Edwin S. Monuki, MD PhD - US grants

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how morphogen gradients pattern the cerebral cortex and other tissues during development. Morphogen gradients are fundamental to animal development, and morphogen defects are primary causes of human birth defects and malformations of the cortex. Nonetheless, tremendous controversy remains about the mechanisms by which morphogen gradients act, which limits our understanding of these human disorders. For the most part, this controversy revolves around a single issue - the inability to distinguish morphogen activities that do not depend on cell-cell communication (the "classical" model) from those that do. To date, insight into this issue has relied on heroic studies using traditional dissociated cell cultures, which are limited both in terms of experimental efficiency and as models of natural morphogen gradients. However, a microfluidic culture device has the potential to address these limitations. This microscale device generates precise and continuous biomolecular gradients with different profiles onto cells, and is designed for time-lapse microscopy. These features should provide several biological and practical advantages over traditional cultures for modeling and studying morphogen gradients. Preliminary studies with cortical precursor cells (CPCs) confirm the promise of this system, but have also identified device design features that need to be optimized in order to answer the basic question driving this proposal - which CPC responses in the normal cortex are determined solely by extracellular morphogen concentration, and which are not? The goals of this R21 proposal are to fabricate optimized microfluidic devices for culturing CPCs (Aim 1) and to develop real time assays with single cell resolution in order to efficiently study CPC responses as functions of morphogen concentration, gradient profile, cell density, and time (Aim 2). To achieve these goals, a multidisciplinary team with primary expertise in bioengineering (Noo Li Jeon, the developer of the original gradient-generating device), morphogen gradients (Arthur Lander) and cortical development (Edwin Monuki) has been assembled. If successful, this proposal should not only advance our understanding of cortical development and malformations, and of morphogen gradients in general, but should also provide a versatile microfluidic tool with a wide range of basic and clinical applications.

Ablation; Address; Adopted; Animals; Area; Automobile Driving; Bathing; Baths; Birth Defects; Bone Morphogenetic Proteins; Brain; Brain Diseases; Brain Disorders; Cell Communication and Signaling; Cell Signaling; Cells; Cerebral cortex; Cerebrospinal Fluid; Choroid Plexus; Congenital Abnormality; Congenital Anatomic Abnormality; Congenital Anatomical Abnormality; Congenital Defects; Congenital Deformity; Congenital Malformation; Consult; Defect; Development; Disease; Disorder; Dorsal; Drivings, Automobile; Embryo; Embryonic; Encephalon; Encephalon Diseases; Encephalons; Family; Family member; Fore-Brain; Forebrain; Gene Expression; Genes; Genetic; Goals; Histology; Holoprosencephaly; Human; Human Resources; Human, General; Individual; Intracellular Communication and Signaling; Intracranial CNS Disorders; Intracranial Central Nervous System Disorders; Life; Mammals, Mice; Man (Taxonomy); Man, Modern; Manpower; Maps; Measures; Mediating; Mice; Modeling; Molecular Genetic Abnormality; Murine; Mus; Nervous System, Brain; Pathogenesis; Patients; Pattern; Phenocopy; Phenotype; Physiology; Programs (PT); Programs [Publication Type]; Prosencephalon; Proteins; Public Health; Range; Reagent; Regulation; Research; Role; Signal Transduction; Signal Transduction Systems; Signaling; Signaling Protein; Structure; Structure of choroid plexus; System; System, LOINC Axis 4; Telencephalon; Testing; Work; biological signal transduction; career; cognitive function; developmental disease/disorder; developmental disorder; disease/disorder; driving; gene product; improved; insight; malformation; morphometry; mouse model; new approaches; novel; novel approaches; novel strategies; novel strategy; personnel; postnatal; programs; public health medicine (field); social role; spinal fluid

DESCRIPTION (provided by applicant): During development, distinctive tissues form in the dorsal telencephalic midline (DTM) that separates the two cerebral cortices. Among these tissues are the cortical hem, which we recently identified as being a hippocampal organizer, and the choroid plexus, the source of cerebrospinal fluid (CSF). The choroid plexus is a well-known tissue with significant therapeutic potential, but its development is quite poorly understood. Moreover, failed DTM development is a central feature of holoprosencephaly (HPE), the most common congenital malformation of the human forebrain. The goal of this proposal is to elucidate the mechanisms and genetic network that govern DTM development, which will inform HPE pathogenesis and the generation of choroid plexus in culture for clinical applications. Previous studies have established central roles for the bone morphogenetic proteins (Bmps) in DTM development. For example, genetic ablation of the Bmp-producing roof plate in mice causes DTM induction deficits that can be rescued with exogenous Bmp4 alone. Nonetheless, fundamental questions about Bmp signaling and morphogenic activity remain unanswered. Genetic roof plate ablation also causes a dorsal form of HPE, which led to new discoveries about human HPE patients and a signaling network model of forebrain development that can explain how distinct human HPE phenotypes arise. However, within this network, insights into Bmp interactions are notably poor, including the identity of factors that inhibit the Bmp pathway to restrict DTM fates and position their borders. We previously used the roof plate ablation model to implicate Bmps in DTM induction in vivo. More recently, we demonstrated responses in cultured cortical neural precursor cells (NPCs) consistent with Bmp4 acting as a DTM morphogen, and identified the LIM homeodomain transcription factor Lhx2 as a cortical selector gene that suppresses cortical hem fate. In Preliminary Studies, we implicate fibroblast growth factor 8 (Fgf8) as a second DTM fate suppressor and describe enabling tools that include a new Bmp activity reporter mouse, a microfluidic culture system, and a mathematical model of DTM development. These findings and tools provide us with a unique opportunity, among vertebrate CNS model systems, to address fundamental questions in morphogen biology, developmental border formation, and Bmp activity regulation in addition to HPE pathogenesis and choroid plexus fate specification. In this proposal, we use validated in vivo, in vitro, microfluidic, and in silico tools to define the molecular mechanisms and genetic network that direct DTM development, focusing on Bmp activity and the factors that modulate it. PUBLIC HEALTH RELEVANCE: The goal for this project is to better understand the network that governs development of the dorsal midline region in the telencephalon. The proposal is based on a signaling network model we developed that can explain holoprosencephaly, the most common congenital malformation of the human forebrain. The relevance of this project to public health derive mainly from the insights into this common birth defects, but also to the increasing number of psychiatric and neurologic diseases associated with neural stem cell defects, and to NSC and other stem cell strategies aimed at treating these brain disorders.

Adhesives; Assay; Bioassay; Biologic Assays; Biological Assay; CRISP; Cell Adhesion; Cellular Adhesion; Computer Retrieval of Information on Scientific Projects Database; Funding; Grant; Image; Institution; Investigators; Mediating; Microscopy; NIH; National Institutes of Health; National Institutes of Health (U.S.); Photons; Property; Property, LOINC Axis 2; Research; Research Personnel; Research Resources; Researchers; Resources; Role; Series; Source; United States National Institutes of Health; imaging; mutant; reconstruction; social role; transcription factor

The forebrain is proportionately larger in humans than in other mammals. Similarly, the forebrain is proportionately larger in parrots and songbirds than in other birds. These species differences in adult brain proportions have been well described and are thought to account for species differences in behavioral complexity and intelligence. Almost completely unknown, however, are the developmental mechanisms that generate such species differences. Previous work from the Striedter laboratory has shown that forebrain enlargement in parrots and songbirds occurs because the forebrain's precursor cells in these species proliferate for a longer period of time, thereby generating a larger forebrain precursor pool. Although this is a powerful mechansim for enlarging a brain region, other species may enlarge a brain region by other mechanisms, such as changing the spatial patterns of gene expression in young embryos or changing the rates at which precursor cells divide. The proposed research explores these alternative mechanisms by comparing brain region sizes, patterns of gene expression, and rates of cell division across young embryos of different bird species, including parakeets, quail, chickens, and ducks. If one or more of these parameters differs between the examined species, then evolution is free to vary brain proportions through several different developmental mechanisms, rather than constrained to utilize just one. More generally, the findings will clarify some of the rules that govern brain evolution. An important long-term goal is to manipulate brain development in ways that follow these rules and, thus, mimic the natural evolutionary changes. Such experiments are exciting because they will allow for the testing of evolutionary hypotheses. Overall, the proposed work will motivate and train at least one graduate student and several undergraduates performing independent research. It will also excite and educate the general public, who will be exposed to it through public lectures and outreach to student groups.

Although famous for secreting the cerebrospinal fluid (CSF), the choroid plexus has drawn relatively little attention from basic and clinical scientists. Every day, human choroid plexus epithelial cells (CPECs) secrete about two cups of protein-rich CSF, which clears out waste products from and delivers beneficial molecules to every cell in the brain and spinal cord. CPEC defects have been implicated in a large number of major CNS disorders, including hydrocephalus, psychiatric conditions, and neurodegenerative diseases. However, almost all of the literature related to CPEC biology and disease is descriptive due to a lack of CPEC-targeted tools. Importantly, if such tools existed, delivery of compounds to CPECs - in either experimental animals or patients - is straightforward, because CPECs exchange freely with the peripheral circulation. This greatly simplifies drug delivery and strengthens the appeal of CPECs as a new target for CNS studies and disease therapies. Despite this appeal, CPEC-targeted drug screens have not been possible due to difficulties in propagating and deriving them in culture. However, we recently developed a method for generating derived CPECs (dCPECs) from mouse and human embryonic stem cells. This method provides, for the first time, a scalable means to produce dCPECs in the large numbers required for high-throughput screening (HTS). In this R21 proposal, we combine the dCPEC technology inventor with experts in assay development and drug screening to develop a high-throughput dCPEC screening platform. In preliminary studies, we describe dCPEC generation, candidate cell lines to be screened for HTS compatibility, and a sensitive 96-well ELISA for secreted human TTR, an ideal proxy for CPEC secretion and an important molecular target in its own right. The TTR ELISA will be converted into an HTS-compatible AlphaLISA, which will be used for a pilot screen to determine assay readiness. Hits and analogs from the pilot screen will then be screened and validated via testing funnels that include new and already-established assays of CPEC function, mechanism, and specificity. This validated high-throughput dCPEC platform will set the stage for full-scale screening proposals to generate first-in-class tool and therapy leads for a brand-new CPEC-based biomedicine.

ABSTRACT Physician-scientists serve as a critical bridge between the research laboratory and the clinic, uniquely positioned to conduct innovative biomedical research and translate relevant insights into meaningful improvements in human health. The success of the physician-scientist within the academic and biomedical research enterprise is clear, as dual-degree trained investigators are disproportionately represented among successful NIH grant awardees and recipients of honorary distinctions such as the Nobel Prize in Physiology or Medicine. Despite these successes, recent trends described in the NIH Physician Scientist Workforce Study predict the future extinction of this highly-specialized cohort, citing obstacles such as the lack of demographic diversity, high rates of career attrition among junior investigators, and limited mentorship. In response, the NIH and other associations have initiated programs to prevent this population crash. One such organization is the American Physician Scientist Association (APSA), a student-driven, non-profit organization dedicated to career development and mentorship of physician-scientist trainees. In addition to its national reach, APSA maintains a strong regional presence through annual meetings in the Southeastern, Northeastern, Midwestern, Southern, and Western regions of the United States. This year we are proud to host the 2017 APSA West Regional Meeting at the University of California, Irvine School of Medicine on December 2nd, 2017. The purpose of the meeting is to provide a framework for bridging the scientific and mentorship gaps in physician-scientist training. Through interdisciplinary scientific talks by early and established clinician-scientists, workshops tailored to academic career development, and extensive outreach efforts to women and individuals underrepresented in science and medicine, the proposed conference will stimulate novel scientific collaborations, address critical voids in the physician-scientist training pipeline, and foster development of a diverse next-generation of scientists.

Core D: Neuropathology Core Project Summary/Abstract The UCI ADRC Neuropathology (NP) Core links clinical evaluations with definitive neuropathological diagnoses. Neuropathology remains the definitive method for diagnosing Alzheimer's disease (AD). The UCI ADRC cohorts consist of a longitudinal cohort, and two unique patient populations: adults with Down syndrome and individuals over 90 years of age ( 90+ ), representing cohorts at high risk of developing dementia. Down syndrome represents the single most prevalent cause of early-onset AD, whereas the 90+ cohort represents subjects with a high rate of conversion to dementia. Both cohorts provide unique opportunities to study the transition of `cognitively normal' to dementia. The overarching goal of the NP Core is to provide the infrastructure to support research on all three ADRC cohorts that aim to elucidate the underlying mechanisms that define normal aging, the transition to MCI, and the subsequent transition from MCI to AD/dementia. As part of its mission, the UCI NP Core disseminates well-characterized tissues, biospecimens and reagents to basic scientists at UCI and abroad to stimulate and facilitate research in AD and other age-associated neurodegenerative diseases. Using standardized methods for processing, the NP Core supports multi-center collaborative studies. Additionally, the NP Core seeks to innovate by establishing infrastructure to support novel technologies, techniques, and data collection to increase the value of the stored tissue and biospecimens. Lastly, the NP Core reports important AD research findings in collaboration with the Education Core. In order to achieve the goals of the NP Core, we propose seven specific aims: (1) Perform an accurate and timely autopsy of the ADRC cohorts (Longitudinal, Down syndrome, and 90+) and provide a standardized neuropathological report to the Data Management and Statistics Core for deposition with NACC, and to the Clinical Core for dissemination to the families of the deceased participants and their physicians; (2) Store, catalog, and disseminate brain tissue and biospecimens from ADRC subjects; (3) Store, catalog, and disseminate novel reagents made by UCI investigators; (4) Support and consult on ADRC projects, ADRC pilot studies, and studies on AD mechanisms by investigators funded by other means, including ADC collaborative projects; (5) Develop and establish new core functions to facilitate innovative research to study the transition of MCI to AD; (6) Participate in the public education of Chinese-Americans and others; (7) Train pathologists, neurologists, neurosurgeons, neuroscientists, neuropsychologists, and students on the neuropathological features of AD and other dementing disorders.

PROJECT SUMMARY We will test the hypothesis that choroid plexus epithelial cells (CPECs) are affected in late onset Alzheimer's Disease (AD) associated with the type 4 isoform of apolipoprotein E (APOE4). At least one copy of APOE4 is present in 56-65% of people with AD, and two copies are highly predictive of AD. APOE4 has been implicated in faulty clearance of the toxic A? peptide associated with AD, as well as in numerous other AD-related functions in various cell types. Rodent CPECs are known to remove toxins from the cerebrospinal fluid (CSF), express high levels of APOE, and take up and transport A? peptides. However, human CPECs have not been well studied, in part due to the difficulty in acquiring healthy human cells. Our laboratory has developed a protocol to derive human CPECs from pluripotent stem cells, which provides the opportunity to study basic human CPEC functions and their roles in AD and other diseases. We propose in Aim 1 to derive CPECs using existing induced pluripotent stem cells (iPSCs) from APOE4/4 AD patients and from their isogenic APOE3/3 counterparts that have been edited using CRISP/Cas9. We will monitor the CPEC derivations for changes in differentiation efficiency, then test the specific hypothesis that A? uptake is impaired in APOE4/4 CPECs. In Aim 2, we will characterize the transcriptomes of individual human CPECs using single cell RNA sequencing (scRNAseq) to look for gene expression changes in CPEC subpopulations that correspond to the A? uptake findings from Aim 1. Based on a prior study that examined neurons, astrocytes, and microglia derived from APOE isogenic iPSCs, we anticipate a broad spectrum of gene expression differences that are unique to CPECs and predictive of altered functions. We will then validate scRNAseq findings by RT-qPCR and immunostaining, then cross-validate in vivo by immunostaining postmortem choroid plexus specimens from patients with known APOE genotypes. We envision this R21 proposal leading to subsequent R01 submissions that extend this work to CPECs derived from additional isogenic pairs involving APOE and other AD risk genes, to test additional emergent hypotheses regarding altered CPEC functions in AD, and to explore potential therapeutic interventions to correct impaired CPEC functions.

Contact PD/PI: Cooper, Dan M NRSA-Training-001 (248) J. NRSA TRAINING CORE PROJECT SUMMARY The importance of the CTSA TL1 program is underscored by a fundamental realization that the future of science is dependent, to a large extent, on the quality of training today. This is especially true within the field of clinical translational science (CTS) where, prior to the advent of the CTSA TL1 program, formal curricula were few and far between. This latter point was certainly true of the environment at UCI. The founding of the ICTS KL2 and TL1 programs has transformed the NRSA training and mentored career development environment at UCI. Our goal in this renewal is to enhance the quality of our TL1 program by pursuing the following specific aims. Specific Aim 1: Champion NRSA training by providing an integrated TL1 program that consists of outstanding leadership and oversight at all levels. Our administrative structure is highlighted by a team that consists of program directors, administrative team, Executive Committee, and External Advisory Committee. Collectively, they are guided by a strong survey, evaluation, and tracking team, and implementation of Quality by Design principles. Additionally, we have begun discussions with our Provost that will result in a campus- wide strategic plan focused on NRSA training and mentored career development. Specific Aim 2: Provide a flexible and innovative curriculum that emphasizes both core and advanced competencies in CTS. We will continue to offer a strong complement of workshops, journal clubs, formal courses, and certificate programs focused on core and advanced competencies in CTS. In this context, we will refine our Focused Flexible Accelerated Studies (FFASt) course on CTS core values and add new FFASt modules focused on: 1) the development of collaboration plans (Team Science curriculum), 2) community engagement studios (Community Engagement curriculum), and 3) network medicine (Big Data and Informatics curriculum). Specific Aim 3: Maximize access to the NRSA training program. One of our main priorities is maximizing opportunities for our trainees to pursue scientific discovery and the promotion of health. We will 1) expand our program by providing institutional support for two additional trainees, 2) continue our Affiliated Scholars Advancement Program, which significantly expands training opportunities for unfunded CTS trainees, and 3) continue to promote diversity. Specific Aim 4: Implement and integrate local, regional, and national strategies to transform NRSA training. Locally, we created a campus-wide ?KT Training Council? whose goal is to promote training and career development (e.g., K and T NIH mechanisms). This council interacts with senior leadership at all levels, including ongoing and evolving discussions with our Provost. Regionally, ICTS will continue to chair and administer the Western CTSA Education Consortium, which includes the CTSAs in California, Oregon, and New Mexico. This consortium is focused on all aspects of the CTSA?s KL2 and TL1 programs. Nationally we will continue to participate in key programs like the Domain Task Force on workforce development and the Common Metrics Turn the Curve program. Page 907 Project Summary/Abstract Contact PD/PI: Cooper, Dan M NRSA-Training-001 (248) J. NRSA TRAINING CORE REFERENCES 1. Gruppen L, Frank JR, Lockyer J, et al. Toward a research agenda for competency-based medical education. Med Teach 2017;39:623?30. 2. Ten Cate O. Competency-Based Postgraduate Medical Education: Past, Present and Future. GMS J Med Educ 2017;34:Doc69. 3. Carraccio C, Englander R, Van Melle E, et al. Advancing Competency-Based Medical Education. Acad Med 2016;91:645?9. 4. Meeker-O?Connell A, Glessner C. Clinical trial quality: From supervision to collaboration and beyond. Clin Trials J Soc Clin Trials 2018;15:23?6. 5. Crump C, Ned J, Winkleby MA. The Stanford Medical Youth Science Program: educational and science- related outcomes. Adv Heal Sci Educ 2015;20:457?66. 6. Duncan GA, Lockett A, Villegas LR, et al. National Heart, Lung, and Blood Institute Workshop Summary: Enhancing Opportunities for Training and Retention of a Diverse Biomedical Workforce. Ann Am Thorac Soc 2016;13:562?7. 7. Cunningham SL, Kunselman MM. University of Washington and partners? program to teach middle school students about neuroscience and science careers. Acad Med 1999;74:318?21. 8. Wilkes M, Feldman MD. Mentoring clinical trainees: a need for high touch. Lancet 2017;389:135?7. 9. Curran MA, Black M, Depp CA, et al. Perceived Barriers and Facilitators for an Academic Career in Geriatrics: Medical Students? Perspectives. Acad Psychiatry 2015;39:253?8. 10. Richardson DM, Keller TE, Wolf DSS, Zell A, Morris C, Crespo CJ. BUILD EXITO: a multi-level intervention to support diversity in health-focused research. BMC Proc 2017;11:19. 11. Feldman MD, Steinauer JE, Khalili M, et al. A mentor development program for clinical translational science faculty leads to sustained, improved confidence in mentoring skills. ClinTranslSci 2012;5:362?7. 12. Romanick M, Ng K, Lee G, Herbert M, Coller BS. The Rockefeller University Graduate Tracking Survey System. ClinTranslSci 2014; 13. Schneider M, Kane CM, Rainwater J, et al. Feasibility of common bibliometrics in evaluating translational science. J Clin Transl Sci 2017;1:45?52. 14. Schneider M, Guerrero L, Jones LB, et al. Developing the Translational Research Workforce: A Pilot Study of Common Metrics for Evaluating the Clinical and Translational Award KL2 Program. Clin Transl Sci 2015;8:662?7. 15. Salazar MR, Lant TK, Fiore SM, Salas E. Facilitating Innovation in Diverse Science Teams Through Integrative Capacity. Small Gr Res 2012;43:527?58. 16. Vogel AL, Hall KL, Fiore SM, et al. The Team Science Toolkit. Am J Prev Med 2013;45:787?9. 17. Olson JS, Olson GM. Working Together Apart: Collaboration Over the Internet. San Rafael: Morgan and Claypool; 2014. 18. Bietz MJ, Abrams S, Cooper D, et al. Improving the Odds Through the Collaboration Success Wizard. TranslBehavMed 2012;2:480?6. 19. Rice RE, Evans SK, Pearce KE, Sivunen A, Vitak J, Treem JW. Organizational Media Affordances: Operationalization and Associations with Media Use. J Commun 2017;67:106?30. 20. Vogel AL, Feng A, Oh A, et al. Influence of a National Cancer Institute transdisciplinary research and training initiative on trainees? transdisciplinary research competencies and scholarly productivity. TranslBehavMed 2012;2:459?68. 21. Vogel AL, Hall KL, Fiore SM, et al. The Team Science Toolkit: enhancing research collaboration through online knowledge sharing. AmJPrevMed 2013;45:787?9. 22. Perkins SM, Bacchetti P, Davey CS, et al. Best Practices for Biostatistical Consultation and Collaboration in Academic Health Centers. Am Stat 2016;70:187?94. 23. Collins FS, Tabak LA. Policy: NIH plans to enhance reproducibility. Nature 2014;505:612?3. 24. Steward O, Balice-Gordon R. Rigor or Mortis: Best Practices for Preclinical Research in Neuroscience. Neuron 2014;84:572?81. Page 908 References Cited

PROJECT SUMMARY ? NEUROPATHOLOGY CORE (CORE D) The UCI ADRC Neuropathology (NP) Core provides autopsy, diagnostic, and biorepository services for the three cohorts of the UCI ADRC - Uniform Data Set (UDS), 90+, and Down syndrome - which span a broad range of ages and susceptibilities to AD. The overarching goal of the NP Core is to support education, outreach, and research on AD and related disorders (ADRD) in collaboration with other ADRC cores and ADRD researchers to ?identify, quantify, and validate factors that influence the risk of AD across the lifespan.? Since its inception, the NP Core has collected and maintained a considerable bank of high-quality biospecimens, which have been distributed broadly to NCRAD and federally-funded local, national, and international ADRD investigators and consortia. Core operations have been continually enhanced, and metrics of core performance are strong. To expand its capacity, three new faculty have joined the NP Core, including a neuropathologist who is also a REC mentee (Mari Perez-Rosendahl). Together with the other UCI ADRC Cores, the NP Core proposes six Aims to enhance its diagnostic, education, and research functions via collaboration, innovation, and education: 1) Provide accurate, timely, comprehensive, and standardized postmortem examinations and diagnostic reports on UDS subjects and special populations, including the addition of postmortem skin collection on selected cases in collaboration with the iPSC Core; 2) Harvest, process, store, catalog, and distribute high-quality brain tissue and biospecimens to ADRD researchers at UCI and beyond, collaborating with the DMS and Biomarker Cores in its data management and biospecimen utilization functions; 3) Incorporate new analysis methods to enable novel microglial studies by UCI investigators at the cutting-edge of a new microglial biology in AD; 4) Incorporate whole slide imaging (WSI) to facilitate diagnoses, research, and education, including the establishment of interfaces with a UC-wide WSI network and UCI artificial intelligence (AI) center; 5) Support novel ADRD research and outreach in collaboration with other UCI ADRC Cores and other ADRCs, including new studies involving postmortem MRI, light sheet microscopy of cleared brain tissues, and AI studies of WSIs; and 6) Contribute unique expertise to the education and training of the next generation of AD clinicians and researchers, including Dr. Perez- Rosendahl, neuropathologist and REC mentee.