Triple-negative breast cancer (TNBC) surpasses other BC subtypes as the most challenging to treat due to its lack of traditional BC biomarkers. Nearly 30% of TNBC patients express the androgen receptor (AR), and the blockade of androgen production and AR signaling have been the cornerstones of therapies for AR-positive TNBC. However, the majority of women are resistant to AR-targeted therapy, which is a major impediment to improving outcomes for the AR-positive TNBC subpopulation. The hypoxia signaling cascade is frequently activated in the tumor microenvironment in response to low oxygen levels; activation of the hypoxia signaling cascade allows tumors to survive despite hypoxia-mediated interference with cellular metabolism. The activation of hypoxia signaling networks in TNBC promotes resistance to most anticancer drugs including AR inhibitors. The activation of hypoxia network signaling occurs more frequently in TNBC compared to other BC subtypes. Herein, we examine the (1) interplay between hypoxia signaling networks and AR and (2) whether hypoxia and hypoxic stress adaptive pathways promote the emergence of resistance to therapies that target AR. We also pose the well-supported question, “Can the efficacy of androgen-/AR-targeted treatments be enhanced by co-targeting hypoxia?” By critically examining the evidence and the complex entwinement of these two oncogenic pathways, we argue that the simultaneous targeting of androgen biosynthesis/AR signaling and hypoxia may enhance the sensitivity of AR-positive TNBCs to AR-targeted treatments, derail the emergence of therapy resistance, and improve patient outcomes. Triple-negative breast cancer (TNBC) continues to be the most difficult-to-treat BC subtype. TNBCs do not express the conventional BC targets currently exploited for therapeutic intervention, including the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) . TNBCs aOver the past few decades, the androgen receptor (AR) has been highlighted as an alternate endocrine target of interest for the subset of TNBC patients positive for AR expression. AR expression is assessed by immunohistochemistry (IHC); about 20%–40% of TNBC tumors express AR . These rHypoxia, or limited oxygen availability due to insufficient blood supply, is a unique feature of most solid tumors . Rapid aThe adaptive response to hypoxia is regulated by hypoxia-inducible factors (HIFs), which modulate hypoxic gene expression and regulate oxygen homeostasis. Heterodimeric HIF is composed of an oxygen-labile α subunit and a stable β subunit; HIF α/β heterodimers are thus comprised of one of three α subunits  and one of two β subunits (HIF-β and aryl hydrocarbon receptor nuclear translocator or ARNT). The HIF-α subunit undergoes proteasomal degradation under normoxic conditions; in contrast, HIF-β and ARNT are constitutively expressed and reside in the nucleus. Under hypoxic conditions, stabilized HIF-α translocates to the nucleus, where the active HIF-α/ARNT or HIF-α/HIF-β complex activates the transcription of a large group of target genes after binding to cognate hypoxia-responsive elements (HREs) ,17,18.Accumulating evidence shows that hypoxia can hinder the success of androgen deprivation therapy (ADT) and AR inhibitors in AR growth-driven prostate cancers by increasing AR expression and/or activity and upregulating other compensatory signaling pathways to promote castration-resistant prostate cancer (CRPC) ,22,23,24Since AR-driven/-dependent TNBC is a potentially actionable subtype, androgen- and AR-targeting therapies have been explored for their ability to stymie disease progression in AR-positive TNBC . ClinicaBicalutamide is FDA-approved for the treatment of advanced PC in combination with a luteinizing hormone-releasing hormone analog. Bicalutamide is orally available and is a competitive inhibitor of AR that permits AR nuclear localization and binding to chromatin but preferentially recruits AR corepressors rather than coactivators . A cliniIn a phase I dose-escalation study in patients with metastatic BC in 2013, enzalutamide was well tolerated at 160 mg daily. In a 2015 phase II study, among the AR-positive patients evaluable for response, the CBR  at 16 weeks was 42% with patients receiving 160 mg enzalutamide daily. About 34% of the participants in the study continued to show clinical benefits after 24 weeks. Furthermore, when a novel, binary, gene signature-based predictive biomarker that was reflective of AR signaling was used to stratify patients, the outcomes for diagnostic-positive patients were more favorable in all response measures, including CBR16 (39% vs 11%), CBR24 (36% vs 6%), and median PFS (16.1 weeks vs 8.1 weeks), when compared with those of patients who tested negative for this companion diagnostic. Interestingly, this companion diagnostic was a stronger predictor of response to enzalutamide than AR expression  alone. These responses to an AR antagonist supported (a) the development of anti-AR therapy for the treatment of a subgroup of patients with TNBC . DarolutAgents that target steroidogenic enzymes to impair intracrine and adrenal androgen biosynthesis and concomitantly reduce downstream estrogen synthesis have also been tested in clinical trials for TNBC; abiraterone acetate, a CYP17 inhibitor, is one such therapeutic, which is a robust, orally available, selective inhibitor of both 17α-hydroxylase and C17, 20-lyase. In a phase II multicenter trial that aimed to assess the safety and efficacy of abiraterone acetate in combination with prednisone  in women with metastatic or inoperable locally advanced AR-positive TNBC, the CBR at 6 months was 20.0% and median PFS was 2.8 months. This study suggested that some TNBC patients with molecular apocrine-like tumors may benefit from the coadministration of abiraterone acetate and prednisone. Seviteronel, an oral, selective CYP17-lyase inhibitor and AR antagonist, is also currently being evaluated as a novel option for the treatment of AR-positive TNBC because this agent does not cause a decrease in cortisol and no steroid supplementation is required. Preliminary pharmacokinetic data from a large phase I/II trial for seviteronel in AR-positive TNBC and ER-positive metastatic BC suggested that the bioavailability of seviteronel may be sex-specific . The phaSince other oncogenic aberrations may co-exist with AR dependency, combination regimens involving AR inhibitors and other targeted therapies are also underway for AR-positive TNBC. Palbociclib, an orally administered and highly selective and reversible CDK4/6 inhibitor, prevents the onset of the S phase, resulting in cytotoxicity and growth arrest. Studies have shown that luminal phenotypes, elevated cyclin D1 and Rb protein expression, and reduced p16 expression are all associated with sensitivity to Palbociclib . AR-posiA significant body of evidence shows that tumor hypoxia interferes with therapies that disrupt AR signaling or reduce androgen levels in PC. A recent study showed that enzalutamide induces hypoxia and microenvironment adaptation and that two cytokines—interleukin-8 (IL8) and vascular endothelial growth factor A (VEGF-A)—upregulated by this treatment-induced hypoxia may contribute to this treatment resistance . This stIt was also discovered that HIF-1α coordinates AR translocation to the nucleus via binding to β-catenin and AR to form a ternary complex that binds to the androgen response elements . Under hSome groups have begun to unravel other mechanisms of intra-tumoral hypoxia-mediated resistance to androgen/AR signaling inhibition. Hypoxia-mediated resistance to the potent second-generation AR inhibitor, enzalutamide, was shown to mechanistically occur in PC via restoration of glucose-6-phosphate isomerase (GPI), which is transcriptionally repressed by AR in hypoxia and maintains glucose metabolism and energy homeostasis under hypoxic conditions . AR inhiSLCA4 and SLC4A5, which regulate tumor pH levels when adapting to hypoxic conditions [Hypoxic regions have been observed in over 50% of breast tumors but more frequently in TNBC. Genomic profiling revealed high HIF-1α signaling activity in basal-like/TNBCs . Specifinditions .The enhanced hypoxic signaling observed in TNBC has been suggested to underlie advanced progression and treatment resistance in TNBC, which may also be undermining the efficacy of AR inhibition, as seen in Hypoxia has also been shown to predict prognosis in TNBC. Positive HIF-1α IHC expression significantly correlated with greater tumor size, higher histological grade, positive lymph node status, and higher tumor TNM stage as well as poorer postoperative survival . The enrAlthough AR therapy is a promising prospect for TNBC, it has so far consistently proven beneficial for only a subset of the participants in the clinical trials. Consequently, researchers have been attempting to identify predictive biomarkers that may indicate a high likelihood of benefit from androgen/AR-targeting therapies. Full-length ER-β protein has been observed in 50%–90% of ER-α-negative BCs ,70. EvidEnzalutamide-resistant tumors have also been shown to upregulate glucocorticoid receptors (GR), which may drive the expression of some AR-regulated genes and thereby decrease dependence on AR . Ligand-As previously discussed, Lehmann and colleagues discovered that AR-positive TNBCs display a greater frequency of PI3KCA and AKT1 mutations ,84. AR pStudies aimed at gaining insight into the basis for the resistance to abiraterone treatment in PC have revealed that the SLCO1B3 gene  is induced by abiraterone . This efTargeting the hypoxia signaling axis in AR-positive TNBC may simultaneously target other mechanisms of resistance induced upon AR signaling inhibition such as angiogenesis, epithelial-mesenchymal transition (EMT), epigenetic reprogramming, and AR-independent lineage plasticity . This upIn the context of PC, the induction of a variety of AR splice variants (AR-Vs) that occurs as an acute response to selection pressures, such as castration, or exposure to therapeutic mainstays, such as enzalutamide and abiraterone, is well documented ,105,106.Another reason it may be beneficial to target hypoxic signaling in AR-positive TNBC is to thwart the induction of the centrosome amplification (CA) that is known to result from hypoxia ,120. TheAR has long been acknowledged as an alternative and actionable endocrine target for AR-positive, ER-negative BCs such as TNBC. Unlike the other BC endocrine targets, ER and PR, the therapeutic inhibition of the AR pathway has been less successful and the reasons for this therapeutic failure are more complex. Although the concept of targeting the androgen/AR axis is not inherently flawed, the data clearly show that we need to better anticipate that hypoxia is likely to diminish the clinical efficacy of these strategies and design customized therapies that address these resistance mechanisms preemptively. Accumulating evidence shows that TNBCs are more enriched in hypoxic signaling than the other BC subtypes and that hypoxia may undergird the increased resistance to anticancer drugs, such as endocrine therapies and monoclonal antibodies against HER2, which is frequently observed in TNBC ,132,133.The afore-mentioned studies provide a sobering reminder that AR inhibition of tumors can lead to the activation or upregulation of adaptive pathways, including the PI3K/AKT, mTOR, GR, and Wnt/β-catenin pathways, which are themselves oncogenic. Although targeting these pathways in combination with AR inhibitors such as enzalutamide has elicited some preclinical and clinical success, the inhibition of these pathways is often met with challenges such as the activation of other compensatory pathways. For example, the inhibition of the PI3K/AKT/mTOR signaling network has led to the upregulation of the interconnected pathways RAS-MEK-ERK and JAK2/STAT5 signaling . The GR Inhibiting hypoxia in combination with AR therapy in AR-positive TNBCs may represent a more effective multipronged approach to circumventing the multiple mechanisms of AR therapy resistance. As mentioned prior, the hypoxia signaling network is interconnected with the compensatory pathways of AR inhibition such as PI3K/AKT and Wnt/β-catenin. Thus, targeting the hypoxia signaling axis may concomitantly target or interfere with the downstream effects of these and other compensatory pathways. Additionally, hypoxic signaling promotes the development of other mechanisms of AR therapy resistance such as increasing alternative splicing, EMT, stem cell regeneration, and angiogenesis. Thus, therapeutically targeting hypoxia may also suppress these additional mechanisms.Therapies aimed at inhibiting the hypoxic signaling axis have primarily been designed to directly or indirectly block HIF-1α and/or HIF-2α expression or function. Specifically, these HIF inhibitors interfere with HIFα mRNA expression, protein synthesis, dimerization, DNA binding, and transcriptional activity, as well as protein stabilization and accumulation . These aTherefore, moving forward, we envisage that the thrust of our efforts would most likely need to be in the following directions: we need to increase pre-clinical and clinical investigations into (a) the AR signaling axis and its rich “conversations” with the tumor microenvironment in TNBC, (b) the molecular mechanisms by which hypoxia engenders and/or exacerbates androgen-/AR-independence and therapy resistance in AR-positive TNBC, (c) the effects of combining AR and HIF inhibitors in pre-clinical models that more accurately recapitulate the hypoxic conditions within solid tumor microenvironments, (d) AR-variant protein expression in TNBC tumor specimens, particularly post-treatment with AR-targeted therapies, alone and in combination with HIF inhibitors, and (e) the prognostic value of HIF-1α, or hypoxia gene signatures as biomarkers could improve risk-stratification for AR-positive TNBC patients as well as patient segmentation for HIF inhibitor treatment. A pre-clinical investigation should include assessing the response of AR-enriched TNBC cell lines and patient-derived xenograft models to dual treatment with current-generation AR and HIF-1α inhibitors compared to each inhibitor administered alone. Above all, and given the fact that AR IHC alone is a poor proxy for AR dependence and is a woefully inadequate patient pre-selection criterion and predictor of response to AR-targeted treatments, we need to focus efforts on identifying better tumor-intrinsic biomarkers that integrate information about true AR dependence and the tumor’s TNBC molecular subtype, that take into consideration and are attuned to the hypoxia status of the tumor, the AR splice variant status, the AR mutational and ER-β status, and the CA landscape of the tumor. Novel biomarker discovery also needs to be pre-planned and built into the design of clinical trials. The identification of superior, multi-omic predictive biomarkers will enable the thoughtful design of better, tumor-informed clinical trials involving carefully selected patient subsets, within which specific treatment combinations may deliver the greatest and most durable clinical benefits and in the end be practice-changing. Since angiogenesis, induced upon hypoxia, is a major culprit of AR therapy resistance, it may be beneficial to evaluate angiogenesis markers and incorporate anti-angiogenic therapies in the clinic.Angiogenesis is often the culprit in hypoxia-induced therapy resistance; it is no wonder then that several targeted anti-angiogenics have been FDA-approved for epithelial malignancies. Anti-angiogenics are credited with decreasing tumor metastasis and promoting the clinical efficacy of chemotherapy, immunotherapy, and radiation therapy . It is wTo facilitate the individualized treatment of tumors with anti-angiogenics, agents and probes that allow direct, non-invasive, and dynamic in vivo visualization of hypoxic regions of breast tumors, can inform clinical decision making by providing real-time information regarding the hypoxia status of the tumor, the extent of the re-normalization of the vessel architecture, as well as the changes in the interstitial delivery of therapeutics . A 2016 The establishment of AR as a robust risk-prognostic and therapeutic target for AR-positive TNBC patients still requires a deeper understanding of the AR signaling axis and the interplay between AR signaling and the TNBC tumor microenvironment. Our discussion aims to encourage further investigation into an important aspect of the tumor microenvironment, hypoxia, and its potential role in undermining current AR-targeted therapies in AR-enriched TNBC. We assert that the increased understanding of the interaction between the AR and hypoxia signaling axes in TNBC could renew interest in exploiting AR for the clinical management of TNBC patients and lead to breakthroughs in AR therapy administration.


O bloqueio atrioventricular (BAV) descreve um comprometimento na condução dos átrios para os ventrículos. Embora o curso clínico do BAV tenha sido avaliado, os achados são de países de alta renda e, portanto, não podem ser extrapolados para a população latina. Avaliar a associação entre BAV e mortalidade. Foram incluídos pacientes do estudo CODE , maiores de 16 anos que realizaram eletrocardiograma (ECG) digital de 2010 a 2017. Os ECGs foram relatados por cardiologistas e por software automatizado. Para avaliar a relação entre BAV e mortalidade, foram utilizados o modelo log-normal e as curvas de Kaplan-Meier com valores de p bicaudais < 0,05 considerados estatisticamente significativos. O estudo incluiu 1.557.901 pacientes; 40,23% eram homens e a média de idade foi de 51,7  anos. Durante um seguimento médio de 3,7 anos, a mortalidade foi de 3,35%. A prevalência de BAV foi de 1,38% (21.538). Os pacientes com BAV de primeiro, segundo e terceiro graus foram associados a uma taxa de sobrevida 24% , 55%  e 64%  menor quando comparados ao grupo controle, respectivamente. Os pacientes com BAV 2:1 tiveram 79%  menor taxa de sobrevida do que o grupo controle. Apenas Mobitz tipo I não foi associado a maior mortalidade . BAV foi um fator de risco independente para mortalidade geral, com exceção do BAV Mobitz tipo I. O estudo CODE  é um grande banco de dados que compreende todos os ECGs realizados majoritariamente em unidades básicas de saúde pela Rede Telessaúde de Minas Gerais, Brasil, no período de 2010 a 2017.12 O banco de dados de ECG foi vinculado ao Sistema de Informação sobre Mortalidade nacional e pode fornecer informações epidemiológicas em uma população que é representativa da população geral. Assim, no presente estudo, objetivamos descrever a prevalência e os fatores de risco do BAV e, principalmente, avaliar a associação entre o BAV e a mortalidade geral nesta grande coorte brasileira de atenção primária.No entanto, não há estudo prospectivo sobre o valor prognóstico de todos os graus de BAV em uma população geral, o que limita a compreensão do significado dessas anormalidades no ambiente ambulatorial. De fato, estudos anteriores do nosso grupo mostraram que anormalidades do ECG consideradas prognosticamente importantes, como a síndrome de pré-excitação, não têm impacto prognóstico em um ambiente comunitário.13 Foi analisado o conjunto de dados do estudo CODE,14 que compreende todos os ECGs válidos realizados em pacientes maiores de 16 anos pela Rede Telessaúde de Minas Gerais de 2010 a 2017. Foram excluídos exames sem traçados válidos ou com problemas técnicos. Nos pacientes que realizaram mais de um ECG, foi analisado apenas o primeiro exame.Foi realizado um estudo retrospectivo utilizando um banco de dados de ECGs digitais da Rede Telessaúde de Minas Gerais.Os dados clínicos foram coletados por meio de um questionário padronizado, que incluiu idade, sexo e comorbidades autorreferidas, tais como: hipertensão, diabetes, tabagismo, doença de Chagas, infarto prévio do miocárdio e doença pulmonar obstrutiva crônica.Os ECGs foram realizados pelo profissional da atenção primária local, utilizando eletrocardiógrafos digitais da Tecnologia Eletrônica Brasileira, modelo ECGPC  ou Micromed Biotecnologia, modelo ErgoPC .15Um software específico, desenvolvido internamente, foi capaz de capturar o traçado do ECG, fazer o upload do ECG e do histórico clínico do paciente e, então, enviá-los para o centro de análise da Rede Telessaúde de Minas Gerais pela internet. As informações clínicas, traçados de ECG e relatórios foram armazenados em um banco de dados específico. Os laudos de ECG foram gerados em modelo de texto livre por cardiologistas e, também, interpretados e codificados automaticamente em códigos de Glasgow e Minnesota pelo programa de análise de ECG de Glasgow 12 derivações .3 .18A coorte eletrônica foi obtida ligando os dados dos exames de ECG  e os do Sistema de Informação sobre Mortalidade nacional,O programa R  foi utilizado para análise estatística. Os dados categóricos foram relatados como contagens e porcentagens; as variáveis contínuas foram relatadas como média e desvio padrão (DP). O desfecho foi mortalidade por todas as causas, incluindo todos os códigos da Classificação Internacional de Doenças na certificação médica da causa de óbito. Foi utilizado o teste de Shapiro-Wilk para verificar a normalidade dos dados. O método de Kaplan-Meier foi utilizado para estimar as curvas de sobrevida para todas as causas de óbito. Utilizamos o teste da razão de verossimilhança (TRV) para ajustar os dados para o melhor modelo paramétrico, uma vez que a suposição proporcional para o modelo de regressão de Cox foi violada. No TRV, o modelo generalizado, representado pelo modelo de regressão gamma generalizado, foi comparado com os demais modelos de interesse . Optamos por trabalhar com o modelo log-normal, pois a log-probabilidade desse modelo era maior e a análise de resíduos indicou que a distribuição log-normal foi a melhor escolha para esses dados. A taxa de sobrevida relativa (SR) foi utilizada como medida de associação, com intervalo de confiança de 95%. SR < 1 significa maior risco de mortalidade e SR > 1 significa risco menor. Valores de p bicaudais < 0,05 foram considerados estatisticamente significativos. Este estudo foi aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de Minas Gerais.Em total, foram incluídos 1.557.901 pacientes; 40,23% eram homens e a média de idade foi de 51,67  anos. Durante um seguimento médio de 3,7 anos, a mortalidade foi de 3,35%. A prevalência de BAV foi de 1,38% (21.538); 1,32% (20.644) correspondendo ao BAV de primeiro grau, 0,02% (273) ao BAV de segundo grau e 0,04% (621) ao BAV de terceiro grau. Dentre esses, 273 casos eram de BAV de segundo grau, 212 eram Mobitz tipo I e 61 eram 2:1. As condições clínicas de todos os pacientes estão descritas na Após ajuste para sexo, idade e condições clínicas, os pacientes com BAV de primeiro, segundo e terceiro graus foram associados a uma taxa de sobrevida 24%, 55% e 64% menor quando comparado ao grupo controle, respectivamente   . Na anáNesta grande coorte eletrônica com mais de um milhão de pacientes, o BAV foi associado a maior risco de mortalidade geral. Em relação ao tipo de BAV, apenas o Mobitz tipo I não apresentou risco aumentado de mortalidade, comparado ao grupo controle.20 Por outro lado, estudos longitudinais anteriores na população geral, que incluíram principalmente homens jovens e de meia-idade, verificaram que o intervalo PR prolongado tem um curso benigno.23 Devemos destacar que esses dados vieram de uma população específica com vigilância limitada e uma amostra relativamente baixa de pacientes com BAV. Mais recentemente, uma publicação da coorte de Framingham4 mudou esse paradigma. Após 20 anos de seguimento, o prolongamento do PR foi associado ao aumento do risco de fibrilação atrial, implante de marcapasso e óbito4 . Um grande estudo de ECG dinamarquês com 288.181 pacientes confirmou o maior risco de fibrilação atrial associado à presença do primeiro BAV.24Em pacientes com cardiopatia estrutural, o BAV de primeiro grau tem sido descrito como um fator de risco para desfechos adversos.25 Devem ser apontadas algumas diferenças entre essas coortes. A coorte brasileira era mais velha  e também incluia pacientes idosos. Analisamos cerca de 1,5 milhão de ECG contra 10 mil. A doença de Chagas foi relativamente prevalente e teve forte associação com a presença de BAV, independente do grau. As diferenças sociais entre os dois países também podem ter contribuído. O acesso aos serviços públicos de saúde e a educação da população são completamente desiguais em países de baixa e média renda e podem ter impacto prognóstico na população.26Em nossa população, foi encontrada uma redução de 24% na sobrevida de pacientes com PR > 200 ms, após ajuste para idade, sexo e condições clínicas, em contraste com um estudo anterior na população finlandesa.3 Sua associação com a mortalidade é esperada,9 pois a lesão da condução AV é mais grave e a cardiopatia está frequentemente relacionada.3 O prognóstico no BAV 2:1 está intimamente relacionado ao local do BAV: nodal ou infranodal.3 No presente estudo, o BAV 2:1 no ECG de 12 derivações foi associado a uma redução de 79% na sobrevida relativa, provavelmente indicando um bloqueio infranodal. O BAV Mobitz tipo I, por outro lado, não foi associado a maior mortalidade em nossa coorte.Está bem estabelecido que BAV Mobitz tipo II irreversível, BAV de alto grau e BAV de terceiro grau são indicações para estimulação permanente, mesmo em pacientes assintomáticos.27 Pode ser um BAV mediado vagal que não apresenta comprometimento anatômico da condução AV,28 não evoluindo, portanto, para BAV de alto grau. Em pacientes mais velhos, a história natural pode ser diferente, e eles podem se beneficiar de um marcapasso permanente.29 Não realizamos subanálise em pacientes idosos e a presença de sintomas é desconhecida.BAV Mobitz tipo I frequentemente tem prognóstico benigno, principalmente em pacientes jovens sem doença cardíaca.30 Em nosso serviço, o BAV de segundo grau foi estatisticamente maior nos ECGs assinalados como eletivos do que naqueles com prioridade de emergência30 . Os desfechos dos pacientes podem mudar com o encaminhamento precoce ao hospital e consequente implante de marcapasso.31 Os dados de hospitalização não estavam disponíveis para toda a nossa coorte e, portanto, não foram incluídos no presente artigo. No entanto, futuros trabalhos estão planejados nesta área para avaliar a jornada dos pacientes em nosso sistema de saúde a partir do diagnóstico de BAV por ECG.Os pacientes com emergências cardiovasculares frequentemente procuram atendimento em unidades básicas de saúde, principalmente em municípios pequenos e remotos. Os serviços de tele-eletrocardiografia desempenham um papel importante nesse cenário, principalmente por reconhecerem anormalidades de ECG potencialmente fatais que são diagnosticadas erroneamente pelo médico local.Lazy Associative Classifier usado para classificar laudos de ECG tem boa precisão, sensibilidade e valor preditivo positivo, mas pode cometer erros. Para minimizar esse problema, incluímos a classificação automática de Glasgow e Minnesota no algoritmo diagnóstico. Além disso, foi realizada a revisão manual de mais de 9.000 ECGs para confirmar a presença de BAV. A ligação probabilística também apresenta alguns problemas, tais como sensibilidade menos que perfeita e a possibilidade de pares falsos. Portanto, definimos um alto ponto de corte para pares verdadeiros e realizamos revisão manualO nosso estudo possui algumas limitações. Os dados clínicos foram autorrelatados e, portanto, podem ter sido subnotificados. O software para os casos duvidosos. Ainda não temos informações sobre sintomas ou dados de internação, mas os dados de procedimentos de marcapasso em cada grupo estarão disponíveis em breve para análise e têm sido planejados trabalhos futuros sobre esse tema.Apesar disso, nosso estudo traz novos dados sobre o prognóstico do BAV, visto que avalia uma população latina de um centro de atenção primária com mais de um milhão de pacientes. Os nossos achados são consistentes e podem constituir uma ferramenta útil para direcionar políticas de saúde pública e recursos de financiamento.A presença de BAV foi associada a um risco aumentado de mortalidade geral na população da Rede Telessaúde de Minas Gerais. Nos pacientes com BAV de segundo e terceiro grau, apenas aqueles com Mobitz tipo I não apresentaram maior risco de mortalidade. 1 The presence of delay or interruption in AV conduction is called atrioventricular block (AVB),2 which is classified into three degrees, according to the electrocardiogram (ECG) presentation.3 There are several known causes of AVB, including ischemic heart disease, degenerative conduction system disease, congenital heart disease, connective tissue disease, inflammatory diseases, medications, and increased autonomic tonus.4The atrioventricular (AV) node is responsible for the electrical connection between the atria and ventricles.6 The prevalence is usually higher in the elderly and in men.5 First-degree AVB is the most common, and can be frequently found in outcome patients.4AVB prevalence varies between 0.6% to 6.04% in the literature, depending on the population studied and the degree of AVB.4 Patients with first-degree AVB have a higher risk of atrial fibrillation,7 death, stroke, or hospitalization for heart failure.8 It is also described that, in patients with acute myocardial infarction, high-degree AVB is associated with an increased risk of morbidity and mortality.9The clinical course of first-degree AVB has been evaluated in studies from community-based samples, such as the Framingham cohort.10 In contrast, the risk of mortality for a patient with right bundle branch block (BBB) is almost as high as that of a patient with left BBB,11 even though the latter is considered a much stronger marker of risk in general cardiology practice. The CODE  study is a large database that comprises all ECGs performed mostly at primary health care facilities by the Telehealth Network of Minas Gerais, Brazil, from 2010 to 2017.12 The ECG database was linked to the Brazilian Mortality Information System and can provide epidemiological information in a population that is representative of the general population. Thus, in the present study, we aim to describe the prevalence and risk factors of AVB and, mainly, to evaluate the association between AVB and overall mortality in this large primary care Brazilian cohort.Nonetheless, there is no prospective study on the prognostic value of all degrees of AVB in a general population, which limits the understanding of the significance of these abnormalities in an outpatient setting. Indeed, previous studies from our group showed that ECG abnormalities that are considered prognostically important, such as pre-excitation syndrome, have no prognostic impact in a community setting.13 The CODE dataset,14 which comprises all valid ECGs performed in patients over 16 years old by the TNMG from 2010 to 2017, was analyzed. Exams without valid tracings or with technical problems were excluded. In patients who underwent more than one ECG, only the first exam was analyzed.We conducted a retrospective study using a database of digital ECGs from the Telehealth Network of Minas Gerais (TNMG),Clinical data were collected using a standardized questionnaire, which included age, sex, and self-reported comorbidities, such as: hypertension, diabetes, smoking, Chagas disease, previous myocardial infarction, and chronic obstructive pulmonary disease.ECGs were performed by the local primary care professional, using digital electrocardiographs by Tecnologia Eletrônica Brasileira, model ECGPC  or Micromed Biotecnologia, model ErgoPC 13 .15Specific software, developed in-house, was capable of capturing ECG tracing, uploading the ECG and the patient’s clinical history, and sending them to the TNMG analysis center through the internet. The clinical information, ECG tracings, and reports were stored in a specific database. The ECG reports were generated in a free text model by cardiologists and, also, automatically interpreted and coded into Glasgow and Minnesota codes by the Glasgow 12-lead ECG analysis program .3 .18The electronic cohort was obtained linking data from the ECG exams  and those from the Brazilian Mortality Information System,R program  was used for statistical analysis. Categorical data were reported as counts and percentages; continuous variables were reported as mean and standard deviation (SD). The endpoint was all-cause mortality, including all International Classification of Diseases codes in the medical certification of cause of the death. The Shapiro-Wilk test was used to verify the normality of the data. The Kaplan-Meier method was used to estimate the survival curves for all causes of death. We used the likelihood ratio test (LRT) to adjust data for the best parametric model, since the proportional assumption for the Cox regression model was violated. In the LRT, the generalized model, represented by the generalized gamma regression model, was compared with the other models of interest . We chose to work with the log-normal model, since the log-likelihood of this model was higher and the residual analysis indicated that log-normal distribution was a better choice for this data. Relative survival rate (RS) was used as the measure of association, with a confidence interval of 95%. RS < 1 means higher risk of mortality, and RS > 1 means lower risk. Two-tailed p-values < 0.05 were considered statistically significant. This study was approved by the Research Ethics Committee of the Federal University of Minas Gerais.A total of 1,557,901 patients were included; 40.23% were men, and mean age was 51.67 (SD ± 17.58) years. In a mean follow-up of 3.7 years, the mortality rate was 3.35%. The prevalence of AVB was 1.38% ; 1.32%  corresponding to first-degree AVB, 0.02% (273) to second-degree AVB, and 0.04% (621) to third-degree AVB. Among these 273 cases of second-degree AVB, 212 were Mobitz type I, and 61 were 2:1. The clinical conditions of all patients are described in After adjustment for sex, age, and clinical conditions, patients with first-, second-, and third-degree AVB were associated with 24%, 55%, and 64% lower survival rate when compared to the control group, respectively   . In theIn this large electronic cohort with more than one million patients, AVB was associated with higher risk of overall mortality. Regarding the type of AVB, only Mobitz type I did not have an increased risk of mortality, compared to the control group.20 On the other hand, previous longitudinal studies in the general population that mainly included young and middle-age men found that prolonged PR interval has a benign course.23 We should highlight that this data came from a specific population with limited surveillance and a relatively low sample of patients with AVB. More recently, a publication from the Framingham cohort4 changed this paradigm. After 20 years of follow up, PR prolongation was associated with increased risk of atrial fibrillation, pacemaker implantation, and death4 . A large Danish ECG study with 288,181 patients confirmed the higher risk for atrial fibrillation associated with the presence of the first AVB.24In patients with structural heart disease, first-degree AVB has been described as a risk factor for adverse outcome.25 Some differences between these cohorts must be pointed out. The Brazilian cohort was older (mean age 51.7 versus 44 years), and it also included elderly patients. We analyzed about 1.5 million ECG versus 10,000. Chagas disease was relatively prevalent and it had a strong association with the presence of AVB, regardless of the degree. The social differences between both countries might also have contributed. Access to public health services and population education are completely unequal in low- and middle-income countries and may have a prognostic impact on the population.26In our population, a 24% reduction in the survival rate of patients with PR > 200 ms, after adjustment for age, sex and clinical conditions were found, contrary to a previous study in the Finnish population.3 Their association with mortality is expected,9 since AV conduction injury is more severe, and heart disease is often related.3 The prognosis in 2:1 AVB is intimately related to the site of the AVB: nodal or infranodal.3 In the present study, 2:1 AVB in the 12-lead ECG was associated with a 79% reduction in relative survival, probably indicating an infranodal block. Mobitz type I AVB, on the other hand, was not associated with higher mortality in our cohort.It is well established that irreversible Mobitz type II, high and third-degree AVB are indications for permanent pacing, even in asymptomatic patients.27 It can be a vagal mediated AVB that does not have an anatomical involvement of AV conduction,28 and it does not, therefore, progress to a high-degree AVB. In older patients, the natural history can be different, and they might benefit from a permanent pacemaker.29 We did not perform a sub-analysis in elderly patients, and the presence of symptoms is unknown.Mobitz type I AVB frequently has a benign prognosis, especially in young patients without cardiac disease.30 In our service, second-degree AVB was statistically higher in the ECGs assigned as elective than in those with emergency priority.30 Patients’ outcomes could change with early referral to the hospital and consequent pacemaker implantation.31 Hospitalization data was not available for our entire cohort and, therefore, was not included in this paper. Nonetheless, further work in this field is planned to evaluate patients’ journey in our healthcare system from the ECG diagnosis of AVB.Patients with cardiovascular emergencies often seek health assistance in primary care units, especially in small and remote counties. Tele-electrocardiography services play an important role in this setting, mainly for recognizing potentially life-threatening ECG abnormalities that are misdiagnosed by the local physician.Our study has limitations. The clinical data was self-reported and, thus, might have been underreported. The Lazy Associative Classifier software used to classify ECG reports has good accuracy, sensibility, and positive predictive value, but it may make errors. In order to minimize this problem, we included the Glasgow and Minnesota automatic classification in the diagnostic algorithm. Furthermore, manual revision of more than 9,000 ECGs was conducted to confirm the presence of AVB. The probabilistic linkage also has some issues, such as less than perfect sensitivity and the possibility of false pairs. Therefore, we defined a high cutoff point for true pairs and conducted manual revision for the doubtful cases. We still do not have information on symptoms or hospitalization data, but data from pacemaker procedures in each group will soon be available for analysis, and future work on this topic has been planned.Nevertheless, our study brings new data on AVB prognosis, as it evaluates a Latinx population from a primary care centers with more than one million patients. Our findings are consistent and might be a useful tool to direct public health policies and funding resources.The presence of AVB was associated with an increased risk of overall mortality in the TNMG population. In patients with second and third-degree AVB, only those with Mobitz type I did not have a higher risk of mortality.


Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein overexpressed in human malignancies, including prostate cancer (PCa). In this study, we aimed to explore the oncogenic function of CIP2A in PCa cells and its underlying mechanism. We showed that 63.3% (38/60 cases) of PCa tissues exhibited a high CIP2A immunostaining, compared to 25% (3/12 cases) of BPH samples (p = 0.023). Furthermore, the protein level of CIP2A was positively correlated with patients’ short survival time and nuclear AR levels in PCa tissues. Compared to PZ-HPV-7, an immortalized prostate cell line, androgen-sensitive LNCaP C-33, androgen-independent LNCaP C-81, or 22Rv1 cells exhibited a high CIP2A level, associated with high protein and phosphorylation levels of AR. While AR expression and activity modulated CIP2A expression, manipulating CIP2A expression in PCa cells regulated their AR protein levels and proliferation. The reduction of CIP2A expression also enhanced the sensitivity of PCa cells toward Enzalutamide treatment. Our data further showed that depletion of polo-kinase 1 (PLK1) expression or activity in C-81 or 22Rv1 cells caused reduced protein levels of c-Myc and AR. Notably, inhibition of PLK1 activity could abolish CIP2A-promoted expressions in c-Myc, AR, and prostate-specific antigen (PSA) in C-33 cells under an androgen-deprived condition, suggesting the role of PLK1 activity in CIP2A-promoted AR expression. In summary, our data showed the existence of a novel regulation between CIP2A and AR protein levels, which is critical for promoting PCa malignancy. Thus, CIP2A could serve as a therapeutic target for PCa. Prostate cancer (PCa) is the most commonly diagnosed cancer among men in the developed world. Like normal prostate cells, most PCa cells require androgens for their growth and survival. Thus, androgen deprivation therapy (ADT) which aims to reduce androgen levels or block the activity of androgen receptor (AR) becomes the standard treatment for locally advanced or metastatic PCa. Most tumors initially respond to ADT but later are refractory to hormonal therapies. Within 2–3 years, the diseases eventually develop the castration-resistant PCa (CRPC), resulting in tumor relapse and cancer-related death . Until rAR gene amplification [The AR is a steroid hormone receptor that functions as a ligand-regulated transcription factor. Upon activation by androgens, AR dimerizes as a homodimer and subsequently binds to androgen-responsive elements (ARE) on target genes, activating gene expression for regulating the growth and survival of prostate epithelial cells. However, deregulated AR portein level or activity plays a key role in the development of CRPC. Accumulated evidence demonstrates that the majority of CRPC is still dependent on the AR signaling and exhibits a reactivated AR even under castration. Numerous mechanisms have been identified to drive the progression from androgen-dependent to CRPC . Interesfication . It indification .KIAA1524 gene, is an oncoprotein overexpressing approximately 39 to 90% of malignant tissues. Its protein level is correlated with aggressive disease and the poor survival rate [CIP2A, encoded by the val rate , 7. Accuval rate . Thus, Cval rate . Furtherval rate . In PCa val rate . KnockdoIn this study, we investigated the novel regulatory mechanism between CIP2A and AR in PCa cells. We showed that positive correlation exhibited between CIP2A and AR expression in PCa tumors. We further provided evidence that AR signaling could transcriptionally regulate CIP2A protein levels. Importantly, CIP2A modulated AR protein level required the activity of PLK1. Our data demonstrated the oncogenic role of CIP2A in promoting AR protein level, which potentially serves as a therapeutic target for CRPC treatment.Antibodies against CIP2A (lot number K2420), PP2Ac (lot number D2309), or β-actin (lot number J0421) and horseradish peroxidase (HRP)-conjugated secondary antibodies were from Santa Cruz Biotechnology . Antibodies against PSA (Lot number 3), PLK1 (lot number 6), phospho-c-Myc (Ser62) (lot number 5), or c-Myc (lot number 15) were purchased from Cell Signaling . Antibodies against phospho-AR (Ser81) (lot number 3497306) or AR (lot number 3256650) were obtained from Millipore . Enzalutamide was obtained from MedChemExpress . LB100 and BI6727 were purchased from BioVision . Okadaic acid, TD52, Bicalutamide, and other chemicals were obtained from Sigma-Aldrich .Cell culture media, FBS, charcoal/dextran-treated FBS (c-FBS), and supplements were purchased from ThermoFisher Scientific . Immortalized prostate cell line PZ-HPV-7 cells, androgen-sensitive LNCaP (passage 28), and androgen-independent 22Rv1 prostate carcinoma cell lines were purchased from the Biosource Collection and Research Center  and cultured as described previously . Cells wKIAA1524 expression vector, the full-length human KIAA1524 cDNA flanked by attL sequences and cloned in the pENTR223.1 vector was purchased from MyBioSource . Using the Gateway LR Clonase II enzyme mix kit , the KIAA1524 cDNA was shuttled from the pENTR223.1 vector to the attR-contained pLX302 lentiviral vector . For gene overexpression or knockdown experiments, the production or infection of vector-contained lentiviruses was performed as described previously [For constructing the eviously .Total RNA content was isolated from cells using an RNA purification kit , and cDNA was synthesized using a GoScript reverse transcription system , following the manufacturer’s protocols. All real-time PCR reactions were conducted by the Bio-Rad CFX96 Real-time PCR Detection System . The amplification was conducted using the iQ SYBR Green Supermix (Bio-Rad). The thermal cycling conditions were 30 cycles . The paired primers used for AR and GAPDH were AR (forward): 5′-CCTGGCTTCCGCAACTTACAC-3′; AR (reverse): 5′-GGACTTGTGCATGCGGTACTC-3′; GAPDH (forward): 5′-TGGTATCGTGGAAGGACTCATGAC-3′; and GAPDH (reverse): 5′-TGCCAGTGAGCTTCCCGTTCAGC-3′. These primers were designed according to the database of RTPrimerDB .5 cells/well were seeded onto 24-well plates. After incubation for 48 h, one set of attached cells was harvested and counted as day 0. The remaining cells were fed with fresh medium, and the cell number was counted on day 3.The virus-infected or inhibitor-treated cells with a density of 0.7–1 × 10The experimental procedure was described previously . BrieflyTissue samples, including 12 benign prostatic hyperplasia (BPH) and 60 PCa, were collected from patients who underwent prostatectomy and whose personal information was de-identified between 2003 and 2006 at Kaohsiung Veterans General Hospital . All specimens from the archival files and reviewed by two experienced pathologists. None of the patients had received chemotherapy, hormonotherapy, or radiotherapy before surgery. The study protocol was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital (#KSVGH21-CT5-01) in accordance with the IRB’s guidelines and regulations.The tissue microarrays (TMA) were prepared from the formalin-fixed paraffin-embedded tissue blocks using a manual TMA Arrayer MTA-1 . Immunohistochemical (IHC) procedures were conducted following the manufacturer’s protocol of the Bond III Autostainer . Briefly, tissue sections were deparaffinized and rehydrated. Subsequently, the slides were immersed in ethylenediaminetetraacetic acid buffer (pH 9.0) for 40 min. The sections were then incubated with an anti-CIP2A  at 1:100 dilution or an anti-AR  at 1:50 dilution at RT for 30 min. The signal was amplified using a BOND Polymer Refine Detection kit (Leica Biosystems). The protein levels of CIP2A and nuclear AR in the prostate epithelia were evaluated independently by two experienced pathologists who scored using a semi-quantitative H-score method, which considered both the staining intensity and the percentage of positively stained cells. The intensity of the staining was divided into three grades: negative (0), weak (1+), moderate (2+), and strong (3+). The percentage (0 to 100%) of staining was determined and a total score ranging from 0 to 300 was obtained .-value of < 0.05 was considered significant.The MedCalc version 19.1.6  and PRISM version 5.0  were used for statistical analyses. A median cut-off immunoscore value of 160 was used to separate cases into low and high CIP2A immunoexpression. A Student’s two-tailed t-test or a one-way ANOVA was applied to determine the significance between groups. Fisher’s exact or chi-square test was applied to evaluate associations between categorical variables. Spearman’s or Pearson’s correlation was used to assess the correlation between variables. The Kaplan–Meier method was used to analyze overall survival (OS), and the log-rank test was used to compare groups. A phttp://xena.ucsc.edu/) showed that the PCa patients with a high expression of the KIAA1524 gene with a low ratio of progression-free survival (PFS) (p = 0.0413)  further indicated a moderate positive correlation between the KIAA524 and AR genes in PCa samples with the Pearson correlation coefficient (r) = 0.6  staining, whereas the BPH sample only showed a weak (1+) immunoreactivity , an androgen-sensitive LNCaP C-33 (C-33), and two androgen-independent PCa cell lines, i.e., LNCaP C-81 (C-81) and 22Rv1 , 17. ComWe next examined whether CIP2A could regulate the AR protein level and cell proliferation in PCa cells. As shown in Fig. Because CIP2A has been shown to inhibit PP2A, thus promoting the stability and activity of c-Myc , a transWe next examined the role of polo-like kinase 1 (PLK1), a serine/threonine kinase, in mediating the CIP2A-promoted AR protein level. As shown in Fig. CIP2A is a well-known oncoprotein in various cancers. Its oncogenic function in modulating castration resistance in PCa cells has not been studied. In this study, we showed a positive correlation between CIP2A and AR protein levels in PCa tumors. While AR signaling could modulate CIP2A expression, CIP2A reciprocally regulated AR levels in mRNA and protein levels. Importantly, our data showed the CIP2A-modulated AR level required the activity of PLK1. To our knowledge, this is the first report showing the novel regulation between CIP2A and AR protein levels in PCa cells, which may contribute to the castration resistance of PCa cells.KIAA1524 gene between normal and PCa samples. However, lines of evidence indicated high CIP2A protein levels in PCa tumors, correlating with high Gleason scores [Based on the TGCA database, no difference is found in the expression of n scores , 24. In n scores . The hign scores  first shn scores , 26, ELKn scores , and E2Fn scores , to inten scores .AR gene amplification or increased protein level is commonly found in CRPC patients . IncreasOur results clearly showed that CIP2A could modulate PLK1 protein levels in immortalized prostate and PCa cells. The functional linkage between CIP2A and PLK1 was further supported by the observation that CIP2A interacts with the polo-box domain of PLK1, which maintains PLK1 stability and enhances its kinase activity . PLK1 isIn summary, we have demonstrated that CIP2A was highly expressed in PCa tumors, associated with AR protein level. While AR signaling could upregulate CIP2A level in an AR-dependent manner, CIP2A inversely modulated AR level. The underlying mechanism was not mediated by PP2A inhibition but was associated with PLK1 activity. Thus, our data supported the oncogenic role of CIP2A in promoting AR expression and castration-resistant growth in PCa cells, potentially serving as a therapeutic target for CRPC treatment.


Counting the number of grape bunches at an early stage of development offers relevant information to the winegrower about the potential yield to be harvested. However, manual counting on the fields is laborious and time-consuming. Remote sensing, and more precisely unmanned aerial vehicles mounted with RGB or multispectral cameras, facilitate this task rapidly and accurately. This dataset contains 40 RGB videos from a 1.06-ha vineyard located in northern Spain. Moreover, the dataset includes mask labels of visible grape bunches. The videos were acquired throughout four UAV flights with an RGB camera tilted at 60 degrees. Each flight recorded one side of a row of the vineyard. The grape berries were between pea-size (BBCH75) and bunch closure (BBCH79) stage, which is two months before harvesting. No operations other than those usual in a commercial vineyard, such as pruning, cane tying, fertilization, and pest treatment, have been carried out, hence, the dataset presents leaf occlusion. The dataset was gathered and labelled to train object detection and tracking algorithms for grape bunch counting. Furthermore, it eases the work of winegrowers to check the sanitary status of the vineyard. Specifications Table•Dataset is useful for researchers interested in instance segmentation, as it allows the detection and tracking of the clusters •Dataset can be employed to count the number of visible grape clusters per row, which can provide insights into the potential yield to be harvested.•Dataset eases the work for winegrowers to visually check the sanitary status of the vineyard.•Data is of importance for researchers with an interest in phenotyping since phenotyping traits such as the bunch length and width can be extracted.1The main purpose of this dataset is to acquire RGB videos of the side of vineyard rows and the labels of the bunches to provide enough information to analyze 1) the application of object detection and tracking algorithms in vineyards for grape cluster detection and tracking, and 2) the counting of the number of visible grape clusters per row for early yield assessment.2Vitis vinifera cv. Loureiro. The vineyard is located in Tomiño, Spain, and belongs to 'Bodegas Terras Gauda, S.A.'    and 2. T2.1owNumber*.*videoclipNumber*_*partNumber*”. For instance, Row4.1_1 refers to the first video clip recorded for row 4, part 1. Therefore, all videos that have the same row number and video clip number are contiguous, starting from part 1 until the last part number. As can be observed in The dataset 2.2A total of 29 videos of the vineyard were annotated for grape cluster detection and tracking using the CVAT software, an annotation tool that was developed by Intel. The videos used the MOTS style The annotations are available in PNG format. The number of frames per video ranges from 15 to 28, with a total number of 681 labelled frames. 3Vitis vinifera cv. Loureiro, using a multi-rotor platform (DJI Matrice 210 RTK). The flight conditions were a clear sky and wind speed lower than 0.5 m/s. The videos were recorded at 3 m above ground level, just above the rows of the vineyard, to get an accurate and close overview of the plants and the grape clusters.The flights were carried out on June 28th 2021 over a 1.06-ha vineyard, The camera (DJI Zenmuse X5S) has a 20.8-megapixel sensor that captures images at a high resolution of 3.4 μm pixel size. The frame width and height of the recorded videos are 4096 × 2160, respectively.In the matter of not reducing the research possibilities and further analysis of the dataset, no processing of the acquired videos is done and hence, the videos are as taken by the RGB sensor.The authors state that the present work meets the ethical requirements for publication in Data in Brief. The work does not involve studies with animals and humans.Mar Ariza-Sentís: Investigation, Methodology, Data curation, Visualization, Writing – original draft. Sergio Vélez: Investigation, Methodology, Data curation, Writing – review & editing. João Valente: Conceptualization, Supervision, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


This study shifts the focus away from demonstrating the existence of the effect toward understanding the mechanism by which the effect of AR operates in museum learning. By uncovering and describing the contingencies of AR from the perspectives of learner control, this study investigates how and when AR affects museum learning experiences, and to give insights into curation with AR. A between-subjects experiment was conducted with 48 college students divided into three groups. This study considered both qualitative and quantitative features of learner control and designed the AR control tools and experiment accordingly, and the findings supported the success of integrating the immersive technology of AR and the theoretical framework of learner control to construct museum exhibits. The results showed that visitors are willing to use the provided tools in museum AR and perform steadily in knowledge acquisition. In addition to offering more learner control in museums, AR promotes positive behaviors and attitudes. This study contributes to the field studies of learner control by linking learner control with the critical dimensions of AR-enhanced museum learning to provide more guidance in exhibit design. Based on the findings, practical suggestions on incorporating learner control in AR-based interactive exhibits are provided. Museums are one of the main informal learning environments where people learn in a particular situation with real objects. As memory institutions, museums not only collect and display culturally meaningful artifacts but also affect visitors’ learning profoundly. Visitors achieve their learning by interpreting and memorizing incoming information during the visits on a more self-regulated basis with more control over the environment , 2. As FAmong the investigations of possible factors on learning effectiveness, studies in educational psychology suggest that the provision of learner control is positively related to learning outcome and process , 15. RelMotivated by the aforementioned issues, this study focused on how AR affects visitors’ museum learning from the perspective of learner control. This study aimed to investigate the effectiveness of an AR-based interactive exhibit designed with a theoretical framework of learner control  on visitThe past decade has witnessed the tremendous growth of augmented reality technologies and applications. With the enhanced version of reality created by AR technology, which can overlay digital information on something being viewed , 24, AR Previous studies that used AR in both formal and informal educational settings have pointed out that AR combines real environments and virtual items, offers more delicate presentation, and promotes learning experiences and impressions , 33. ForTo understand visitors’ museum experiences in general and learning experiences in detail, researchers in related fields have adopted different approaches and measurements, including more interpretivist or qualitative paradigms in visitor studies, and the positivist or quantitative paradigm in educational research communities –40. The Learners’ interactions within the AR environment are highly involved with their autonomy and control over the learning situation. All interactions need to be actively triggered and linked by the users, making AR a highly user-controlled environment. In instructional technology studies, the idea of learner control refers to design features of the instructional interface or content that enable learners to autonomously decide and choose the path, rate and feedback while learning , 52. GenGiven the essence of learner control as a design feature of the learning environment, it is therefore closely related to the technology used . CompareWith reference to the abovementioned features of both the instructional interface and content for designing learner control, this study adopted AR technology as the platform to construct a museum exhibit and provided various tools for interacting with the physical context, including the artifacts, content and environment, to achieve different levels of learner control. By employing a qualitatively and quantitatively different learner control design, this study investigated the impact of the AR tools on participants’ museum experiences by examining their flow state, learning performance and visiting behaviors.To discover how learner control affects the museum learning experience, an AR-based interactive exhibit of a special collection of herbarium specimens was designed to provide different levels of learner control. The exhibit was technically developed with augmented reality technology, and the exhibit content was designed and presented as a story that incorporated a scenario, narrative and interactive elements to provide learners with sufficient environmental context , 24. TheThis study followed general principles of constructivist theory and multimedia learning , 32 to sTo investigate whether different levels of learner control affect museum learning experiences, this study designed and developed 4 types of AR tools, with reference to Lawless and Brown . As showIn addition to the qualitative nature of the control level, this study further investigated the quantity of learner control and developed three versions of the exhibit with different numbers of AR learner control tools. As shown in With these AR learner control tools in hand, visitors were guided by Professor Tanaka to use the tools to observe and record the information about the specimens during their visits. For example, in the task of observing the specimen, Professor Tanaka would suggest that the visitor imitate him in using the AR tools to accomplish four learning activities, from observing a whole specimen to noticing the specific features of citrus fruits. The learning units were namely (1) observing the materials of the setting paper, (2) looking carefully at the components of the herbarium specimen, (3) reading the information on the original identification cards, and (4) observing the petiole wings of the plant. After the observation task, Professor Tanaka would present a situated quiz and ask the participant to match the correct specimens and identification cards.A total of 48 college students voluntarily participated in the experiment. Their prior knowledge and experiences of the museum, augmented reality technology, and herbarium specimens were investigated in advance, and the participants were divided and assigned into three groups to experience the three different versions of the AR exhibit. The Research Ethics Committee of the University approved all procedures, the protocol, and the methodology (NTU-REC 201807HS006). All participants’ signed written consent forms were obtained before the experiment.This study adopted quantitative and qualitative measurements with the Personal Context Questionnaire, the Flow State Scale, the Performance Test, and the Behavior Mapping Log to investigate participants’ museum experiences, learning processes and outcomes in the AR-based interactive exhibit.The Personal Context Questionnaire was distributed before the experiment to investigate participants’ personal contexts, including their prior experiences with the museum, AR, and herbarium specimens. Prior experience included four aspects: awareness, reaching frequency, purpose, and familiarity. Each aspect was transformed into a score, and the total score was calculated to represent the personal context of each participant.As a measurement to understand the constructs of participants’ flow experiences, the Flow State Scale (FSS) proposed by Jackson and Marsh  was adopParticipants’ learning outcomes were assessed by a multiple-choice quiz consisting of measurements of memory and comprehension performance . The PerTo prevent interference, the observation of visitors’ behaviors during the experiment was conducted remotely through video monitoring by two researchers outside of the experimental galley. A Behavior Mapping Log was composed with reference to Griffin’s  indicatoDuring the participants’ visits, the researchers used a video camera and two 360-degree cameras to remotely monitor the environment as well as the participants’ interactions with the AR. Participants signified their completion of the visit by leaving the gallery, at which point they were invited to another room to take the Performance Test and reflect on their visits using the Flow State Scale. A follow-up interview was conducted at the end to investigate participants’ subjective perceptions and feelings about the exhibit and their experiences.In all, 48 subjects were recruited to participate in the experiment, including 15 males and 33 females aged from 18 to 45 years old . Based on the responses in the pre-test Personal Context Questionnaire, subjects with different backgrounds and prior knowledge were equally assigned to the three groups such that all had similar compositions. Two participants in the Medium Control Group dropped out of the experiment at the end, and their data were therefore excluded from the analysis. As shown in Flow experience, measured with the FSS, showed that average scores of the three groups of participants were all above 4.00/6. The global scores of the flow experiences indicated whether the participants had fewer tools , had a moderate number of tools  or had the most abundant tools , they all experienced the state of flow when using the AR tools. This finding is consistent with previous studies that illustrated the advantages of AR in improving participants’ engagement in especially the hands-on interaction with the physical objects because of the integrated virtual and real-world representations . DespiteI just followed my inclinations. I watched and did what I liked. It was fun to choose the positions of the magnifier and the scanning specimen” (H-5). “Tools and operations made me feel I was operating the real equipment” (H-8). “I liked using these tools when operating the magnifier. It provided instant feedback that allowed me to engage when using it” (H-9).However, the qualitative investigation with the triangulation of interviews and behavior logs, focused on the perceptions of the tool interactivity of the participants, showed that the participants’ visit experiences were very different among the three groups in terms of the flow constructs. 12 subjects in the high control group and 9 in the medium group reported that they perceived a greater sense of control by actually manipulating the virtual tools to interact with the physical objects of specimens during their visits: “All I could do was put the specimen under the observation desk and start playing with the magnifier. I spent most of the time listening to the narration, so it was difficult for me to concentrate on the AR” (L-14). Participants also felt powerless due to poor action–awareness merging [I could not decide what to look at or how many times to repeat it. I don’t think I was controlling the tablet, AR or content. It did not feel like observing the specimen and it distracted me” (L-9).In contrast, 10 of the 16 participants in the low control group pointed out that they were distracted by their inability to operate the AR tools autonomously. Many of them felt frustrated when they realized that they had little control over the learning process and AR content in the first few attempts. They also reported their inability and distraction in catching up with the program control. “ merging , 45 whenI could handle these tools, and I felt I was interacting with AR. What it showed told me what I should do at the moment, and these many tools I could use also increased my engagement” (M-16). “The way to move the magnifier to a specific position and reach the instruction was new to me. It made the learning content less boring. I felt I was interacting with the content” (H-13).The results of the interviews revealed two major factors that affected the participants’ flow experiences. First, more interaction with the exhibit content provided the participants with more immediate and clear feedback and resulted in better flow experiences. Eleven participants in the low control group complained about being distracted from the visit because there was no interaction. Another 8 participants in the medium and high control groups said that the interaction increased their concentration and pleasure of experiencing the exhibit. “I felt I was observing a specimen in the real field when I used the magnifier to zoom in; everything became so real. (The specimen) was no longer just something hanging on the wall” (M-7).Second, the context-specific operation of AR control tools positively affected users’ flow experiences by enhancing physical immersion. This finding is consistent with previous studies of immersive technology, wherein the participants experienced the virtual tools as actual physical equipment and enhanced their physical presence accordingly . The desThe process of attaching the identification card was similar to what was done by Professor Tanaka, so I could imagine the situation of specimen identification” (M-14). “When I attached the identification card to the specimen, I suddenly realized that this was what Professor Tanaka had done decades ago” (H-9). This finding is consistent with previous studies of learner control, as active control aids participants’ acquisition of the abstract concepts of the rationale behind specimen identification [In addition, not only the number of tools but the contextual ways the tools were used affected participants’ flow experiences. Taking the specimen identification card as an example, participants within the low control group had no control over the tool. They could only see how the character had used the tool on the display with narration. They also reported more frequently that the tool interrupted, rather than facilitated, their visits. Participants who were given a higher level of learner control with more tools and interaction, on the other hand, reflected a more plausible illusion and tended to be more engaged. “fication .Both the quantitative and qualitative data showed how the participants positively engaged in the AR exhibit with different levels of learner control. The results suggested that the number, affordance and context of the learner control tools affected participants’ flow experiences. It was worthy of note that the affordance of the learner control tools was seen by the participants as more important than the availability of more tools. They valued the responses of the learner control tools to their situated cognition and actions in the context.Participants’ learning outcomes were divided into two aspects, namely, memory and comprehension, and measured by the Performance Test. The results showed that participants within higher learner control groups had better performance in both memory and comprehension, but the differences were not statistically significant see . This fiWhile the statistical test did not reveal significance, the qualitative analysis of data on participants’ learning behaviors and processes supported a positive association between learner control and generic learning performance. First of all, participants within higher learner control groups spent significantly more time on their visits see . SpendinThe behavior logs of participants’ viewing times also suggested similar findings. Participants with more learner control tools tended to interact more actively with the AR exhibit. The system just showed a Magnifier and kept explaining. I was distracted because I could not handle what I was doing” (L-13).Generally, the participants were aware of whatever was available due to the guidance on the screen provided by AR, but they paid special attention to and actively used the Magnifier and Camera, which provided immediate and direct feedback to accomplish basic interaction, and controls like browsing and connecting to the content. Some participants were rather unfamiliar with the exhibit theme of herbarium specimens. Most of these relied heavily on the physical context for guidance to develop a general understanding. As shown in When the exhibition content was very long, I preferred to write down keywords in the Notebook. It reminded me of important things” (H-13).It was also found that the participants used the control tools as an external cognition mechanism to relieve their cognitive load and help them process the content. Over half  of the participants in the high control group took notes about the narration, the specimen, and even their own reflections. For example, one participant reported “I was very curious about these tools. They looked interesting, so I tried to use them. I felt like I was playing a game, and I clicked these buttons spontaneously” (M-11). On the other hand, participants used the control tools mainly for tasks of reciting and summarizing. “I immediately took photos and recorded important scenes. It helped me understand and learn” (H-11).Interviews with the participants indicated that those who possessed higher learner control were more highly motivated to learn and explore the unfamiliar content with the facilitation of the AR control tools. When a participant in the medium control group followed the narration and used the Magnifier to look closely at the petiole wings of the citrus fruit, she spontaneously picked up another specimen sheet for comparison. “This was not just listening to what Professor Tanaka said when using the Magnifier. I could actually play by myself” (H-2). “I focused on using the Magnifier and liked to find out what I hadn’t observed. I was eager to know what else I could see and why I couldn’t complete the task” (H-1).Further examination of the amounts of time the participants spent using each tool see  revealedCombining quantitative and qualitative findings, it was evident that the multimodal presentation and immediate feedback provided by augmented reality technologies facilitated the learners’ control over the exhibit content and context, thereby enhancing their museum experiences. All participants, regardless of group, were able to achieve the state of flow in the AR environment. In addition, the control tools and features enabled control over pacing, content and access to learning support, in turn providing learners with powerful guidance as they explored the AR-based environment. They could engage through not only the senses of sight and hearing but also hands-on activities. Those who were given higher learner control in terms of tool numbers and interactivity considered their visits and learning to be of better quality. They not only were more likely to engage in the museum exhibit but also actively pursued more opportunities for learning. Their active engagement, indicated by the dwelling time and frequent accessing of learning materials, also facilitated their memory and comprehension of the exhibit content.In this study, an interactive exhibit using AR technology to provide different levels of learner control was designed and developed for a special specimen collection of a university herbarium. Given the shortage of empirical studies on the learning mechanisms within museum AR applications, this study designed the contextual AR tools for museum learning on the theoretical basis of learner control. A visitor experiment study was conducted in an authentic museum exhibit to investigate how the mechanism of learner control affected visitors’ museum learning. The results clearly indicated that the AR-based interactive exhibit with learner control provided visitors with more volitional choice and resulted in their enriched encoding process and museum experiences. The findings from this study suggested that the affordance of AR technology enhanced the situated feature of the learner control tools such that they blended into the museum environment seamlessly for visitors’ intuitive and effective uses, resulting in visitors’ engagement and learning. The multimodal representations in the AR-based exhibit improved visitors’ coordination of attention with the flow of experience, and the situated control tools provided the visitors with additional metacognitive processing to achieve meaningful interactions with the exhibit content. While all participants achieved medium to high states of flow, those who possessed higher levels of learner control tended to be more engaged in inquiry and in acquiring factual knowledge. This finding echoed those of previous studies using AR in museums, as the visitors tended to become engaged in interactive and concrete exhibit content , 61, andMore importantly, given the highly situated nature of museum learning, this study makes a specific contribution to the field research and practices by taking into account both qualitative and quantitative features of learner control in designing the AR control tools and the visitor study. The findings provide empirical support that the visitors valued functionality over quantity of the control tools during their museum visits. They were highly motivated by the contextually appropriate virtual tools, which allowed them to apply real-world schemata to their operation. The visitors viewed the learner control tools not only as a navigation feature but also as an external cognition mechanism to help them process the exhibit content. They used the AR tools more extensively in basic learning tasks, such as in reciting by Magnifier and mnemonics by Notebook. Those who regarded themselves as having lower learner control reported more recital uses of copying materials by Camera or verbatim note-taking in Album and Notes, while those who perceived higher learner control reflected more elaborate uses of summarizing and generative note-taking in Notebook. This finding echoed previous studies on learner control in school settings , 20, andThe findings supported the success of integrating the immersive technology of AR and the theoretical framework of learner control to construct museum exhibits. Several practical suggestions for curation are made based on the findings directly available to museum educators, designers and curators. First, when introducing AR technology in museums, it is important to focus on the nature of the immersive experience in addition to the technical features of the simulation technology. Visitors must be required to employ their active perception throughout the AR experiences in order to retain awareness of the physical and virtual worlds, which makes the active learner control even more important. Second, more design considerations should be placed on the qualitative features of learner control, such as the defining features of learner interaction with the exhibit content  in this Methodologically, this study contributes to the field studies of learner control by linking learner control with the critical dimensions of museum learning to provide more guidance in exhibit design. In-depth efforts to observe visitor behaviors alone with the analytic investigations through different instruments were made to form a systemic understanding of how the AR affected museum learning from the learner control perspective. However, several limitations of our investigation should be noted. First, the research intention to conduct the visitor experiment in a real museum setting resulted in a relatively small sample size due to the limitations of space and facilities. Therefore, the results should be interpreted with caution. Second, this study conducted a one-off session with voluntary participants. Although their background and experiences were considered in the research design, some participants might have different inherent levels of motivation than the general museum visitors, and some participants might not have had sufficient spare time to achieve flow experiences with the mindset of research participants under the researchers’ observation. Our further work is therefore incorporating separate interactive sessions with ambient observation technologies in order to acquaint visitors with the treatment. So as not to interrupt participants’ visits, this current study adopted qualitative measures of remote observation and interviews. More direct behavioral measurements with physiological devices will be considered and recommended.


Despite the rapid advance in cancer therapies, treatment-resistant relapse remains a significant challenge in cancer treatment. Acquired resistance arises during or after treatment administration, and is usually the main contributor to relapse. For example, prostate cancer, the most frequent type of cancer in the elderly male population, frequently develops into aggressive forms resistant to chemical and hormonal therapies. In this condition, the so-called “cholinic phenotype” that is characterized by the overexpression of choline kinase alpha (CHKA) and increased phosphocholine levels leads to aberrant lipid metabolism. Our work demonstrates that CHKA, which is necessary for membrane phospholipid synthesis, is a target of the long non-coding RNA MALAT1. This study helps to further decipher how MALAT1 affects the regulation of crucial phospholipid/sphingolipid metabolic enzymes, as well as how the androgen receptor pathway is involved in MALAT1-dependent transcriptional regulation.Background. Choline kinase alpha (CHKA), an essential gene in phospholipid metabolism, is among the modulated MALAT1-targeted transcripts in advanced and metastatic prostate cancer (PCa). Methods. We analyzed CHKA mRNA by qPCR upon MALAT1 targeting in PCa cells, which is characterized by high dose-responsiveness to the androgen receptor (AR) and its variants. Metabolome analysis of MALAT1-depleted cells was performed by quantitative High-resolution 1 H-Nuclear Magnetic Resonance (NMR) spectroscopy. In addition, CHKA genomic regions were evaluated by chromatin immunoprecipitation (ChIP) in order to assess MALAT1-dependent histone-tail modifications and AR recruitment. Results. In MALAT1-depleted cells, the decrease of CHKA gene expression was associated with reduced total choline-containing metabolites compared to controls, particularly phosphocholine (PCho). Upon MALAT1 targeting a significant increase in repressive histone modifications was observed at the CHKA intron-2, encompassing relevant AR binding sites. Combining of MALAT1 targeting with androgen treatment prevented MALAT1-dependent CHKA silencing in androgen-responsive (LNCaP) cells, while it did not in hormone-refractory cells (22RV1 cells). Moreover, AR nuclear translocation and its activation were detected by confocal microscopy analysis and ChIP upon MALAT1 targeting or androgen treatment. Conclusions. These findings support the role of MALAT1 as a CHKA activator through putative association with the liganded or unliganded AR, unveiling its targeting as a therapeutic option from a metabolic rewiring perspective. The average age in Western countries is increasing rapidly, and with it the incidence of aging-associated cancers . FindingCHKA and CHKB, and are active in homodimeric, heterodimeric, and oligomeric forms 18], respCHKAprom (−3500) 5′-GGAAAAGGTTTGGTAATTGGAACA-3′ and 5′-CTGTGCACAAGTAGACGAGTTTGA-3′CHKAprom (−1500) 5′-CGCCACAGCAGCCTTACAA-3′ and 5′-CCCAAAGTGCTGGGATTACAG-3′CHKAprom  5′-CAGGACTTAGGGAGCCTGAACA-3′ and 5′-GGCAAGATGGACTTCTGCAATAT-3′.hCHKA  5′-GATGGGAGTAATGGAGGGTTCTG-3′ and 5′-CGTTAGTGACATGTGGCTGATGA-3′CERKprom (TSS) 5′-ACAAGACGGACTGTGGATGGA-3′ and 5′-CATCTGTTCTTGGAGTAAACTGCAA-3′CERKprom (−8600) 5′-GCCACTCTGTTCTGCGATCAC-3′ and 5′-CTGTTGGAGCCTCCGTTTTC-3′CERKprom  5′-CTTGGGAGACGGGTCTTCTG-3′ and 5′-GGTTGGTGTGCCTGATGAGA-3′Chromatin Immunoprecipitation (ChIP). ChIP was performed in PC3 (N = 2) and LNCaP (N = 2) cells as in ,20. BriePutative AR-binding sites on the CHKA gene  were identified using both MatInspector  and LASAGNA-Search 2.0 . In addiProliferation assay: Assessment of proliferation was conducted using the IncuCyte system after MALAT1 gapmer delivery or CHKA inhibitor treatment   at timesCell death assay: Apoptosis was determined using a Cell Death Detection ELISA PLUS kit  with cell lysates following the manufacturers’ instructions. Absorbance at 405 nm and 490 nm was measured using VICTOR X4 (Perkin Elmer).g at 4 °C for 30 min. Next, the polar methanol/water phase containing water-soluble cellular metabolites was lyophilized using a rotary evaporator (Savant RTV 4104 freeze dryer). In contrast, the organic phase (lipid phase) was collected in a tube, evaporating chloroform under nitrogen gas flow. Both phases of extracted cells were stored at −20 °C. For extracellular metabolome, cell culture medium extraction was performed by adding ice-cold extraction solvent ) to each tube and stored at −20 °C for at least 24 h. Afterward, the samples were centrifuged at 14,000× g for 30 min and the supernatant obtained was then freeze-dried in a Savant RTV 4104 freeze dryer. The aqueous fractions from cells and extracellular media were reconstituted in 700 μL D2O using TSP (0.1 mM) as NMR internal standards. In contrast, lipid fractions from cells were resuspended in a CD3OD/CDCl3 solution (2:1 v/v) with 0.05% of tetramethylsilane (TMS) as an internal reference [Intracellular and extracellular samples for NMR Analysis: Cell pellets and related cell culture media were stored at −80 °C until metabolomic analysis was performed by NMR spectroscopy. Then, the shots were resuspended in ice-cold extraction solvents (methanol/chloroform/water (1:1:1)) and vigorously vortexed for intracellular metabolome. At least 24 h later, polar and lipid phases were separated by centrifugation at 20,000× eference . 3OD), chloroform (CDCl3) and deuterium oxide (D2O) , and 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid sodium salt (TSP)  were used for NMR analyses. High-resolution 1H-NMR analysis was performed at 25 °C at 400 MHz  on aqueous and organic cell extracts using acquisition pulses, water pre-saturation, data processing, and peak area deconvolution, as previously described [6 cells (nmol/106) [6 cells/sample) for statistical analyses, we considered a pool (n = 3) of PCa cell lines . We reported the mean concentration (nmol/106 cells) of individual metabolites for both cell lines in http://metaboanalyst.ca, accessed on 13 April 2022) was used to identify the biochemical pathways affected by MALAT1 depletion determined by quantitative enrichment analyses. Metabolomics analysis by NMR spectroscopy: Deuterated reagents (methanol (CDescribed . The absmol/106) . The reln = 2) and HUVEC (n= 2) cells as described in [RNA ImmunoPrecipitation (RIP): RIP was performed in LNCaP . Cells were incubated overnight with anti-AR antibody , diluted 1:50 in PBS 5% Goat Serum, and then 45 min at room temperature with anti-mouse Alexa Fluor 546 , diluted 1:200 in 5% Goat Serum in PBS, Nuclei were counterstained with DAPI. Images were taken using a Nikon Eclipse Ti2 confocal microscope and Z stack images were processed by NIS Elements AR 5.30 software (Nikon Europe B.V.) using the same acquisition settings. Representative fields were further analyzed with ImageJ software  to show red fluorescence intracellular distribution in control conditions and after DHT treatment or exposure to LACZ and MALAT1 gapmers.t-test and a one-way ANOVA test (Bonferroni’s multiple comparisons test). Statistical analysis was performed using GraphPad Prism 8.0 software or Excel. p-values ≤ 0.05 were considered significant. Statistical analysis: Data were expressed as mean ± SEM or fold change, as indicated in figure legends. Statistical significance was calculated using a parametric paired two-tailed Student’s p = 0.02). In addition, we found a significant decrease in glutathione (p = 0.03) and its precursor L-glutamic acid (p = 0.04), a decrease in the average content of glycine, which is involved in the redox balance, and a reduction in the antioxidant taurine  spectroscopy in MALAT1-depleted vs. LACZ PCa control cells after 48 h from gapmer delivery. In the presence of MALAT1 gapmers, we found a significant decrease in the intracellular levels of total choline-containing metabolites (tCho) A. The reConsistent with the metabolites screening, CHKA, which catalyzes the conversion of choline to phosphocholine along the Kennedy pathway, emerged among the modulated genes identified in a MALAT1-dependent transcriptome analysis performed in advanced and metastatic PCa cell lines . In ordeBecause CHKA is involved in the phosphatidylcholine biosynthesis, we evaluated whether other genes belonging to the phospholipid and sphingomyelin metabolism were modulated under the same experimental conditions. Specifically, the analysis included phospholipase C gamma 1 (PLCG1), sphingomyelin phosphodiesterase 1 . As shown in In order to dynamically map the most common epigenetic modifications along the specific genomic regulatory regions of CHKA , has been recently identified as an AR target . TherefoOverall, these data indicate that in MALAT1-depleted cells the chromatin landscape enriches repressive signals, contributing to CHKA mRNA reduction.Previous studies ,22 have As CERK kinase is under the control of AR, we measured the expression of CERK before and after MALAT1 targeting in cells cultured in a hormone-deprived medium. CERK mRNA was efficiently and stably silenced upon MALAT1 targeting in LNCAP. In 22RV1, however, this effect was evident at 24 h upon MALAT1 gapmer delivery A. A simi−8 M to 10−6 M was carried out in both cell lines. The expression of CHKA and CERK, the latter having been recently identified as an androgen-repressed gene, and of PSA, a classical AR-positive regulated gene, was analyzed by qRT-PCR. In LNCaP cells, CHKA mRNA increased more than three-fold, although only at the highest dose of DHT, whereas PSA and CERK were significantly modulated at a physiological concentration (10−8 M). In 22RV1 cells, a defective androgen sensitivity was observed, which can reasonably be attributed to the presence of the ARv7 variant. Consequently, we observed a small increase in CHKA and PSA regardless of DHT concentration in this model, while CERK was modulated at the lowest dosage  for 16 h, focusing on CHKA gene modulation. As a control, we examined the effect of MALAT1 targeting and androgen treatment on the CERK gene as an androgen-repressed target, and in parallel on PSA mRNA as an androgen-induced target. In this setting, LNCaP cells transfected with LacZgapmers preserved androgen responsiveness in terms of CHKA and PSA mRNA increase (2.5- and six-fold induction by LacZgapmers DHT vs. LacZgapmers NT) and CERK mRNA downmodulation (40% repression LacZgapmers DHT vs. LacZgapmers NT), which is in line with the data reported by Camacho et al. [In the combined treatment , LNCaP ao et al. . MALAT1 targeting reduced basal level of CHKA and CERK mRNA, as shown in In 22RV1 cells (expressing the AR-V7 variant), we noted an overall reduced androgen responsiveness compared to LNCaP cells in all experimental conditions B. In 22RUpon MALAT1 targeting, CHKA and CERK protein levels paralleled those observed in mRNA in terms of inhibition in both LNCaP and 22RV1 cells . HoweverAltogether, these results suggest that the androgen responsiveness characterized by a complete response with AR full length or by a weak response with the ARv7 variant was preserved regardless of MALAT1. However, the consequence of MALAT1 depletion was particularly evident on the androgen-repressed target gene, CERK, and on the high-dose androgen positive-regulated gene, CHKA.−8 M and 10−6 M, increased the AR recruitment at the same genomic regulatory regions in both genes. This suggests that in cells depleted of MALAT1, AR recruitment is facilitated.In order to investigate this aspect further, we performed ChIP experiments for AR recruitment on CHKA and CERK regulatory regions at the 16 h time point upon MALAT1 or LacZ gapmer delivery A. The noThese data prompted us to evaluate whether MALAT1 might associate with AR to act as a co-regulator and eventually affect the transcription of AR-dependent genes. To address this possibility, we immunoprecipitated the AR in LNCaP cells treated with androgens and analyzed whether MALAT1 was co-immunoprecipitated by RNA immunoprecipitation (RIP). AR appeared to associate with MALAT1 in basal conditions B. Howeve–8 M and 10–6 M, respectively). We queried whether the AR might translocate into the nucleus upon MALAT1 targeting in order to deepen insight into the underlying mechanism. We analyzed the AR shuttling from cytoplasm to nucleus in LNCaP cells before/after MALAT1 targeting using confocal microscopy C. As negThe present work significantly establishes CHKA, an essential gene in phospholipid metabolism, as a MALAT1 target. In our previous work , the traMoreover, CHKA is involved in antioxidant cellular defenses by regulating glutathione, cysteine content, and reactive oxygen species level. In ovarian cancer, the antioxidant role of CHKA, which is exerted by decreasing glutathione cysteine and methionine content, contributes to sensitizing cancer cells to chemotherapy ,31. In oIn hepatocellular carcinoma , MALAT1 Conversely, sphingomyelin synthases (SGMSs) use Phosphatidylcholine and Ceramide to form SM and diacylglycerol (DAG) . In partOn the other hand, the CERK-dependent phosphorylation of Ceramide enhances its role in proliferation and migration. Furthermore, CERK activity and its metabolites support cancer progression, particularly PCa cell aggressiveness . In thisSphingoMyelin (PC/SM) homeostasis.Lipogenesis and lipid catabolism is controlled by AR signaling. Lipid biosynthesis promotes CRPC development, counteracting the effect of AR antagonist treatment ,39. FurtSphingolipid metabolism is under the control of AR  through This work demonstrates that AR signaling might interact with MALAT1 in PCa cells. This evidence adds a layer of complexity to the role of MALAT1 in cancer, particularly in hormone-driven cancers such as PCa. In our hands, AR/MALAT1 interaction occurs predominantly without androgens B. Intereper se, as well as upon response to androgens. In this light, therapeutic intervention to reduce MALAT1 intracellular content might restore a physiological metabolism in highly aggressive transformed cells, with resulting detrimental consequences on tumor growth.Interestingly, AR is released from MALAT1 either by adding the ligand or after MALAT1 targeting B. In thiIn conclusion, this work reports an unprecedented role of MALAT1 in Choline metabolism with implications in Sfingolipid and Ceramide biosynthesis, as suggested by the downregulation of CHKA and CERK upon MALAT1 targeting (see Moreover, dysregulation of sphingolipids, including Ceramide, Sphingomyelin, or Sphingosine 1-phosphate, has been linked to drug resistance in different types of cancer. Hence, MALAT1 targeting might have essential antitumor consequences affecting these critical biosynthetic pathways. In conclusion, this manuscript describes a novel aspect of MALAT1 in cellular metabolism, possibly leading to innovative therapeutic strategies.


AR gene mutations in MAIS.Androgen receptor gene (AR) is essential for male growth and fertility. Its mutations are responsible for androgen insensitivity syndrome (AIS) that usually shows the phenotype of azoospermia resulting in male infertility. This study reported the first case of mild AIS with complete normal serum hormones in a Chinese family. The proband referred for infertility because of azoospermia. His uncle and two cousins are both infertile and have azoospermia. Whole-exome sequencing in the genetic analyses showed that the proband carries a novel hemizygous AR missense mutation, NM_000044.6: c.2051G>C (p.Gly684Ala), in exon four within the ligand-binding domain. His mother and maternal aunt are heterozygous carriers, while his father and brother are wildtype, indicating that the mutation in the proband was inherited from his mother. This pattern is consistent with the genetic model of the X-linked recessive inheritance of AR in AIS pathogenesis. HOPE predicts that p.Gly684Ala increases the hydrophobicity of AR but does not change the AR conformation. PolyPhen-2 predicts that p.Gly684Ala is harmful. This study provides the new knowledge to understand the  AR gene is located in Xq11-12 and encodes 920 amino acids, composed of the N-terminal transactivation domain (NTD), the DNA binding domain (DBD), and the C-terminal ligand binding domain (LBD) (AR gene are the main reason of androgen insensitivity syndrome (AIS), which is divided into complete (CAIS), partial (PAIS), and mild (MAIS) according to the degree of feminization of patients (Androgen receptor (AR) binds androgen and plays a role in regulating male reproductive development . The AR in (LBD) . Mutatiopatients . Becausepatients . In patipatients . Howeverpatients . But it patients .AR in Hep3B cells , in exon four within the ligand-binding domain were identified in the proband using whole-exome sequencing (WES). HOPE predicts that p.Gly684Ala increases the hydrophobicity of AR but does not change the AR conformation. PolyPhen-2 predicts that p.Gly684Ala is harmful. However, a previous study has shown that this mutation cannot affect the transactivation activity of AR when expressing this site-mutated 3B cells . This stA 32-year-old man was referred to the Reproductive Medical Center of Jiangxi Maternal and Child Health Hospital for evaluation of subfertility. The proband had been married for 10 years and had been unsuccessful in attempting to father a child. He had no significant medical or surgical history and was not on any long-term medications. Chromosomal analysis demonstrated a normal karyotype . Y chromosome microdeletions were not detected in the proband. And the proband with normal hormone. The proband (III-2) is from a family with a history of infertility . Semen ahttps://evs.gs.washington.edu/EVS/), gnomAD data  calculation in PLINK . The Agilent SureSelect Human All ExonV6 Kit  and Illumina Novaseq 6,000 platform  were used for exome capture and genomic DNA sequencing. The resulting fastq data were submitted to in-house quality control software to remove low-quality reads, and then were aligned to the reference human genome (hs37d5) using the Burrows–Wheeler Aligner . Single mAD data , and in mAD data , PolyphemAD data , MutatiomAD data , and CADmAD data , and themAD data , there win PLINK . The IBD△△Ct method.Total RNA extraction from testis tissue was extracted by Trizol , and cDNA was got by reverse transcription with RT kit . Taq II quantitative kit  was used to perform Real-time quantitative PCR with StepOnePlus™ Real-Time PCR System instrument . Relative transcriptional levels of target genes was determined with the 2−https://www3.cmbi.umcn.nl/hope/) was accessed to analyze the effect of variation (http://missense3d.bc.ic.ac.uk/∼missense3d/) was used to structure and assess missense variants (Annotations about AR was obtained from UniProtKB entry P10275. HOPE SERVER(ariation . The strvariants .The means ± SEM of the data were expressed. T tests was used to assessed differences between the control and the sample.WES showed that the proband carries the hemizygous AR missense mutation NM_000044.6: c.2051G > C (p.G684A), which showed in AR as that of a fertile control  showed that the testis of the proband transcribes an equivalent level of  control . Therefo control . HOPE an control . PolyPhe control . These rAR , luteinizing hormone (LH), and testosterone levels could be found in MAIS patients  Karuppi. But AboHormone replacement therapy has been used to treat AIS , Weidle. Two stuIn conclusion, the present study reported that an infertile man with MAIS with azoospermia but normal serum hormone levels. The AR NM_000044.6: c.2051G>C mutation in this case is responsible for the phenotype of azoospermia independently of the transactivation activity of AR. This mutation provides a candidate target for studying the mechanism of AR in regulating spermatogenesis using the site-mutated mouse model.