions, mainly due to the abrupt entry of oxygen and ionic deregulation in cardiac cells. The ability of these cells to protect themselves against injury including ischemia and reperfusion (I/R), has been termed “cardioprotection”. This protective response can be stimulated by pharmacological agents  and non-pharmacological procedures . Several intracellular signaling pathways mediated by chemical messengers  and cytoplasmic organelles  are involved in cardioprotective responses. Therefore, advancement in understanding the cellular and molecular mechanisms involved in the cardioprotective response can lead to the development of new pharmacological and non-pharmacological strategies for cardioprotection, thus contributing to increasing the efficacy of IHD treatment. In this work, we analyze the recent advances in pharmacological and non-pharmacological strategies of cardioprotection. Althougradicals . As this2+ influx through L-type voltage-dependent Ca2+ channels (VDCC) [2+ influx stimulates the Ca2+ release from sarcoplasmic reticulum (SR) through the activation of Ca2+ channels regulated by the ryanodine receptors (RyR) [2+-release Ca2+-induced (CICR) mechanism transiently amplifies the cytosolic Ca2+ concentration ([Ca2+]c), which stimulates the activity of contractile proteins, unleashing the contraction of cardiac cells c during the contraction of cardiac cells is restored to basal levels (resting) by Ca2+ sequestration SR and other cytoplasmic organelles, and Ca2+ extrusion by ionic transporters located in plasma membrane of cardiac cells [2+]c promotes the relaxation of cardiac cells (diastole) c in cardiac cells.The SR has an important role in the regulating contraction-relaxation cycle in cardiac cells, acting as cytoplasmic reservoir of Ca2+ . SR sequ SERCA2A . The exce (PMCA) ,9. The Nse mode) . Thus, S2+ during transient elevations of [Ca2+]c in cardiac cells, contributing to the contraction-relaxation cycle m) m) . In carder (MCU) , and itsr (mNCX) ,14. Thus2+]c in cardiac cells are responsible for fine control of the heart rate. Thus, changes in Ca2+ extrusion or buffering may precipitate the spontaneous release of Ca2+ from the SR, and generate triggered activity as the delayed after depolarization m, increasing the activity of dehydrogenases of Krebs cycle, and consequently stimulating the ATP synthesis c, which acts as driving force to reverse the transport of Na+ and Ca2+ by plasma membrane NCX, causing cytosolic Ca2+ overload, which favors an increase in the incidence of arrhythmias during ischemia and early reperfusion [2+ and mitochondrial influx of Na+ on reperfusion in hearts submitted to cardiac I/R injury [Oxygen deprivation caused by cardiac I/R injury leads to a deficit in ATP production in cardiac cells due to inhibition of ATP-dependent ion transporters, such as Naerfusion . TreatmeR injury ,105,106.2+ and Na+ currents by LMWH attenuates the Na+ and Ca2+ overload in cardiac cells, reducing the incidence of arrhythmias and lethality caused by cardiac I/R injury [+ channels [Low molecular weight heparins (LMWH), such as trisulfated disaccharide derived from heparin (TDH), have antiarrhythmic and cardioprotective properties ,113,114.2+ homeostasis and excitation-contraction coupling, producing cardioprotection [In cardiac cells, NO is synthetized by action of NOS isoforms on L-arginine. NO participate in cellular Caotection ,117,118.otection ,120. Admotection .+ and Ca2+ cytosolic overload during reperfusion by phosphorylating of fosfoleman (Na+/K+ ATPase modulating protein), increasing the Na+ efflux via Na+ pump, and reduce the Ca2+ influx via plasma membrane NCX [2+ to cytosol and phosphorylates fosfolambam, increasing the sequestration of cytosolic Ca2+ by SR via SERCA [2+ [NO formed from L-arginine regulates different cellular processes by activation of soluble GC, increase in cGMP formation and PKG activation . The PKGrane NCX . This PKrane NCX . PKG stirane NCX . In the ia SERCA . PKG phoERCA [2+  and phosERCA [2+ .Our studies have shown that the administration of micromolar concentrations of L-arginine or sodium nitroprusside reduces the heart rate and contraction force of the isolated heart ventricle in rats submitted to prolonged tissue hypoxia ,82,127. 2+ homeostasis  and NO biosynthesis in cardiac cells.Resveratrol  is a naturally occurring polyphenol antioxidant compound found in Polygonum cuspidatum, grapes, peanuts and berries, as well as their manufactured products, especially red wine, with cardioprotective effects. Resveratrol is a pharmacologically active compound that interacts with multiple targets in a variety of cardiovascular disease models to exert protective effects or induce a reduction in cardiovascular risks . Studies2+ overload [The cardioprotective effects of resveratrol result from multiple biological actions. This polyphenol exerts protective effects against mitochondrial oxidative stress and apoptosis in neonatal rat cardiac cells induced by hypoxia/reoxygenation injury , and incoverload . Resveraoverload . These f2+ channels function [Methylene blue is a redox drug with reported protective effects on mitochondria. The methylene blue ameliorates cyanide toxicity by normalizing the oxidation-reduction state and Cafunction . Methylefunction ,143. TheVasoplegia is a term that describes excessive loss of vascular muscle tone leading to a distributive shock status . Vasople2+ homeostasis disrupted by hydrogen sulfide poisoning [2+ homeostasis and L-type VDCC), reduction in the risk of arrhythmias by stabilization of membrane potential, and preservation of cellular bioenergetics [The methylene blue counteracts the effects of hydrogen sulfide cardiotoxicity by improving cardiac cells contractility and intracellular Caoisoning . In vivooisoning . The proergetics . These fMetabolic syndrome is a complex disorder represented by a set of cardiovascular risk factors usually related to central fat deposition and insulin resistance, and it is highly associated with cardiovascular disease, increasing overall mortality by 1.5 times and cardiovascular mortality by 2.5 times ,147,148.Since the discovery that cardiac cells possess intracellular signaling pathways that, when stimulated, protect these cells against the damage caused by I/R injury, several cardioprotective strategies have been proposed for the treatment of IHD, including acute myocardial infarction. This review focused on the recent advances in non-pharmacological and pharmacological strategies to protect the myocardium of lesions caused by I/R injury. Several non-pharmacological procedures stimulate the cellular signaling pathways involved in the cardioprotective response. In this review, we focused on the cardioprotection stimulated by ischemic pre- and post-conditioning, remote ischemic conditioning, hypothermia, and some physiological and pathological conditions that potentially stimulate the cardioprotective response such as the regular practice of physical exercise and sympathetic hyperactivity. In addition, different classes of drugs stimulate the cellular signaling pathways involved in cardioprotective response. In this review, we focused on the cardioprotection stimulated by agonists of β-AR or adenosine receptors, L-type VDCC or MCU blockers, modulators of NO biosynthesis or NCX, resveratrol, methylene blue and intestinal lipase inhibitors. Although the translation of ischemic postconditioning and remote ischemic conditioning protocols to patients with acute myocardial infarction has been fairly successful, the pharmacological recruitment of cardioprotective signaling has been largely disappointing to date due to the complexity involved in this process. It is important to mention that the most of the cardioprotective drugs are effective when administrated before ischemia, and only in experimental assays performed under controlled conditions. Thus, no definitive human cardioprotective strategy exist.Although a lot of scientific information about the cellular and molecular mechanisms involved in the cardioprotective responses has been acquired, there are two major outstanding issues to be addressed in the future: (1) understanding the spatiotemporal relationship between chemical messengers and receptors involved in intracellular signaling responsible to cardioprotective response, and (2) devising therapeutic strategies against myocardial diseases based on cardioprotective signaling. Future research is necessary for an adequate understanding of the molecular mechanisms involved in cardioprotection and clinical studies are required to properly test the clinical efficacy and safety of potential cardioprotective strategies.


Here we ask about the ion channels that are the effectors of DA actions. This work studies their Ca2+ currents.Striatal fast-spiking interneurons (FSI) are a subset of GABAergic cells that express calcium-binding protein parvalbumin (PV). They provide feed-forward inhibition to striatal projection neurons (SPNs), receive cortical, thalamic and dopaminergic inputs and are coupled together by electrical and chemical synapses, being important components of the striatal circuitry. It is known that dopamine (DA) depolarizes FSI via D2+ channel classes: CaV1, CaV2.1, CaV2.2, CaV2.3 and CaV3. However, CaV1 Ca2+ channel carries most of the whole-cell Ca2+ current in FSI. Activation of D1-like class of DA receptors by the D1-receptor selective agonist SKF-81297 (SKF) enhances whole-cell Ca2+ currents through CaV1 channels modulation. A previous block of CaV1 channels with nicardipine occludes the action of the DA-agonist, suggesting that no other Ca2+ channel is modulated by D1-receptor activation. Bath application of SKF in brain slices increases the firing rate and activity of FSI as measured with both whole-cell and Ca2+ imaging recordings. These actions are reduced by nicardipine.Whole-cell recordings in acutely dissociated and identified FSI from PV-Cre transgenic mice were used to show that FSI express an array of voltage gated CaV1 Ca2+ channels positive modulation.The present work discloses one final effector of DA modulation in FSI. We conclude that the facilitatory action of DA in FSI is in part due to Ca Inhibitory GABAergic interneurons are part of striatal circuitry. They control striatal projection neurons output (SPNs), are a part of neuronal ensembles and participate in cognition, procedural learning and motor performance –8. Among+, Ca2+ and synaptic channels, neurons and neuronal ensembles [2+ entry through somatic CaV1, CaV2.1 and CaV2.2 currents [2+ current regulating firing properties, as well as the time course and shape of action potentials (AP) [2+ channel classes expressed in FSI, (2) if there is dopaminergic modulation of Ca2+ currents in FSI, and finally, (3) whether there are particular Ca2+ channels modulated by dopamine receptors. Accordingly, as a first approach, we use whole-cell recording in acutely dissociated striatal and identified FSI obtained from transgenic PV-cre mice in order to avoid indirect actions. Besides, whole cell current clamp recordings in slices as well as dynamic Ca2+ imaging with single cell resolution were performed. All techniques confirmed the hypothesis that D1-class receptor agonists enhance Ca2+ current carried by CaV1 channel leading to an increase in excitability of striatal FSI.Striatal neurons receive massive dopaminergic innervation from the substantia nigra pars compacta (SNc) –16. In vnsembles –19. In Fnsembles , 21. Actnsembles , 21, as nsembles , 23 and nsembles . Althougcurrents , 26 regucurrents , 27. In als (AP) . HoweverPvalbtm1(cre)Arbr/J , called PV+ mice from now on. Experimental subjects were housed in acrilic cages  with wood-based bedding and cardboard cylinders, kept on a 12:12 light/dark (light beginning at 8 am) period with a temperature maintained at 20–21 °C in IFC vivarium after surgery (see below) until used for experiments. All animals had standard rodent chow and water ad libitum. In order to identify isolated PV+ interneurons, PV-Cre transgenic mice at PD 21 , were anesthetized i.p. with ketamine (Bayer 75 mg/kg) and xilazine (Bayer 10 mg/kg) and injected stereotaxically in a laminar flow hood (Telsar technologies. Model PV-30/60) in a dedicated, sterile room, with the following viral constructs (University of Pennsylvania Vector Core): AAV2/1.CAG.Flex.tdTomato.WPRE.bGH (Honguki Zeng) for whole cell recordings in isolated cells, AAV1.Syn.Flex.GCaMP6f.WPRE.SV40 [2+ channels classes . Using a vibratome , sagittal brain slices of 300 µm thick were cut and placed in the same saline solution for 1 h at 34 °C. When recordings were done in slices, they were transferred to a submerged chamber and superfused at 5 ml/min with saline solution. When recordings were done in dissociated cells, the dorsal striatum was dissected from the slices and returned into the saline solution containing 10 mM HEPES plus 0.5 mg/ml of papain  at 34 °C. After 20–25 min of digestion, the striatum slices were transferred to a low Ca2+ (0.4 mM CaCl2) saline solution. To obtain individual cells, the striatal slices were mechanical dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension (1 ml) was plated into a Petri dish mounted on the stage of an inverted microscope . Cells were left for 10–15 min for neurons to adhere to the bottom of the dish. The dish contained 1 ml of the whole-cell recording saline solution (in mM): 0.001 tetrodotoxin (TTX), 140 NaCl, 3 KCl, 5 BaCl2, 2 MgCl2, 10 HEPES, and 10 glucose . Thereafter, the cells were superfused at 1 ml/min with saline of the same composition at room temperature (approximate 25 °C). Tomato-positive neurons were visualized using a UV lamp , 40 HEPES, 10 EGTA, 4 MgCl2, 2 ATP, 0.4 GTP and 0.1 leupeptin . Whole-cell recordings used electrodes with D.C. resistance of 3–6 MΩ in the bath. Liquid junction potentials (5-10 mV) were corrected. Recordings of Ca2+ currents were obtained with an Axopatch 200B patch-clamp amplifier  and controlled and monitored with pClamp  and a 125 kHz DMA interface . We recorded currents passing through Ca2+ channels using Ba2+ as a charge carrier as shown in previous articles [2+ is a potent K+ blocker. In addition, intracellular K+ was replaced by 180 mM NMDG. Na+ channels were blocked with 1 µM TTX. Currents isolated in this way were completely blocked by 200–400 µM Cd2+  were generated before and after drug application. Figure 2+ currents evoked with 20 ms rectangular voltage commands from − 80 to 50 mV in 10 mV steps. Figure 2+ current in response to a voltage ramp command (0.7 mV/ms) from − 80 to 50 mV. When I–V plot from both methods coincide, space-clamp was considered acceptable . Recordings were made with an Axopatch 200A amplifier  and data were acquired with the Im-Patch© software designed in the Lab View environment (freely available for download at im-patch.com). Evoked firing responses at different depolarizing membrane potentials were obtained before and after a selective dopamine receptor agonist was administered. Current–voltage relationships made in current-clamp mode superimposed tightly with those performed in voltage-clamp mode at steady state, suggesting that neither bridge balance, nor series resistance, represented a problem in our recordings.Current clamp recordings were performed with the patch clamp technique in the whole cell configuration of PV+ neurons of infected mice ranging in age 28–60 days. Sagittal slices (250–300 μm thick) were cut using a vibratome , transferred to a recording chamber and superfused continuously with oxygenated saline solution (5 ml/min) at room temperature (~ 25 °C). Neurons within the striatum were visualized with infrared differential interference contrast videomicroscopy and PV+ neurons were identified using epifluorescent illumination with a 40 × immersion objective . Micropipettes were pulled  from borosilicate glass tubes  to an outer diameter of 1.5-mm for a final D.C. resistance of 4–6 MΩ when filled with internal saline. The internal solution contained (in mM): 120 KSOT test and Friedman, one-way ANOVA with post hoc Dunn’s tests were used to assess statistical significance between paired or unpaired samples comparisons. Statistical significance was defined by P-values below 0.05.Digitalized electrophysiological data were imported and analyzed into Origin v8, Microcal , and MatLab . Data are presented as the mean ± standard error (SEM). Firing rate plots were made by taking firing rate at rheobase in the different pharmacological conditions Fig. c. Free-d3, 1.2 NaHPO4, 1 CaCl2, 1.3 MgCl2, 10 glucose, 0.2 thiourea and 0.2 of ascorbic acid . For recordings, a microscope equipped with a 20 × 0.95 NA water-immersion objective  which has an image field of 750 × 750 μm, was used. To observe spontaneous changes in GCamp6f fluorescence intensity, light pulses at 488 nm (15–50 ms exposure) were delivered to the preparation with a Lambda LS illuminator  connected to the microscope via optic fiber. Brief image sequences or movies (~ 180 s per epoch) were acquired with open access Im-Patch© software [2+ entry was seen as spontaneous neuronal intrasomatic Ca2+ transients in PV+ neurons whose first time derivative reflects the time of electrical activity [Calcium imaging recordings were obtained from PV+ neurons of mice infected with a Cre-dependent GCamp6f expression. Recordings were performed in saline solution containing (in mM): 126 NaCl, 2.5 KCl, 26 NaHCOactivity . ActivitPV-Cre mice were infected as described earlier. Mice were deeply anesthetized (see above) and perfused transcardially with a solution of 4% paraformaldehyde in PBS. Thereafter, animals were decapitated and their brains removed from the skull and fixed overnight with 4% paraformaldehyde in PBS. The brains were then cut on a vibratome into 40 μm slices that were incubated 30 min with 1% bovine albumina to block nonspecific binding sites and for 36 h with a rabbit polyclonal antibody against parvalbumin (anti PV 1:2000 Abcam dissolved in PBS containing 0.25% Triton-X). The slices were then rinsed thrice with PBS and incubated with a goat versus rabbit secondary antibody  during 1 h. This antibody was conjugated with FITC . Samples were mounted with vectashield  and observed in a confocal microscope ZEISS LSM 700 (10 ×/1.0 NA) (n = 10).2+ CaV1 antagonist nicardipine (Cat# N7510) all from Sigma-Aldrich-RBI ; Ca2+ CaV2.2 blocker ω-conotoxin GVIA (Cat# C-300), Ca2+ CaV3 blocker TTA-P2 (Cat# T-155), Ca2+ CaV2.3 blocker SNX-482 (Cat# RTS-500), Na+ blocker tetrodotoxin (TTX) (Cat# T-550) from Alomone Laboratories (Israel) and Ca2+ CaV2.1 blocker ω-agatoxin TK (Cat# 4294-s) from Peptides International .For dissociated cell recordings, drugs were applied with a gravity-fed system that positioned a glass capillary tube 100 μm from the recording cell in the direction of superfusion flow. Solution changes were performed with a D.C. controlled microvalve system . This method allowed reversible drug applications , 33. For2+ currents changes for dissociated FSI recordings without assuming normal distributions. We also used the 5, 25, 50 (median), 75 and 95 percentile ranges of absolute current values represented as Tukey box plots. Friedman, Kruskal–Wallis or Wilcoxon test with post hoc Dunn for multiple comparisons tests were used . Friedman and Wilcoxon test were used when we compared the same samples in two or three different conditions . P< 0.05 was used as significance threshold. Analysis was conducted by GraphPad Prism 6.01 . Here, Ba2+ currents are reported as Ca2+ currents and graphs summarizing sampling results are illustrated. For current clamp recordings, we report mean ± SEM of firing rate. For calcium imaging experiments, activity of each FSI was determined as the total number of active frames/total number of frames. Finally, to quantify the amount of activity on each experiment, a cumulative activity plot was built on each condition.Collected digitalized data were analyzed and plotted using commercial software . We report mean ± SEM of peak Ca2+ channel to the whole cell Ca2+ current was described in previous work [2+ channel, the amount of Ca2+ current blocked by a given antagonist: nicardipine, ω-conotoxin GVIA (ω-CgTx), ω-agatoxin TK (ω-AgTx), TTA-P2 (TTA) and SNX-482 (SNX) was obtained by subtraction in the same or different experiments. Hardly all antagonists could be tested in a single experiment, but the amount blocked by each antagonist was taken no matter the number or order of the antagonists tested. This amount of blocked current was defined as the contribution of that specific channel class to the whole-cell control Ca2+ current normalized to 100% without any antagonist. Thereafter the data was introduced in the following system of linear equations:L, PQ, N, T and R are the contributions in percentage (± SEM) of each channel class: CaV1, CaV2.2, CaV2.1, CaV3 and CaV2.3 to the whole-cell Ca2+ current. For example, PQ refers to the current blockade by the selective P/Q type Ca2+ channel antagonist (ω-AgTx). Zero in the linear equation system means a blockade of a given Ca2+ channel class, thus, coefficients L, N, PQ, T or R were replaced by zero when the corresponding Ca2+ channel class was blocked. A, B, C, D or E stand for the mean percentage of Ca2+ current in the control (100%) with one channel class blocked (< 100%). Subscripts X1–X5 are the unknown variables, in other words, the values that multiply the coefficients L, N, PQ, T and R in order to determine percentage contribution of each channel to the whole-cell Ca2+ current.The method to obtain the average contribution of a given class of Caous work . Briefly2+ binding protein PV [2+ currents in FSI is unknown. With the help of PV-Cre transgenic mice we explored whether DA receptor actions in acutely dissociated striatal FSI modulate Ca2+ currents. In our experiments Na+ and K+ channels were blocked . To determine whether CaV1 (L-type) contributed to the whole-cell current, application of nicardipine, a specific CaV1 Ca2+ channels antagonist was examined. As shown in Fig. 2+ current without any drugs). This large amount of current flux through CaV1 Ca2+ channel might contribute to neuronal depolarization and AP generation after addition of D1-like agonist, since this current have a slow voltage-dependent inactivation [FSI were identified using mice expressing Cre-recombinase under the control of the PV promoter (PV-Cre) and stereotaxic injections of an adeno-associated virus into the dorsal striatum allowed expression of a fluorescent protein (td-Tomato) only in striatal FSI. Acutely dissociated neurons were used in the first part of this study to avoid any indirect inputs from afferents, gap junctions, dendritic or axonal inputs. First, we estimated which Cativation , 39, 40;V2.2 (N) contribute to 23.4 ± 0.7% as revealed by 1 µM of ω-conotoxin GVIA (ω-CgTx) blockade, a specific CaV2.2 channel antagonist  was 11.1 ± 1.4% disclosed by ω-agatoxin TK  Ca2+ channels: 20 ± 2%  Ca2+ channels contribute with 7.4 ± 2.3% . Note that Ca2+ current returns to values similar to the control when the agonist is washed-off. Representative I–V plots . Note that in the presence of nicardipine subsequent addition of 10 µM SKF fail to elicit any change in the remaining Ca2+ current . We conclude that the action of nicardipine occluded the action of SKF and therefore CaV1 Ca2+ channels are the final effectors of DA receptor modulation; without excluding other classes of channels . This action is similar to that found in striatonigral projection neurons expressing D1-like receptors in both cell bodies and terminals [2+ currents, there is no other effector for D1- receptor modulation in FSI.To further investigate the role of DA on Caked them . Time coerminals , 41, 42.V1 Ca2+ channels modulation is robust enough to explain, in part, the increase in firing rate due to D1-like receptor modulation in FSI [V1 antagonist, nicardipine (20 µM), reversed in part the increase in firing rate induced by SKF. The same was true for a D1-receptor antagonist SCH . This result indicates that DA increases the number of FSI firing within the striatal microcircuit in agreement with data from dissociated neurons and slice experiments. 20 µM nicardipine . These results were obtained in voltage-clamp mode in identified dissociated FSI. Specific channel subtypes are still in need of investigation. (2) Contributions in percentage of each Ca2+ channel class are reported. CaV1 channels represent much of the whole cell Ca2+ current. (3) DA D1-class receptors, probably D5-type [V1 carried current. (4) The CaV1 class is the only Ca2+ channel modulated by DA in FSI. This modulation is occluded by a previous administration of nicardipine or blocked by the antagonist SCH [V1 Ca2+ channels is reflected in an increase in firing rate of FSI. These data were obtained in slices in current clamp mode. (6) Ca2+-imaging recordings of several identified FSI obtained from different slices/animals showed that DA increases the number of FSI that are active in the striatal microcircuit. To our knowledge these are the first evidences of a molecular final effector for the DA-dependent modulation in striatal FSI, leading to a better understanding of the DA actions in the striatum.A summary of original data and findings of the present work follow: (1) all major classes of voltage gated Ca D5-type , up-modunist SCH . (5) Mod2+ currents [2+ channels, HVA  and LVA (CaV3), although, their contributions vary  except that in the present case there was no need to block intracellular phosphatases [A selective D1-class DA receptor agonist, SKF-81297, was used to investigate dopaminergic modulation. The DA receptor agonist enhanced Caphatases .V1 channels has been associated with enhanced evoked depolarization and discharge facilitation [V1 also induces short-term synaptic depression and facilitates GABA release in SNr [V1 channels are in part responsible for these functions in striatal FSI. It would be interesting to know if FSI from other nuclei express this modulation or if it is particular for striatal FSI.Current through Calitation , 52, 53.litation . CaV1 ale in SNr . Blockad2+ imaging of identified FSI with single cell resolution showed that the firing of these interneurons is enhanced in the striatal microcircuit by dopaminergic modulation. This action was partially blocked by nicardipine, lasted for several minutes without overt desensitization, suggesting that D1-class receptor activation, probably D5-type, increases feed-forward inhibition in the microcircuit [In addition, dynamic Caocircuit , 56. Netocircuit . Given tocircuit , it may ocircuit , 58–60. ocircuit , 61.V1 Ca2+ channels are blocked. There could be various reasons for this behavior. One is that the DA receptor agonist not only affects FSI within the circuit, but turns on network activity in a way that does not return to control even after blocking CaV1 in FSI [However, the number of dopamine activated FSI within the striatal microcircuit does not return to control conditions after Ca1 in FSI , 62. Ano1 in FSI . Finally1 in FSI . This laV1 channels are final effectors of DA modulation in FSI. In addition, we show the classes of Ca2+ channels that striatal FSI express and show evidence that CaV1 are the only ones modulated by D1-class receptors activation. Enhancement of CaV1 channels is a main cause for the increase in excitability of these interneurons due to DA receptors signaling, and collectively, this modulation increases the number of active FSI during striatal microcircuit operation. The demonstration that identified interneurons can be isolated for recording opens the pathway for future studies such as: to study other current classes  and their modulation in interneurons in space clamp conditions, it also suggests comparisons between current phenotypes between FSIs from the striatum and other nucleus such as the cortex, and finally, quantitative single cell PCR may be used to prove whether different regions of the striatum possess different types of FSIs.To our knowledge this is the first demonstration that Ca


TRP proteins are classified into six subfamilies: TRPC , TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin), which are activated by numerous physical and/or chemical stimuli. TRP channels participate to the handling of the intracellular Ca2+ concentration in cardiac myocytes and are mediators of different cardiovascular alterations. This review provides an overview of the current knowledge of TRP proteins implication in the pathologic process of some frequent cardiac diseases associated with the adverse cardiac remodeling such as cardiac hypertrophy, fibrosis, and conduction alteration.Calcium is an important second messenger required not only for the excitation-contraction coupling of the heart but also critical for the activation of cell signaling pathways involved in the adverse cardiac remodeling and consequently for the heart failure. Sustained neurohumoral activation, pressure-overload, or myocardial injury can cause pathologic hypertrophic growth of the heart followed by interstitial fibrosis. The consequent heart’s structural and molecular adaptation might elevate the risk of developing heart failure and malignant arrhythmia. Compelling evidences have demonstrated that Ca Cardiac relaxation starts when intracellular Ca2+ concentration ([Ca2+]i) decreases as a result of the activity of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) responsible for the Ca2+ reuptake into the sarcoplasmic reticulum and the Na+/Ca2+ exchanger responsible of the Ca2+ extrusion out of cardiomyocytes  channels contribute to the Ca2+ influx induced by a wide spectrum of physico-chemical stimuli from cellular microenvironments, such as thermal, mechanical stresses, and neurohormonal (2+-permeable cation channel superfamily showing a common architecture. They contain six transmembrane domains (TM1–TM6) and the cation-permeable pore region formed by a loop between TM5 and TM6  that ranges from channels selective for monovalent cations, such as TRPM4 and TRPM5 with a ratio PCa/PNa < 0.05, to highly Ca2+ selective channels, as TRPV5 and TRPV6, which exhibit a ratio PCa/PNa close to 100  (TRP channels display diverse cation selectivity and activation mechanisms . Most ofe to 100 . TRPM6 and Mg2+) , intracend Mg2+) , phosphond Mg2+) ], and vand Mg2+) .2+]i or by the interplay with Ca2+-dependent regulatory proteins. Actually, they contribute to Ca2+ homeostasis by directly conducting Ca2+ or indirectly via membrane depolarization and modulation of voltage-gated Ca2+ channels  required for diverse cardiac physiopathological process  release  is the most sustained clinical arrhythmia, which occurs due to structural remodeling, involving prominent fibrotic changes . TRP chaIn vitro experiments showed that 9-phenanthrol decreased Ca2+ oscillations in atrial HL-1 mouse cardiac myocytes, thought to play a critical role in arrhythmias  to characterize and consider as therapeutic tools. More specific inhibitors/activators of TRPs are eagerly awaited to shed a light on the complex mechanism of cardiac diseases associated with remodeling. Of hope, the increasing amount of available information related to TRP-drug interaction sites and gating processes of TRP channels is expected to facilitate the development of novel therapeutic concepts by pharmaceutical companies. Overall, and in the light of the reported studies, TRP channels are still considered promising therapeutic targets to regulate pathological cardiac plasticity.A growing body of evidence has demonstrated that, by controlling CaTS conceived the concept of the review. TS, IG-O, DF, RT, MM-B, and EC-S wrote the review. IG-O, DF, AH and JR designed and formatted the figures. TS, IG-O, RT, DF, AH, and JR read and edited the review manuscript.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.


While rare high-risk variants affecting specific transcription factors account for a proportion of myelodysplastic syndrome (MDS) and AML associated with a family history, the contribution of other germline variants conferring low-intermediate risk has not yet been determined, partly because these are more difficult to identify from pedigree analysis. Here we use an Australian AML patient cohort to analyze rare, deleterious variants affecting genes involved in the rare recessive bone marrow failure syndrome Fanconi Anemia (FA). FA is caused by bi-allelic germline mutations in any of the 22 FANC genes , and is associated with profoundly increased risk of AML2. The proteins encoded by the FANC genes participate in the removal of interstrand crosslinks (ICL) and the protection and resolution of stalled replication forks, an essential step for faithful DNA replication3. Deficiency for these genes, combined with other mutations, results in pre-leukemia or leukemia in mouse models4.Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy caused by somatically acquired changes affecting a well-defined set of genes1, MAF < 0.001) were identified using whole exome sequencing (WES) of 131 samples from adult Caucasian AML patients (cohort characteristics shown in Supplementary Table n = 323)5. Following pathogenicity filtering, we identified a total of 53 heterozygous FANC variants in 45 patients  .Sanger sequencing of selected FANC tumor variants, for which matched non-tumor material was available, showed that the majority were present in the non-tumor material  and breast cancer (kConFab: http://www.kconfab.org/Index.shtml and BIC: https://research.nhgri.nih.gov/bic/) databases 7 . The frequency of damaging variants for several FANC genes , consistent with a previous report8. Taken together, the elevated frequencies of D-C and damaging FANC variants suggest that subtle changes to FA pathway function in HSC may play a role in AML predisposition and pathogenesis. There are a number of important differences that need to be considered when relating our study to previous studies of FA families which have not found increased incidence of AML in FA carriers10. Firstly, such familial studies are biased towards analysis of only 3 FANC genes , which represent the vast majority of FA cases. It is possible that bi-allelic loss of function for some FANC genes may not be tolerated at the germline level  while rare heterozygous mutations may still confer a cancer risk for carriers. Secondly, while familial studies address the question of high-risk D-C FANC variants in carriers, modest-risk or low-risk variants in these genes would be predicted to induce AML with low penetrance, masking the familial pre-disposition. Thus, further analysis focusing on assessing familial cancer and AML incidence for families across all FA complementation groups, and additional prospective sequencing studies in larger multi-center AML and control cohorts, will help to clarify this issue. Such studies are also important to establish the implications for FA families, particularly for selection of sibling donors for stem cell transplantation of FA patients, and for identification of carriers who may have modest-intermediate risk of AML or other cancers. Other studies have reported damaging germline FANC variants in MDS/sAML, therapy related AML, and in cohorts of patients with a range of other familial cancers  variants in the AML and healthy cohorts by cross-referencing to pathogenic variants in the FA  for a single WT clone, 3 independent heterozygous FANCL-mutant clones, and a bi-allelic FANCL-mutant clone carrying a nonsense mutation on one allele and a single amino acid deletion on the other , play a key role protecting replication forks and preventing genomic instability during replication3. Thus, AML cells with partial FA pathway deficiency may display enhanced sensitivity to agents that boost replicative stress and DNA damage, particularly when combined with PARP1 inhibitor; this combination has been proposed as a potential therapeutic approach in AML15.In summary, our findings suggest that decreased function of the FA DNA repair pathway in HSC is due to deleterious germline heterozygous FANC variants which may result in a reduced capacity to maintain genome integrity, which may in turn contribute to increased risk of AML. This may be particularly significant under conditions associated with increased replicative stress or DNA damage. Such a deficiency would be predicted to have important consequences during emergency hematopoiesisSupplementary InformationSupplementary table S2


The maximum duration of a single oscillatory event is about 300 s. A major question arises as to how such a long-duration Ca2+ elevation is maintained. Store-operated calcium channels (SOCCs) are one of the calcium (Ca2+)-permeable ion channels. SOCCs are opened by activating the metabotropic glutamate receptor type 5 and inositol 1,4,5-trisphosphate receptor (mGluR5-IP3R) signal transduction cascade and are related to the pathophysiology of several neurological disorders. However, the functions of SOCCs in striatal neurons remain unclear. Here, we show that SOCCs exert a functional role in striatal GABAergic neurons. Depletion of calcium stores from the ER induced large, sustained calcium entry that was blocked by SKF96365, an inhibitor of SOCCs. Moreover, the application of SKF96365 greatly reduced the frequency of slow Ca2+ oscillations. The present results indicate that SOCCs contribute to Ca2+ signaling in striatal GABAergic neurons, including medium spiny projection neurons (MSNs) and GABAergic interneurons, through elevated Ca2+ due to spontaneous slow Ca2+ oscillations.The striatum plays an important role in linking cortical activity to basal ganglia output. Striatal neurons exhibit spontaneous slow Ca Ca2+-free ACSF was made by omitting CaCl2 and adding 7.5 mM NaCl. Corticostriatal sagittal slices (300 μm thick) were prepared using a vibratome tissue slicer  and incubated at room temperature in a submerged chamber containing gassed ACSF for at least 60 min prior to the Ca2+-sensitive Fura-2 LR/AM  fluorescent dye, loading.The methods for Ca2+]i elevation and manganese ion (Mn2+) quenching was measured in striatal cells loaded with the ratiometric Ca2+ sensitive dye Fura-2 LR/AM. The dye-loading methods used were as previously described  to avoid Ca2+ elevations caused by the opening of voltage-gated Ca2+ channels due to action potentials. The experiments were performed at 30 ± 1°C.As previously described  from each imaged cell. The criterion for identifying neurons with the slow Ca2+ oscillations was whether they had a frequency of occurrence of spontaneous Ca2+ elevation above 0.001 Hz. The total recording duration was more than 4,200 s. All equipment was controlled by iQ software . The analyses of the imaging data were performed with ImageJ software  by observing green fluorescence excited at 488 nm  and quantified the average fluorescence (F340 and F380) within the region of interest (ROI) of these cells as a function of time. i  and 0.0042 ± 0.0002 , respectively. These results indicate that SOCCs exist in striatal GABAergic neurons and exert some functional role.To deplete Ca [Ca2+]i . The [Castration . The [Ca2+]i in striatal GABAergic neurons exhibiting spontaneous slow Ca2+ oscillations, we first tested the contribution of Ca2+-influx from the extracellular space in striatal GABAergic neurons with and without slow Ca2+ oscillations by quantifying the time constant of fluorescence quench  administration in the neurons with slow Ca2+ oscillations (q of fluorescence quench were 211 ± 4 s (n = 23) in the neurons without slow Ca2+ oscillations and 193 ± 4 s (n = 9) in those with these oscillations (p < 0.01). These results suggest that more Ca2+ enters those striatal neurons with spontaneous slow Ca2+ oscillations than those without the oscillations.To examine whether SOCCs are responsible for maintaining high [Cae quench . More Mnllations . The tim2+ oscillations we blocked the channels with SKF96365 . Thus, SOCCs clearly contribute to the occurrence of spontaneous slow Ca2+ oscillations in striatal GABAergic neurons.To clarify the contribution of SOCCs to spontaneous slow CaSKF96365 . To avoiSKF96365 . The fre2+ entry (SOCE) are STIM (STIM1 and 2) and Orai ” channels . We also examined the expression of STIM1 and 2, the ER Ca2+ sensor activating CRAC channels on the plasma membrane . These results suggest that in striatal GABAergic neurons, Orai2 is a major molecular component of SOCCs, and both STIM1 and STIM2 contribute to the activation of this Orai protein.The major molecular components of the store-operated Cas Kraft, , in stris Kraft, . The pere Kraft, . Both ofe Kraft, . The per2+entry greatly reduces the frequency of spontaneous slow Ca2+ oscillations in these neurons  and Orai  proteins Kraft, . Therefo2+ oscillations in striatal neurons and astrocytes , Huntington’s disease (HD), and Parkinson’s disease , USA.SK, YI, TK, KK, and MO conceived and performed experiments. SK, YI, and MO analyzed data. YY provided the GAD67-GFP mice and information for maintaining them. SK, YI, YY, MT, and MO wrote the manuscript.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.


However, the mechanisms used by enamel cells to handle bulk Ca2+ safely remain unclear. Our previous work contradicted the dogma that Ca2+ is ferried through the cytosol of Ca2+-transporting cells and instead suggested an organelle-based route across enamel cells. This new paradigm involves endoplasmic reticulum (ER)-associated Ca2+ stores and their concomitant refilling by store-operated Ca2+ entry (SOCE) mediated by Ca2+ release activated Ca2+ (CRAC) channels. Given that Ca2+ handling is maximal during the enamel-mineralization stage (maturation), we anticipated that SOCE would also be elevated then. Confirmation was obtained here using single-cell recordings of cytosolic Ca2+ concentration ([Ca2+]cyt) in rat ameloblasts. A candidate SOCE agonist, cholecystokinin (CCK), was found to be upregulated during maturation, with Cck transcript abundance reaching 30% of that in brain. CCK-receptor transcripts were also detected and Ca2+ imaging showed that CCK stimulation increased [Ca2+]cyt in a dose-responsive manner that was sensitive to CRAC-channel inhibitors. Similar effects were observed with two other SOCE activators, acetylcholine and ATP, whose receptors were also found in enamel cells. These results provide the first evidence of a potential regulatory system for SOCE in enamel cells and so strengthen the Ca2+ transcytosis paradigm for ER-based transport of bulk Ca2+. Our findings also implicate enamel cells as a new physiological target of CCK and raise the possibility of an auto/paracrine system for regulating Ca2+ transport.Dental enamel is formed by specialized epithelial cells which handle large quantities of Ca Enamel formation occurs in two main stages, termed secretory and maturation, with Ca2+ transport increasing fourfold during the latter. Determining how these specialized epithelial cells handle and transport Ca2+ in bulk while avoiding the toxic effects associated with high cytosolic concentration is an important issue (2+ predominantly via a transcellular (active) route. Commonly in epithelia, such active systems require logical steps of Ca2+ entry, transit and extrusion while changes in cytosolic calcium ([Ca2+]cyt) are retained for cell signaling. In many tissues the safe transit step has been ascribed to the vitamin-D regulated family of cytosolic Ca2+-binding proteins known as calbindins  mediated by the Ca2+ release activated Ca2+ (CRAC) channels is in agreement with the calcium transcytosis model in enamel cells  and requires a substantial supply of Cant issue . Amelobllbindins . Howeverlbindins . Insteadturation  leading cytosis” , 2000. Olasts andel cells .2+ release activated Ca2+ channels mediate Ca2+ influx in many cell types  because several IP3R subtypes were found to be upregulated during maturation  that promote IP3 production by phospholipase C  cells from secretory and maturation stage, we identified cholecystokinin (CCK) as being highly up-regulated in maturation . CCK is ar cells , we hypoAll animal procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of New York University College of Dentistry (protocol # s16-00625).2 incubator being manually pipetted every 5 min to mechanically separate the cells. The enzymatic reaction was stopped by adding X-VivoTM 15  cell media containing 10% FBS and 1% penicillin/streptomycin (solution 2). Cells were centrifuged at 500 ×g for 5 min and washed twice with solution 2. Cells were plated on glass cover slips coated with Poly-L-lysine or Cell-Tak (Corning) bathed in solution 2 and maintained overnight at 37°C in a 5%-CO2 incubator. Cells were used within 24–48 h after isolation.Rats (100–140 gram) were euthanized and mandibles were dissected out. The surrounding soft tissues  were removed to expose the body of the mandible as described  to be ab2+]cyt measurements of secretory and maturation EO cells  for 20 min at room temperature. After blocking for 30 min with 2% bovine serum albumin in PBS, sections were incubated overnight at 4°C with appropriate antibodies . After washing in PBS, samples were incubated with secondary antibodies  for 30 min, washed, and mounted using Prolong Gold Mounting Media containing Dapi .2+concentration ([Ca2+]cyt) measurements, isolated secretory and maturation EO cells were plated on coated cover slips for 24 h after isolation, and loaded with Fura-2/AM  and CD90  for 30 min at room temperature. Fluorescence measurements were performed every 7 s, using a Nikon Eclipse fluorescence microscope . Cells were excited alternatively at 340 or 380 nm and emitted fluorescence intensity was recorded at 505 nm. Data acquisition was performed by using computer software . We first measured SOCE by monitoring changes in cytosolic Ca2+ upon passively depleting intracellular Ca2+stores with thapsigargin  to block sarco-endoplasmic reticulum Ca2+ pumps (SERCA). Experiments were carried out prior to, and during exposure of the cells to the Ca2+-free solution (see below). Re-addition of extracellular Ca2+allowed us to make assessments of SOCE activity in secretory and maturation EO cells.For cytosolic Ca2+, changes in [Ca2+]cyt were monitored prior to and following addition of ACh, CCK and ATP to the Ringer solution. [Ca2+]cyt was estimated from calibration curves by using the Calcium Calibration Buffer Kit#2  as described . For the Ca2+-free solution, the Ringer solution was supplemented with 0.5 mM EGTA. Moreover, the effect of ATP, CCK and ACh and their ability to elicit Ca2+release from intracellular stores was measured following addition of acetylcholine chloride , adenosine 5′-triphosphate disodium salt hydrate  or sulfated CCK8 (CCK8S)  to the Ca2+-free solution. Secretory and maturation EO cells were also pre-treated with the CRAC channel inhibitors Synta-66  or GSK-7975A  for 30 min before initiating the experiment and then Synta-66 or GSK-7975A were present throughout the experiment. This was done to ascertain their effect in modulating changes in [Ca2+]cyt when blocking CRAC channels. Both Synta-66 and GSK-7975A are potent pharmacological blockers of ORAI1, the pore subunit of the CRAC channel  according to the manufacturer’s specifications and converted to cDNA using iScriptTM cDNA Synthesis Kit . RT-PCR amplifications were done using SsoAdvancedTMUniversal SYBR Green Supermix  on a CFX ConnectTM Real-Time System . Primer specificity was confirmed by analysis of a melting curve. All experiments were done in triplicates. Beta-actin was amplified to standardize the amount of sample RNA. Relative quantification of gene expression was achieved using the ΔCT method  overnight at 4°C, and decalcified in 10% EDTA (pH 7.3) for 7–10 days and washed. Brain was used as a control and processed as for dental tissues but without the decalcification step. Tissues were embedded in Paraplast + and ∼5 μm sections collected on a glass slide, deparaffinized and hydrated, and hybridized overnight at 60°C in a humidified chamber with DIG-labeled Cck probes. Sections were then washed in 2x SSPE . Probes were detected using an anti-DIG antibody conjugated to alkaline phosphatase at a 1:2000 dilution for 2 h at room temperature. After several washes, the chromogenic reaction was performed overnight by incubation in BM purple . The reaction was stopped by fixation in formalin. The sections were briefly counterstained with eosin, dehydrated and mounted in Permount .Rat Cck (NCBI accession #NM_012829.2) was amplified using probes shown in Supplementary Table n represents the number of independent experiments. For the Ca2+ imaging experiments, a minimum of three independent experiments were performed. Each experiment consisted of pools of secretory and maturation EO cells from three rats. The number of cells used per measurement is detailed in each Figure. For RT-PCR experiments, we used a total of six rats. In all experiments, males and females were used. Cells were pooled from a pair of animals. Differences were tested for significance using 2-tailed unpaired Student’s t-test. P < 0.05 was considered statistically significant.Data collected were sorted, coded, and entered into a Microsoft Excel spreadsheet for analysis using GraphPad Prism 5.0 . Data are provided as means ± SEM, 2+ requirements are higher during enamel maturation than in the preceding secretory stage, a primary requisite was to determine whether SOCE is also elevated during maturation. In a previous study, we reported increased SOCE during maturation in a mixed cell population of rat primary EO cells analyzed in a plate reader . Stimulation of each cell type with thapsigargin to passively deplete the ER/Ca2+ stores revealed higher Ca2+ release from maturation cells . The re-addition of Ca2+ to the extracellular medium to stimulate SOCE elicited significantly higher SOCE in maturation ameloblasts , validating our previous findings from the mixed cell populations . To reinforce these gene expression findings, we analyzed maturation-stage ameloblasts for Cck expression by in situ hybridization . We also investigated the expression of gastrin, a homolog of CCK that can also bind to CCKRs. However, no gastrin transcripts were detected by RT-PCR in enamel cells (data not shown).Our previous genome-wide analysis of enamel cells revealed a ∼200-fold upregulation of turation . Recogniar cells  led us t2+]cyt acting via their respective cell surface receptors. Muscarinic ACh receptors (CHRM) are GPCRs with the CHRM1, CHRM3, and CHRM5 subtypes being linked to IP3 production and a consequent rise in [Ca2+]cyt . ATP is known as an intracellular energy source that also functions as an extracellular messenger by binding to purinergic cell surface receptors . Collectively, these results suggest that CCK, ACh, and ATP might contribute to Ca2+ handling in enamel cells, possibly via regulation of SOCE.Two other well-known physiological agonists, acetylcholine (ACh) and ATP, can also stimulate a rise in [CaCa2+]cyt . Using Receptors . In rat,Ca2+]cyt . SecretoFollowing our observation that ameloblasts express CCK and its receptors as well as the receptors for ACh and ATP, we next asked whether these agonists participate in SOCE in enamel cells and if they do so by modulating the CRAC channel.2+, stimulation with CCK8S led to a transient increase in [Ca2+]cyt representing ER/Ca2+ release . Re-addition of extracellular Ca2+ resulted in a rapid elevation in [Ca2+]cyt via activation of SOCE, which was significantly higher in maturation . These effects of CCK8S on SOCE were concentration-dependent at both developmental stages. To further assess if the changes elicited by CCK were directly associated with SOCE and not with other calcium pathways, we repeated these experiments using two pharmacological inhibitors, Synta-66 and GSK-7975A, to block the CRAC channels . These results indicate that SOCE mediated by CRAC channels is responsive to CCK in ameloblasts.CCK8 comprises the 8 C-terminal amino acids of CCK and is one of its most biologically potent forms . CCK8 exchannels . Both in2+]cyt. It was found that, in the presence of external Ca2+, the application of ATP (100 μM) and ACh (10 μM) elevated [Ca2+]cyt with higher increase in maturation . Addition of Synta-66 and GSK-7975A resulted in a significant reduction of Ca2+ influx suggesting that the effects of ACh and ATP on [Ca2+]cyt were mediated by CRAC channels. Together with those obtained with CCK, these results provide the first evidence of a SOCE-regulatory system that is overexpressed in maturation stage ameloblasts.Next we similarly tested whether exposure to physiological levels of ACh and ATP could stimulate a rise in cyt increase triggered by CCK is mediated via the CRAC channel. The collective evidence that CCK modulates bulk Ca2+ entry into enamel cells via CRAC channels introduces the EO as a potential target of CCK regulation. Although no dental defects were reported in Cck-deficient mice . ACh acts via specific muscarinic GPCRs (Chrm gene products) to elevate [Ca2+]cyt in many cell types . Similarly, the purinergic class of GPCRs responsible for ATP-induced [Ca2+]cyt increase . Again, although no dental defects have been reported to date in P2ry2 and P2yr6 knockout mice . Data presented here and in our previous studies (2+ entry into maturation ameloblasts. The SOCE upregulation in maturation points to a system that modulates SOCE activity to accommodate Ca2+-signaling and Ca2+-transport needs. Here, we have shown that the physiological agonists CCK, ACh and ATP all stimulate CRAC channel mediated Ca2+ entry in ameloblasts, thus providing the first evidence of such a SOCE-regulatory system in enamel development.Enamel mineralization requires a major supply of Cafourfold . In amelr, NCKX4 . This ’C studies  firmly e2+]cyt. This provides the first evidence of a regulatory system for SOCE, a Ca2+ entry mechanism linked to the ER. Moreover, we showed significantly higher capacity for SOCE in maturation ameloblasts than secretory cells. This study is also the first to identify enamel cells as a target of the peptide hormone CCK, raising the possibility of an autocrine/paracrine role of CCK in enamel formation. Additionally, CCK, ACh, and ATP are positive modulators of CRAC channel-mediated Ca2+ influx in ameloblasts, as evidenced by sensitivity to pharmacological inhibitors. Taken together, these findings contribute to a better understanding of enamel cell physiology in particular the regulation of Ca2+ homeostasis.Molecular and functional data presented here show that ameloblasts can be stimulated by the physiological agonists CCK, ACh, and ATP to elicit elevations in [CaMN and RL designed the project. MN, ME, and AD collected the data. MN, ME, J-PS-J, MH, DY, and RL analyzed the data. MN, ME, DY, MH, and RL wrote the paper.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.