Combined functions of two RRMs in dead-end1 mimic helicase activity to promote nanos1 translation in the germline

Dead-end1 expression is restricted to the vertebrate germline where it is believed to activate translation of mRNAs required to protect and promote that unique lineage. nanos1 is one such germline mRNA whose translation is blocked by a secondary mRNA structure within the ORF. Dead-end1 contains a canonical mRNA Recognition Motif (RRM1) in its N-terminus but also contains a less conserved RRM2. Here we provide a mechanistic picture of the nanos1 mRNA-Dead-end1 interaction in the Xenopus germline. We show that RRM1, but not RRM2, is required for binding nanos1. Similar to the zebrafish homologue, Xenopus Dead-end1 possesses ATPase activity. Surprisingly, this activity appears to be within the RRM2, different from the C-terminal region where it is found in zebrafish. More importantly, we show that RRM2 is required for nanos1 translation and germline survival. Further, Dead-end1 functions as a homodimer and binds nanos1 mRNA just downstream of the secondary structure required for nanos1 repression. We propose a model in which the RRM1 is required to bind nanos1 mRNA while the RRM2 is required to promote translation through the action of ATPase. Dead-end1 appears to use RRMs to mimic the function of helicases.Fil: Agüero, Tristán Horacio. University of Miami; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Jin, Zhigang. University of Illinois; Estados UnidosFil: Owens, Dawn. University of Miami; Estados UnidosFil: Malhotra, Arun. University of Miami; Estados UnidosFil: Newman, Karen. University of Miami; Estados UnidosFil: Yang, Jing. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados Unido

High-throughput analysis reveals novel maternal germline RNAs crucial for primordial germ cell preservation and proper migration

During oogenesis, hundreds of maternal RNAs are selectively localized to the animal or vegetal pole, including determinants of somatic and germline fates. Although microarray analysis has identified localized determinants, it is not comprehensive and is limited to known transcripts. Here, we utilized high-throughput RNA sequencing analysis to comprehensively interrogate animal and vegetal pole RNAs in the fully grown Xenopus laevis oocyte. We identified 411 (198 annotated) and 27 (15 annotated) enriched mRNAs at the vegetal and animal pole, respectively. Ninety were novel mRNAs over 4-fold enriched at the vegetal pole and six were over 10-fold enriched at the animal pole. Unlike mRNAs, microRNAs were not asymmetrically distributed. Whole-mount in situ hybridization confirmed that all 17 selected mRNAs were localized. Biological function and network analysis of vegetally enriched transcripts identified protein-modifying enzymes, receptors, ligands, RNA-binding proteins, transcription factors and co-factors with five defining hubs linking 47 genes in a network. Initial functional studies of maternal vegetally localized mRNAs show that sox7 plays a novel and important role in primordial germ cell (PGC) development and that ephrinB1 (efnb1) is required for proper PGC migration. We propose potential pathways operating at the vegetal pole that highlight where future investigations might be most fruitful.Fil: Owens, Dawn A.. University of Miami; Estados UnidosFil: Butler, Amanda M.. University of Miami; Estados UnidosFil: Agüero, Tristán Horacio. University of Miami; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Newman, Karen M.. University of Miami; Estados UnidosFil: Van Booven, Derek. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados Unido

BAP1 regulates epigenetic switch from pluripotency to differentiation in developmental lineages giving rise to BAP1-mutant cancers

The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.Fil: Kuznetsov, Jeffim N.. University of Miami; Estados UnidosFil: Agüero, Tristán Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina. University of Miami; Estados UnidosFil: Owens, Dawn A.. University of Miami; Estados UnidosFil: Kurtenbach, Stefan. University of Miami; Estados UnidosFil: Field, Matthew G.. University of Miami; Estados UnidosFil: Durante, Michael A.. University of Miami; Estados UnidosFil: Rodriguez, Daniel A.. University of Miami; Estados UnidosFil: King, Mary Lou. University of Miami; Estados UnidosFil: Harbour, J. William. University of Miami; Estados Unido

Individual epigenetic status of the pathogenic D4Z4 macrosatellite correlates with disease in facioscapulohumeral muscular dystrophy

BACKGROUND: Both forms of facioscapulohumeral muscular dystrophy (FSHD) are associated with aberrant epigenetic regulation of the chromosome 4q35 D4Z4 macrosatellite. Chromatin changes due to large deletions of heterochromatin (FSHD1) or mutations in chromatin regulatory proteins (FSHD2) lead to relaxation of epigenetic repression and increased expression of the deleterious double homeobox 4 (DUX4) gene encoded within the distal D4Z4 repeat. However, many individuals with the genetic requirements for FSHD remain asymptomatic throughout their lives. Here we investigated family cohorts of FSHD1 individuals who were either affected (manifesting) or without any discernible weakness (nonmanifesting/asymptomatic) and their unaffected family members to determine if individual epigenetic status and stability of repression at the contracted 4q35 D4Z4 array in myocytes correlates with FSHD disease. RESULTS: Family cohorts were analyzed for DNA methylation on the distal pathogenic 4q35 D4Z4 repeat on permissive A-type subtelomeres. We found DNA hypomethylation in FSHD1-affected subjects, hypermethylation in healthy controls, and distinctly intermediate levels of methylation in nonmanifesting subjects. We next tested if these differences in DNA methylation had functional relevance by assaying DUX4-fl expression and the stability of epigenetic repression of DUX4-fl in myogenic cells. Treatment with drugs that alter epigenetic status revealed that healthy cells were refractory to treatment, maintaining stable repression of DUX4, while FSHD1-affected cells were highly responsive to treatment and thus epigenetically poised to express DUX4. Myocytes from nonmanifesting subjects had significantly higher levels of DNA methylation and were more resistant to DUX4 activation in response to epigenetic drug treatment than cells from FSHD1-affected first-degree relatives containing the same contraction, indicating that the epigenetic status of the contracted D4Z4 array is reflective of disease. CONCLUSIONS: The epigenetic status of the distal 4qA D4Z4 repeat correlates with FSHD disease; FSHD-affected subjects have hypomethylation, healthy unaffected subjects have hypermethylation, and nonmanifesting subjects have characteristically intermediate methylation. Thus, analysis of DNA methylation at the distal D4Z4 repeat could be used as a diagnostic indicator of developing clinical FSHD. In addition, the stability of epigenetic repression upstream of DUX4 expression is a key regulator of disease and a viable therapeutic target

Studies on the synthesis and structure of cordycepin monophosphate

Cordycepin monophosphate has been synthesized by two methods. One of these was chemical and involved treatment of cordycepin with cyanoethyl phosphate in the presence of N,N'-dicyclohexylcarbodiimide with subsequent alkaline hydrolysis. The other involved a phosphate transfer from uridine 5'-phosphate to cordycepin utilizing an enzyme prepared from fresh wheat sprouts. In each instance, the product isolated proved to be the monophosphate derivative and the position of attachment of the phosphate group was shown to be on the primary hydroxyl group of the sugar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32257/1/0000319.pd

Studies on the synthesis and structure of cordycepin monophosphate

Cordycepin monophosphate has been synthesized by two methods. One of these was chemical and involved treatment of cordycepin with cyanoethyl phosphate in the presence of N,N'-dicyclohexylcarbodiimide with subsequent alkaline hydrolysis. The other involved a phosphate transfer from uridine 5'-phosphate to cordycepin utilizing an enzyme prepared from fresh wheat sprouts. In each instance, the product isolated proved to be the monophosphate derivative and the position of attachment of the phosphate group was shown to be on the primary hydroxyl group of the sugar.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32256/1/0000318.pd

Analysis of Myogenic and Candidate Disease Biomarkers in FSHD Muscle Cells

The UMMS Wellstone Program is a foundation and NIH-funded cooperative research center focusing on identifying biomarkers for facioscapulohumeral muscular dystrophy (FSHD) to gain insight into the molecular pathology of the disease and to develop potential therapies. FSHD is characterized by progressive wasting of skeletal muscles, with weakness often initiating in facial muscles and muscles supporting the scapula and upper arms. While the genetics associated with FSHD are complex, the major form of the disease, FSHD1, is linked to contraction of the D4Z4 repeat region located at chromosome 4q. Recently, a transcript encoded at the distal end of the repeat region, Dux4-fl, normally expressed in embryonic stem cells and germ cells, was also detected in differentiated muscle cells and biopsies from FSHD subjects, giving rise to the hypothesis that DUX4-FL function contributes to muscle weakness. We established a repository of high quality, well-characterized primary and immortalized muscle cell strains from FSHD and control subjects in affected families to provide biomaterials for cell and molecular studies to the FSHD research community. qPCR and immunostaining analyses demonstrate similar growth and differentiation characteristics in cells from FSHD and control subjects within families. We detected Dux4-fl transcript and protein in FSHD cells as recently described; interestingly, we also detected Dux4-fl in muscle cells from a subset of control individuals, suggesting that any Dux4-fl-mediated myopathy would require additional modifying elements. Microarray analysis of FSHD and control muscle cells demonstrated that several genes were upregulated in FSHD cells, including genes that were concurrently identified as downstream targets of Dux4-fl and as candidate FSHD disease genes. Future studies will further characterize the RNA and protein expression of candidate disease genes in cells from FSHD and control subjects, including nonmanifesting subjects with the D4Z4 lesion but no muscle weakness, and utilizing whole transcriptome sequencing (RNAseq) to identify additional candidates

An interdisciplinary knowledge translation intervention in long-term care: Study protocol for the vitamin D and osteoporosis study (ViDOS) pilot cluster randomized controlled trial

BACKGROUND: Knowledge translation (KT) research in long-term care (LTC) is still in its early stages. This protocol describes the evaluation of a multifaceted, interdisciplinary KT intervention aimed at integrating evidence-based osteoporosis and fracture prevention strategies into LTC care processes. METHODS AND DESIGN: The Vitamin D and Osteoporosis Study (ViDOS) is underway in 40 LTC homes (n = 19 intervention, n = 21 control) across Ontario, Canada. The primary objectives of this study are to assess the feasibility of delivering the KT intervention, and clinically, to increase the percent of LTC residents prescribed ≥800 IU of vitamin D daily. Eligibility criteria are LTC homes that are serviced by our partner pharmacy provider and have more than one prescribing physician. The target audience within each LTC home is the Professional Advisory Committee (PAC), an interdisciplinary team who meets quarterly. The key elements of the intervention are three interactive educational sessions led by an expert opinion leader, action planning using a quality improvement cycle, audit and feedback reports, nominated internal champions, and reminders/point-of-care tools. Control homes do not receive any intervention, however both intervention and control homes received educational materials as part of the Ontario Osteoporosis Strategy. Primary outcomes are feasibility measures (recruitment, retention, attendance at educational sessions, action plan items identified and initiated, internal champions identified, performance reports provided and reviewed), and vitamin D (≥800 IU/daily) prescribing at 6 and 12 months. Secondary outcomes include the proportion of residents prescribed calcium supplements and osteoporosis medications, and falls and fractures. Qualitative methods will examine the experience of the LTC team with the KT intervention. Homes are centrally randomized to intervention and control groups in blocks of variable size using a computer generated allocation sequence. Randomization is stratified by home size and profit/nonprofit status. Prescribing data retrieval and analysis are performed by blinded personnel. DISCUSSION: Our study will contribute to an improved understanding of the feasibility and acceptability of a multifaceted intervention aimed at translating knowledge to LTC practitioners. Lessons learned from this study will be valuable in guiding future research and understanding the complexities of translating knowledge in LTC. TRIAL REGISTRATION: ClinicalTrials.gov NCT01398527

Standardized Definitions for Efficacy End Points in Neoadjuvant Breast Cancer Clinical Trials: NeoSTEEP.

PURPOSE: The Standardized Definitions for Efficacy End Points (STEEP) criteria, established in 2007 and updated in 2021 (STEEP 2.0), provide standardized definitions of adjuvant breast cancer (BC) end points. STEEP 2.0 identified a need to separately address end points for neoadjuvant clinical trials. The multidisciplinary NeoSTEEP working group of experts was convened to critically evaluate and align neoadjuvant BC trial end points. METHODS: The NeoSTEEP working group concentrated on neoadjuvant systemic therapy end points in clinical trials with efficacy outcomes-both pathologic and time-to-event survival end points-particularly for registrational intent. Special considerations for subtypes and therapeutic approaches, imaging, nodal staging at surgery, bilateral and multifocal diseases, correlative tissue collection, and US Food and Drug Administration regulatory considerations were contemplated. RESULTS: The working group recommends a preferred definition of pathologic complete response (pCR) as the absence of residual invasive cancer in the complete resected breast specimen and all sampled regional lymph nodes (ypT0/Tis ypN0 per AJCC staging). Residual cancer burden should be a secondary end point to facilitate future assessment of its utility. Alternative end points are needed for hormone receptor-positive disease. Time-to-event survival end point definitions should pay particular attention to the measurement starting point. Trials should include end points originating at random assignment (event-free survival and overall survival) to capture presurgery progression and deaths as events. Secondary end points adapted from STEEP 2.0, which are defined from starting at curative-intent surgery, may also be appropriate. Specification and standardization of biopsy protocols, imaging, and pathologic nodal evaluation are also crucial. CONCLUSION: End points in addition to pCR should be selected on the basis of clinical and biologic aspects of the tumor and the therapeutic agent investigated. Consistent prespecified definitions and interventions are paramount for clinically meaningful trial results and cross-trial comparison