Tissue-Engineered Disease Modeling of Lymphangioleiomyomatosis Exposes a Therapeutic Vulnerability to HDAC Inhibition.

Lymphangioleiomyomatosis (LAM) is a rare disease involving cystic lung destruction by invasive LAM cells. These cells harbor loss-of-function mutations in TSC2, conferring hyperactive mTORC1 signaling. Here, tissue engineering tools are employed to model LAM and identify new therapeutic candidates. Biomimetic hydrogel culture of LAM cells is found to recapitulate the molecular and phenotypic characteristics of human disease more faithfully than culture on plastic. A 3D drug screen is conducted, identifying histone deacetylase (HDAC) inhibitors as anti-invasive agents that are also selectively cytotoxic toward TSC2-/- cells. The anti-invasive effects of HDAC inhibitors are independent of genotype, while selective cell death is mTORC1-dependent and mediated by apoptosis. Genotype-selective cytotoxicity is seen exclusively in hydrogel culture due to potentiated differential mTORC1 signaling, a feature that is abrogated in cell culture on plastic. Importantly, HDAC inhibitors block invasion and selectively eradicate LAM cells in vivo in zebrafish xenografts. These findings demonstrate that tissue-engineered disease modeling exposes a physiologically relevant therapeutic vulnerability that would be otherwise missed by conventional culture on plastic. This work substantiates HDAC inhibitors as possible therapeutic candidates for the treatment of patients with LAM and requires further study.

Differentiation and single-cell RNA-seq analyses of human pluripotent-stem-cell-derived renal organoids.

Here, we present a protocol for the maintenance and differentiation of human pluripotent stem cells into renal organoids. We describe steps for using a series of readily made differentiation media, multiplexed sample single-cell RNA-seq analysis, quality control, and validation of organoids using immunofluorescence. This provides a rapid and reproducible model of human kidney development and renal disease modeling. Finally, we detail genome engineering using CRISPR-Cas9 homology-directed repair for the generation of renal disease models. For complete details on the use and execution of this protocol, please refer to Pietrobon et al.1.

Tuberous Sclerosis Complex Kidney Lesion Pathogenesis: A Developmental Perspective.

The phenotypic diversity of tuberous sclerosis complex (TSC) kidney pathology is enigmatic. Despite a well-established monogenic etiology, an incomplete understanding of lesion pathogenesis persists. In this review, we explore the question: How do TSC kidney lesions arise? We appraise literature findings in the context of mutational timing and cell-of-origin. Through a developmental lens, we integrate the critical results from clinical studies, human specimens, and genetic animal models. We also review novel insights gleaned from emerging organoid and single-cell sequencing technologies. We present a new model of pathogenesis which posits a phenotypic continuum, whereby lesions arise by mutagenesis during development from variably timed second-hit events. This model can serve as a conceptual framework for testing hypotheses of TSC lesion pathogenesis, both in the kidney and in other affected tissues.

Overview of The Canadian Clinician Investigator Trainees' Research Presented at CSCI-CITAC Joint Meeting.

The 2021 Annual Joint Meeting (AJM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherches Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was hosted virtually on November 14-16th, 2021. The theme of the AJM was "Communication, Collaboration, and Tools for the Next Generation of Clinician Scientists", and emphasized lectures, panels and interactive workshops designed to provide knowledge and skills for professional development of clinician investigator trainees. The opening remarks were given by Nicola Jones (President of CSCI/SCRC) and Adam Pietrobon (Past President of CITAC/ACCFC). The keynote speaker was Dr. Timothy Caulfield, who delivered the presentation titled "Communication in the Era of Misinformation". Dr. Michael Hill (University of Calgary) received the CSCI Distinguished Scientist Award and Dr. Philippe Campeau (Université de Montréal) received the CSCI Joe Doupe Young Investigator Award. Each of the scientists delivered award winning talks during the symposium titled "All the King's Horses and All the King's Men" and "Understanding Growth Plate Disorders to Better Treat Them", respectively. The three interactive workshops included "Data Visualization", "Science Communication on Social Media" and "Mentorship in Action". The two panels were "CIHR Engagement: Challenges and Opportunities in the Clinician Investigator Career Path" and "Early Career Investigator Panel". The AJM also included presentations from clinician investigator trainees from across the country. Over 60 abstracts were showcased at this year's meeting, most of which are summarized in this review. Six outstanding abstracts were selected for oral presentations during the President's Forum.

Renal organoid modeling of tuberous sclerosis complex reveals lesion features arise from diverse developmental processes.

Tuberous sclerosis complex (TSC) is a multisystem tumor-forming disorder caused by loss of TSC1 or TSC2. Renal manifestations predominately include cysts and angiomyolipomas. Despite a well-described monogenic etiology, the cellular pathogenesis remains elusive. We report a genetically engineered human renal organoid model that recapitulates pleiotropic features of TSC kidney disease in vitro and upon orthotopic xenotransplantation. Time course single-cell RNA sequencing demonstrates that loss of TSC1 or TSC2 affects multiple developmental processes in the renal epithelial, stromal, and glial compartments. First, TSC1 or TSC2 ablation induces transitional upregulation of stromal-associated genes. Second, epithelial cells in the TSC1-/- and TSC2-/- organoids exhibit a rapamycin-insensitive epithelial-to-mesenchymal transition. Third, a melanocytic population forms exclusively in TSC1-/- and TSC2-/- organoids, branching from MITF+ Schwann cell precursors. Together, these results illustrate the pleiotropic developmental consequences of biallelic inactivation of TSC1 or TSC2 and offer insight into TSC kidney lesion pathogenesis.

Fall 2021: Clinician Investigator Trainee Association Of Canada (CITAC).

I hope you're taking care and found some time to relax this summer. A new semester may mean a big transition­some folks are starting their graduate studies, re-entering clerkship, starting residency or entering a fellowship. For some, there will be little or no change at all; but just a continuation of one of the many phases of the physician-scientist training pathway. Whatever stage you're at, the Clinical Investigator Trainee Association of Canada (CITAC) community is here to support and advocate for you!

Overview Of The Canadian Clinician Investigator Trainees' Research Presented At The 2020 CSCI-CITAC Joint Meeting.

The 2020 Annual General Meeting (AGM) and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherches Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada/Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was the first meeting to be hosted virtually. The theme was "Navigating Uncertainty, Embracing Change and Empowering the Next Generation of Clinician-Scientists", and the meeting featured lectures and workshops that were designed to provide knowledge and skills for professional development of clinician investigator trainees. The opening remarks were given by Jason Berman (President of CSCI/SCRC), Tina Marvasti (President of CITAC/ACCFC) and Nicola Jones (University of Toronto Clinician Investigator Program Symposium Chair). Dr. Michael Strong, President of the Canadian Institutes of Health Research, delivered the keynote presentation titled "CIHR's COVID-19 Response and Strategic Planning". Dr. John Bell (University of Ottawa) received the CSCI Distinguished Scientist Award, Dr. Stanley Nattel (Université de Montréal) received the CSCI-RCPSC Henry Friesen Award (RCPSC; Royal College of Physicians and Surgeons of Canada) and Dr. Meghan Azad (University of Manitoba) received the CSCI Joe Doupe Young Investigator Award. Each scientist delivered talks on their award-winning research. The interactive workshops were "Developing Strategies to Maintain Wellness", "Understanding the Hidden Curriculum: Power and Privilege in Science and Medicine", "Hiring a Clinician Scientist Trainee: What Leaders Are Looking For" and "COVID-19: A Case Study for Pivoting Your Research". The AGM included presentations from clinician investigator trainees nationwide. Over 70 abstracts were showcased, most are summarized in this review, and six were selected for oral presentations.

Spring 2021: Clinician Investigator Trainee Association Of Canada (CITAC).

The Clinician Investigator Trainee Association of Canada (CI) trainees across the country around the common goal of improving training conditions for those pursuing a career at the junction of research and medicine. Since then, the CI training landscape has shifted dramatically. The number of Canadian CI trainees enrolled totaling 289 MD-PhD trainees and 389 Clinical Investigator Program (CIP) trainees as of 2019 [1]. Alumni outcome data have presented conclusive evidence that MD-PhD training programs are effective in producing CI careers [2-4].

International training considerations of Canadian Clinician-Scientist Trainees - A national survey.

PURPOSE: Canadian clinician-scientist trainees enrolled in dual degree programs often pursue an extended training route following completion of MD and MSc or PhD degrees. However, the proportion, plans and reasoning of trainees who intend to pursue training internationally following dual degree completion has not been investigated. In this study, we assessed the international training considerations of current clinician-scientist trainees. METHODS: We designed an 11-question survey, which was sent out by program directors to all current MDPhD program and Clinician Investigator Program (CIP) trainees. Responses were collected from July 8, 2019 to August 8, 2019. RESULTS: We received a total of 191 responses, with representation from every Canadian medical school and both MD-PhD program and CIP trainees. The majority of trainees are considering completing additional training outside Canada, most commonly post-doctoral and/or clinical fellowships. The most common reasons for considering international training include those related to quality and prestige of training programs. In contrast, the most common reasons for considering staying in Canada for additional training are related to personal and ethical reasons. Irrespective of intentions to pursue international training, the majority of trainees ultimately intend to establish a career in Canada. CONCLUSION: While most trainees are considering additional training outside of Canada due to prestige and quality of training, the majority of trainees intend to pursue a career as a clinician-scientist back in Canada. Trainees would likely benefit from improved guidance and mentorship on the value of international training, as well as enhanced support in facilitating cross-border mobility.

Overview of the Canadian Clinician Investigator Trainees' research presented at the 2019 CSCI-CITAC Joint Meeting.

The 2019 Annual General Meeting and Young Investigators' Forum of the Canadian Society for Clinical Investigation / Société Canadienne de Recherche Clinique (CSCI/SCRC) and Clinician Investigator Trainee Association of Canada / Association des Cliniciens-Chercheurs en Formation du Canada (CITAC/ACCFC) was held in Banff, Alberta on November 8-10th, 2019. The theme was "Positioning Early Career Investigators for Success: Strategy and Resilience". Lectures and workshops provided knowledge and tools to facilitate the attendees' development as clinician investigators. Dr. Jason Berman (President of CSCI/SCRC), Elina Cook (President of CITAC/ACCFC) and Drs. Doreen Rabi and Zelma Kiss (University of Calgary Organizing Co-Chairs) gave opening presentations. The keynote speakers were Dr. William Foulkes (McGill University) (Distinguished Scientist Award winner) and Dr. Andrés Finzi (Université de Montréal) (Joe Doupe Young Investigator Award winner). Dr. Robert Bortolussi (Dalhousie University) received the Distinguished Service Award for his work as the Editor-in-Chief of Clinical and Investigative Medicine and for being instrumental in the development of the Canadian Child Health Clinician Scientist Program. This meeting was the first to host a panel discussion with Drs. Stephen Robbins and Marcello Tonelli from the Canadian Institutes of Health Research. Workshops on communication, career planning and work-life balance were hosted by André Picard and Drs. Todd Anderson, Karen Tang, William Ghali, May Lynn Quan, Alicia Polachek and Shannon Ruzycki. The AGM showcased 90 presentations from clinician investigator trainees from across Canada. Most of the abstracts are summarized in this review. Eight outstanding abstracts were selected for oral presentation at the President's Forum.

Newsletter Fall 2020: Clinician Investigator Trainee Association of Canada (CITAC).

Message from the CITAC president To say that 2020 has been an unprecedented year is an understatement. The coronavirus disease 2019 (COVID-19) global pandemic and the major societal awakening on racial equity and justice have led us to reflect on our direction, goals and mission. Thanks to our talented and dedicated executive team, we were able to pivot our efforts and adapt to the changing landscape of research and advocacy. In April, we provided our members with a list of resources to help facilitate a smooth transition to working from home. In June, we published Clinician Investigator Trainee Association of Canada's (CITAC) press release on our role in combating anti-Black discrimination and racial injustice and have outlined specific advocacy efforts that we will be committing to over the next years (the full statement can be found on our website, https://www.citac-accfc.org).

Performance report for a 10-year-old MD/PhD Program: A survey of trainees at the University of Ottawa.

PURPOSE: Integrated MD/PhD programs are relatively new in Canada and represent a platform to train the next generation of clinician-scientists. However, MD/PhD programs vary substantially by structure, funding and mentorship opportunities, and there exists a paucity of data on the overall students' successes and challenges. The purpose of this study is to assess objective and subjective metrics of the MD/PhD Program at the University of Ottawa. METHODS: Students in all years of the program were invited to complete a 58- question survey, and the resulting data were analyzed by descriptive statistics. RESULTS: Our survey had an 88.5% (23/26) participation rate. The program has been gaining interest and the number of applications increased by 178% between 2013 and 2018. Tuition support was considered an essential element in accepting the admission offer, as 47.8% of students would have declined admission without full tuition coverage. The MD/PhD students were heavily engaged in scholarly activities, with an average of 8.3 presentations/ publications per respondent. Respondents indicated low satisfaction with formal career planning advice (28.6% satisfied/very satisfied) and program transition guidance (22.2%). When delivered informally by peers, both career planning advice and program transition guidance were experienced as more satisfying (65.2% and 63.6%, respectively). Only 34.8% of survey respondents identified as female, highlighting the challenge of achieving diversity in clinician-scientist training programs. CONCLUSION: Our report contributes to the body of knowledge on concrete obstacles experienced by students within MD/PhD programs and key areas that can be improved upon-locally, provincially and nationally-to further advance student success.

Newsletter Spring 2020: Clinician Investigator Trainee Association of Canada (CITAC).

Message from Tina Marvasti, President of the Clinician Investigator Trainee Association of Canada: National and international collaborations and evidence based advocacy for the Canadian MD/PhD and MD+ trainees The overarching goals of the Clinician Investigator Trainee Association of Canada (CITAC) for 2020 are to expand its reach to increase membership and to continue to build strong relationships with our national and international partners. These goals will be achieved by applying the following three strategies: • Continue to develop and strengthen our relationships with national and international key partners and with stake holders • Better understand the needs of clinician-scientist trainees for evidence-based advocacy • Increase membership, improve communication with all members and highlight the accomplishments of trainees.

Newsletter 2018: Clinician Investigator Trainee Association of Canada (CITAC).

Message from the President: Optimism for the Future The Clinician-Investigator Trainee Association of Canada (CITAC) was established in 2006 to address issues relevant to Canadian trainees seeking dual training in medicine and research. As clinician-investigator (CI) trainees, we comprise but a fraction (less than 5%) of all medical trainees. Our 'bilingual' careers render our individual paths less straightforward and more challenging. As a community, we have had to confront several disappointments, perhaps most notably the cessation of funding support for MD/PhD programs in 2015, previously offered by the Canadian Institutes of Health Research (CIHR). Despite these individual and collective challenges, I remain optimistic and incredibly excited about our future. In my own work, I am reminded constantly that being trusted with the dual responsibility of patient care and innovation in medicine is a privilege to be cherished, rather than a burden to be feared. That which makes our path doubly challenging also makes it doubly rewarding. The progress that CITAC has made over the years only adds to my optimism, and I wish to take this opportunity to remind you of how far we have come and how much further we hope to go.

PRL2 links magnesium flux and sex-dependent circadian metabolic rhythms.

Magnesium (Mg2+) plays pleiotropic roles in cellular biology, and it is essentially required for all living organisms. Although previous studies demonstrated intracellular Mg2+ levels were regulated by the complex of phosphatase of regenerating liver 2 (PRL2) and Mg2+ transporter of cyclin M (CNNMs), physiological functions of PRL2 in whole animals remain unclear. Interestingly, Mg2+ was recently identified as a regulator of circadian rhythm-dependent metabolism; however, no mechanism was found to explain the clock-dependent Mg2+ oscillation. Herein, we report PRL2 as a missing link between sex and metabolism, as well as clock genes and daily cycles of Mg2+ fluxes. Our results unveil that PRL2-null animals displayed sex-dependent alterations in body composition, and expression of PRLs and CNNMs were sex- and circadian time-dependently regulated in brown adipose tissues. Consistently, PRL2-KO mice showed sex-dependent alterations in thermogenesis and in circadian energy metabolism. These physiological changes were associated with an increased rate of uncoupled respiration with lower intracellular Mg2+ in PRL2-KO cells. Moreover, PRL2 deficiency causes inhibition of the ATP citrate lyase axis, which is involved in fatty acid synthesis. Overall, our findings support that sex- and circadian-dependent PRL2 expression alter intracellular Mg2+ levels, which accordingly controls energy metabolism status.