Extending the clinical spectrum of X-linked Tonne-Kalscheuer syndrome (TOKAS): new insights from the fetal perspective.

INTRODUCTION: Tonne-Kalscheuer syndrome (TOKAS) is a recessive X-linked multiple congenital anomaly disorder caused by RLIM variations. Of the 41 patients reported, only 7 antenatal cases were described. METHOD: After the antenatal diagnosis of TOKAS by exome analysis in a family followed for over 35 years because of multiple congenital anomalies in five male fetuses, a call for collaboration was made, resulting in a cohort of 11 previously unpublished cases. RESULTS: We present a TOKAS antenatal cohort, describing 11 new cases in 6 French families. We report a high frequency of diaphragmatic hernia (9 of 11), differences in sex development (10 of 11) and various visceral malformations. We report some recurrent dysmorphic features, but also pontocerebellar hypoplasia, pre-auricular skin tags and olfactory bulb abnormalities previously unreported in the literature. Although no clear genotype-phenotype correlation has yet emerged, we show that a recurrent p.(Arg611Cys) variant accounts for 66% of fetal TOKAS cases. We also report two new likely pathogenic variants in RLIM, outside of the two previously known mutational hotspots. CONCLUSION: Overall, we present the first fetal cohort of TOKAS, describe the clinical features that made it a recognisable syndrome at fetopathological examination, and extend the phenotypical spectrum and the known genotype of this rare disorder.

USP27X variants underlying X-linked intellectual disability disrupt protein function via distinct mechanisms.

Neurodevelopmental disorders with intellectual disability (ND/ID) are a heterogeneous group of diseases driving lifelong deficits in cognition and behavior with no definitive cure. X-linked intellectual disability disorder 105 (XLID105, #300984; OMIM) is a ND/ID driven by hemizygous variants in the USP27X gene encoding a protein deubiquitylase with a role in cell proliferation and neural development. Currently, only four genetically diagnosed individuals from two unrelated families have been described with limited clinical data. Furthermore, the mechanisms underlying the disorder are unknown. Here, we report 10 new XLID105 individuals from nine families and determine the impact of gene variants on USP27X protein function. Using a combination of clinical genetics, bioinformatics, biochemical, and cell biology approaches, we determined that XLID105 variants alter USP27X protein biology via distinct mechanisms including changes in developmentally relevant protein-protein interactions and deubiquitylating activity. Our data better define the phenotypic spectrum of XLID105 and suggest that XLID105 is driven by USP27X functional disruption. Understanding the pathogenic mechanisms of XLID105 variants will provide molecular insight into USP27X biology and may create the potential for therapy development.

Chromatin targeting of the RNF12/RLIM E3 ubiquitin ligase controls transcriptional responses.

Protein ubiquitylation regulates key biological processes including transcription. This is exemplified by the E3 ubiquitin ligase RNF12/RLIM, which controls developmental gene expression by ubiquitylating the REX1 transcription factor and is mutated in an X-linked intellectual disability disorder. However, the precise mechanisms by which ubiquitylation drives specific transcriptional responses are not known. Here, we show that RNF12 is recruited to specific genomic locations via a consensus sequence motif, which enables co-localisation with REX1 substrate at gene promoters. Surprisingly, RNF12 chromatin recruitment is achieved via a non-catalytic basic region and comprises a previously unappreciated N-terminal autoinhibitory mechanism. Furthermore, RNF12 chromatin targeting is critical for REX1 ubiquitylation and downstream RNF12-dependent gene regulation. Our results demonstrate a key role for chromatin in regulation of the RNF12-REX1 axis and provide insight into mechanisms by which protein ubiquitylation enables programming of gene expression.

Roles of the Rlim-Rex1 axis during X chromosome inactivation in mice.

In female mice, the gene dosage from X chromosomes is adjusted by a process called X chromosome inactivation (XCI) that occurs in two steps. An imprinted form of XCI (iXCI) that silences the paternally inherited X chromosome (Xp) is initiated at the 2- to 4-cell stages. As extraembryonic cells including trophoblasts keep the Xp silenced, epiblast cells that give rise to the embryo proper reactivate the Xp and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI require the lncRNA Xist, which is expressed from the X to be inactivated. The X-linked E3 ubiquitin ligase Rlim (Rnf12) in conjunction with its target protein Rex1 (Zfp42), a critical repressor of Xist, have emerged as major regulators of iXCI. However, their roles in rXCI remain controversial. Investigating early mouse development, we show that the Rlim-Rex1 axis is active in pre-implantation embryos. Upon implantation Rex1 levels are downregulated independently of Rlim specifically in epiblast cells. These results provide a conceptual framework of how the functional dynamics between Rlim and Rex1 ensures regulation of iXCI but not rXCI in female mice.

Therapeutic validation and targeting of signalling networks that are dysregulated in intellectual disability.

Intellectual disability (ID) represents a major burden on healthcare systems in the developed world. However, there is a disconnect between our knowledge of genes that are mutated in ID and our understanding of the underpinning molecular mechanisms that cause these disorders. We argue that elucidating the signalling and transcriptional networks that are dysregulated in patients will afford new therapeutic opportunities.

Lesiones iatrogénicas del nervio radial en la osteosíntesis de la diáfisis humeral / Intraoperative iatrogenic injury of the radial nerve in humerus osteosynthesis

Introducción: Los objetivos de este estudio fueron determinar la incidencia de lesión iatrogénica intraquirúrgica del nervio radial durante la osteosíntesis de la diáfisis y el extremo distal del húmero, distinguir factores de riesgos asociados y reconocer elementos pronósticos que participan de su recuperación. Materiales y Métodos: Se evaluó, en forma retrospectiva, a 82 pacientes con osteosíntesis de húmero entre 2005 y 2021, sin parálisis radial preoperatoria. Se consideraron los sistemas de fijación utilizados, y se compararon las cirugías primarias con las reoperaciones y el tiempo transcurrido entre estas. El diagnóstico de parálisis radial posoperatorio fue clínico. Todos los pacientes fueron tratados con férula en extensión de muñeca, electroestimulación, kinesiología y vitaminas B1, B6, B12. La electromiografía se solicitó a los fines del pronóstico. Resultados: Nueve pacientes tuvieron déficit motor del nervio radial en el posoperatorio inmediato. El sistema de fijación era una placa (7 casos), sistema de cable-placa (1 caso) y clavo endomedular acerrojado anterógrado (1 caso). Siete ocurrieron en cirugías primarias y dos en reoperaciones. El 88% recuperó su función motora completamente antes de los 6 meses después de la parálisis. La electromiografía reveló un nervio radial no excitable en el 22% restante con parálisis definitiva. Conclusiones: El uso de placa de osteosíntesis, la disección intraoperatoria del nervio radial y las reoperaciones aumentan la incidencia de parálisis. Un nervio radial no excitable se relaciona con un peor pronóstico de recuperación espontánea. Nivel de Evidencia: IV

Introduction: The purpose of this study is to determine the incidence of intraoperative iatrogenic radial nerve injury after osteosynthesis of the diaphysis and distal end of the humerus, identify associated risk factors, and determine the prognostic factors involved in its recovery. Materials and Methods: We retrospectively assessed 82 humerus osteosynthesis cases between 2005 and 2021 who had normal radial nerve function before surgery. We evaluated the fixation systems used, the type of surgery (primary versus revision), and the intervals between surgeries. The diagnosis of postoperative radial palsy was made by clinical examination. All patients were treated with wrist extension splint, physiotherapy, and vitamins B1, B6, and B12. Results: After humerus fixation, 9 patients developed motor palsy. Seven cases were fixed with plates, one with a cable-plate system, and one with an anterograde locking intramedullary nail. Seven cases (22%) occurred after primary procedures, while two occurred during revisions. Within 6 months, 88% had regained full motor function. In the remaining 22% of patients with definite palsy, electromyography revealed no excitability of the radial nerve. Conclusions: The use of an osteosynthesis plate, as well as intraoperative dissection and neurolysis of the radial nerve, were identified as risk factors for the development of radial palsy. Reoperations on the humerus, on the other hand, are a risk factor that increases the likelihood of postoperative radial nerve palsy. A radial nerve with no excitability on the postoperative electromyogram has a poor prognosis of spontaneous radial nerve function recovery. Level of Evidence: IV

An RNF12-USP26 amplification loop drives germ cell specification and is disrupted by disease-associated mutations.

The E3 ubiquitin ligase RNF12 plays essential roles during development, and the gene encoding it, RLIM, is mutated in the X-linked human developmental disorder Tonne-Kalscheuer syndrome (TOKAS). Substrates of RNF12 include transcriptional regulators such as the pluripotency-associated transcriptional repressor REX1. Using global quantitative proteomics in male mouse embryonic stem cells, we identified the deubiquitylase USP26 as a putative downstream target of RNF12 activity. RNF12 relieved REX1-mediated repression of Usp26, leading to an increase in USP26 abundance and the formation of RNF12-USP26 complexes. Interaction with USP26 prevented RNF12 autoubiquitylation and proteasomal degradation, thereby establishing a transcriptional feed-forward loop that amplified RNF12-dependent derepression of REX1 targets. We showed that the RNF12-USP26 axis operated specifically in mouse testes and was required for the expression of gametogenesis genes and for germ cell differentiation in vitro. Furthermore, this RNF12-USP26 axis was disrupted by RLIM and USP26 variants found in TOKAS and infertility patients, respectively. This work reveals synergy within the ubiquitylation cycle that controls a key developmental process in gametogenesis and that is disrupted in human genetic disorders.

Activity-based probe profiling of RNF12 E3 ubiquitin ligase function in Tonne-Kalscheuer syndrome.

Ubiquitylation enzymes are involved in all aspects of eukaryotic biology and are frequently disrupted in disease. One example is the E3 ubiquitin ligase RNF12/RLIM, which is mutated in the developmental disorder Tønne-Kalscheuer syndrome (TOKAS). RNF12 TOKAS variants largely disrupt catalytic E3 ubiquitin ligase activity, which presents a pressing need to develop approaches to assess the impact of variants on RNF12 activity in patients. Here, we use photocrosslinking activity-based probes (photoABPs) to monitor RNF12 RING E3 ubiquitin ligase activity in normal and pathogenic contexts. We demonstrate that photoABPs undergo UV-induced labelling of RNF12 that is consistent with its RING E3 ligase activity. Furthermore, photoABPs robustly report the impact of RNF12 TOKAS variants on E3 activity, including variants within the RING domain and distal non-RING regulatory elements. Finally, we show that this technology can be rapidly deployed in human pluripotent stem cells. In summary, photoABPs are versatile tools that can directly identify disruptions to RING E3 ubiquitin ligase activity in human disease, thereby providing new insight into pathogenic mechanisms.

A novel RLIM/RNF12 variant disrupts protein stability and function to cause severe Tonne-Kalscheuer syndrome.

Tonne-Kalscheuer syndrome (TOKAS) is an X-linked intellectual disability syndrome associated with variable clinical features including craniofacial abnormalities, hypogenitalism and diaphragmatic hernia. TOKAS is caused exclusively by variants in the gene encoding the E3 ubiquitin ligase gene RLIM, also known as RNF12. Here we report identification of a novel RLIM missense variant, c.1262A>G p.(Tyr421Cys) adjacent to the regulatory basic region, which causes a severe form of TOKAS resulting in perinatal lethality by diaphragmatic hernia. Inheritance and X-chromosome inactivation patterns implicate RLIM p.(Tyr421Cys) as the likely pathogenic variant in the affected individual and within the kindred. We show that the RLIM p.(Tyr421Cys) variant disrupts both expression and function of the protein in an embryonic stem cell model. RLIM p.(Tyr421Cys) is correctly localised to the nucleus, but is readily degraded by the proteasome. The RLIM p.(Tyr421Cys) variant also displays significantly impaired E3 ubiquitin ligase activity, which interferes with RLIM function in Xist long-non-coding RNA induction that initiates imprinted X-chromosome inactivation. Our data uncover a highly disruptive missense variant in RLIM that causes a severe form of TOKAS, thereby expanding our understanding of the molecular and phenotypic spectrum of disease severity.

Functional Diversification of SRSF Protein Kinase to Control Ubiquitin-Dependent Neurodevelopmental Signaling.

Conserved protein kinases with core cellular functions have been frequently redeployed during metazoan evolution to regulate specialized developmental processes. The Ser/Arg (SR)-rich splicing factor (SRSF) protein kinase (SRPK), which is implicated in splicing regulation, is one such conserved eukaryotic kinase. Surprisingly, we show that SRPK has acquired the capacity to control a neurodevelopmental ubiquitin signaling pathway. In mammalian embryonic stem cells and cultured neurons, SRPK phosphorylates Ser-Arg motifs in RNF12/RLIM, a key developmental E3 ubiquitin ligase that is mutated in an intellectual disability syndrome. Processive phosphorylation by SRPK stimulates RNF12-dependent ubiquitylation of nuclear transcription factor substrates, thereby acting to restrain a neural gene expression program that is aberrantly expressed in intellectual disability. SRPK family genes are also mutated in intellectual disability disorders, and patient-derived SRPK point mutations impair RNF12 phosphorylation. Our data reveal unappreciated functional diversification of SRPK to regulate ubiquitin signaling that ensures correct regulation of neurodevelopmental gene expression.

Resultados funcionales y radiográficos de la prótesis reversa y la reinserción del troquíter en pacientes con fracturas de húmero proximal / Functional and radiologic outcomes of reverse shoulder arthroplasty and greater tuberosity reattachment in proximal humerus fractures

Objetivo: Comunicar los resultados funcionales y radiográficos de pacientes tratados con prótesis reversa por fracturas complejas. El objetivo secundario fue determinar la relación entre rangos de movilidad y puntaje ASES con la evolución radiográfica del troquíter. Materiales y Métodos: Se incluyeron 16 pacientes >65 años, tratados con prótesis reversa y reinserción del troquíter, entre 2013 y 2017, operados antes de las 4 semanas del trauma y con un seguimiento mínimo de 2 años. Se consignaron el puntaje ASES y el rango de movilidad activa. En las radiografías, se evaluaron la posición y la consolidación del troquíter, y se registraron las complicaciones y su tratamiento. Resultados: La media de la edad fue 74.5 años (RIC 66-78.5), 11 (69%) eran mujeres. Once fracturas (69%) eran a 4 fragmentos y 5, luxofracturas a 4 fragmentos. La media entre el trauma y la cirugía fue 9.4 días y el seguimiento, 29.5 meses. En 9 casos (56%), el troquíter presentó consolidación. Rotación interna: 5 pacientes alcanzaron la región glútea con el pulgar; 4, la vértebra T12; 4, la vértebra L3; 3, la T7. Las medianas de rotación externa y flexión anterior fueron 30° (RIC 17,5-40) y 100° (RIC 87,5-160). El puntaje ASES promedio fue 78,3 (RIC 63,3-87,4). No hubo una asociación estadísticamente significativa entre la evolución del troquíter y la flexión anterior y el puntaje (p = 0,24 y 0,52, respectivamente). Conclusión: La prótesis reversa en fracturas agudas con reinserción de las tuberosidades puede llevar a buenos resultados funcionales. No se encontró relación entre la consolidación del troquíter y el puntaje ASES. Nivel de Evidencia: IV

Objective: To report functional and radiologic outcomes of reverse shoulder arthroplasty (RSA) in patients with complex proximal humeral fractures. A second objective was to assess the relation between the greater tuberosity healing and the range of motion (ROM) and the American Shoulder and Elbow Surgeons (ASES) score. Materials and Methods: Sixteen patients treated between 2013 and 2017, older than 65 years old, operated before 4 weeks after the trauma, and with a minimum of 2-year follow-up were included. ASES scores and active ROMs were recorded. Greater tuberosity and the prosthesis position and healing were radiologically evaluated, and the complications and treatment were recorded. Results: The median age was of 74.5 years (IQR 66-78.5), 11 patients were females (69%). According to Neer classification, 11 cases were four-part fractures and 5 were four-part fracture-dislocations. The average time between trauma and surgery was 9.4 days, and the average follow-up was of 29.5 months. The greater tuberosity was healed in 9 cases (56%). Internal rotation: 5 patients (31.25%) were able to reach up with their thumbs to gluteal level, 4 (25%) to T12, 3 (18.75%) to T7, and 4 (25%) to L3. The medians for external rotation and forward flexion were 30° (IQR 17.5°-40°) and 100° (IQR 87.5°-160°). The average ASES score was of 78.3 (IQR 63.3-87.4). There was no significant statistical relation between greater tuberosity healing and forward flexion or ASES score (P=0.24 and P=0.52, respectively). Conclusion: The use of reverse prostheses for complex fractures with greater tuberosity reattachment could lead to good functional outcomes, low complication rates and reoperations. There was no significant statistical relation between ASES score and greater tuberosity healing or failure to heal. Level of Evidence: IV

Phosphoproteomics identifies a bimodal EPHA2 receptor switch that promotes embryonic stem cell differentiation.

Embryonic Stem Cell (ESC) differentiation requires complex cell signalling network dynamics, although the key molecular events remain poorly understood. Here, we use phosphoproteomics to identify an FGF4-mediated phosphorylation switch centred upon the key Ephrin receptor EPHA2 in differentiating ESCs. We show that EPHA2 maintains pluripotency and restrains commitment by antagonising ERK1/2 signalling. Upon ESC differentiation, FGF4 utilises a bimodal strategy to disable EPHA2, which is accompanied by transcriptional induction of EFN ligands. Mechanistically, FGF4-ERK1/2-RSK signalling inhibits EPHA2 via Ser/Thr phosphorylation, whilst FGF4-ERK1/2 disrupts a core pluripotency transcriptional circuit required for Epha2 gene expression. This system also operates in mouse and human embryos, where EPHA receptors are enriched in pluripotent cells whilst surrounding lineage-specified trophectoderm expresses EFNA ligands. Our data provide insight into function and regulation of EPH-EFN signalling in ESCs, and suggest that segregated EPH-EFN expression coordinates cell fate with compartmentalisation during early embryonic development.

Avance tendinoso no convencional para lesiones en las zonas I y II de Verdan. Nueva técnica quirúrgica / Non-conventional tendon advancement for injuries in Verdan zones I and II. A new surgical technique

Objetivo: Presentar una nueva técnica quirúrgica y los resultados clínicos comparativos de pacientes con roturas de tenorrafia primaria o lesiones crónicas del flexor profundo en las zonas I y II de Verdan, tratados con avance tendinoso no convencional. Ma-teriales y Métodos: Se incluyó a pacientes >18 años, con antecedente de lesiones en las zonas I y II de Verdan y un seguimiento mínimo de 36 meses. La serie estaba formada por 13 pacientes (edad promedio 29 años) que fueron divididos en dos grupos según el tipo de lesión (7 con roturas de tenorrafia primaria y 6 con lesiones crónicas del flexor profundo) y la técnica quirúrgica utilizada (alargamiento en Z más sutura término-terminal y sutura lateral del tendón alargado a un flexor vecino indemne en la zona V, respectivamente). Se empleó la Clasificación de Strickland para la evaluación. Resultados: El seguimiento promedio fue de 51 meses. El intervalo promedio entre la lesión y la cirugía fue de 11.7 semanas. Según la Clasificación de Strickland, 8 pa-cientes tuvieron resultados excelentes; 4, buenos y uno, pobre. El avance tendinoso promedio fue de 20,5 mm en ambos grupos.Conclusiones: El avance tendinoso no convencional para lesiones en las zonas I y II de Verdan, sea en roturas de tenorrafia primaria o lesiones crónicas del flexor profundo, resultó un tratamiento reproducible y eficaz. Nivel de Evidencia: III

Objective: To report a new surgical technique and the comparative clinical outcomes in patients with primary tenorrhaphy rupture or chronic flexor digitorum profundus (FDP) injuries in Verdan zones I and II, treated with non-conventional tendon advancement. Materials and Methods: The study population consisted of 13 patients over 18 years (averaging 29 years) who had had injuries in Verdan zones I and II and at least a 36-month follow-up. Patients were divided into two groups according to injury type (7 cases of primary tenorrhaphy rupture and 6 cases of chronic FDP injuries) and surgical technique (Z-plasty lengthening plus end-to-end suture or lateral suture to an adjacent undamaged FDP tendon at zone-V level, respectively). The results were evaluated according to Strickland's scoring system. Results: The average follow-up was 51 months. Time period between injury and surgery averaged 11.7 weeks (range, 2-24 weeks). Strickland scoring system results: excellent in 8 patients, good in 4 patients, and poor in 1 patient. Study average tendon advancement was 20.5mm. Conclusions: Non-conventional tendon advancement for primary tenorrhaphy ruptures or chronic FDP injuries in Verdan zones I and II proved to be a reproducible and effective treatment. Level of evidence: III

RNF12 X-Linked Intellectual Disability Mutations Disrupt E3 Ligase Activity and Neural Differentiation.

X-linked intellectual disability (XLID) is a heterogeneous syndrome affecting mainly males. Human genetics has identified >100 XLID genes, although the molecular and developmental mechanisms underpinning this disorder remain unclear. Here, we employ an embryonic stem cell model to explore developmental functions of a recently identified XLID gene, the RNF12/RLIM E3 ubiquitin ligase. We show that RNF12 catalytic activity is required for proper stem cell maintenance and neural differentiation, and this is disrupted by patient-associated XLID mutation. We further demonstrate that RNF12 XLID mutations specifically impair ubiquitylation of developmentally relevant substrates. XLID mutants disrupt distinct RNF12 functional modules by either inactivating the catalytic RING domain or interfering with a distal regulatory region required for efficient ubiquitin transfer. Our data thereby uncover a key function for RNF12 E3 ubiquitin ligase activity in stem cell and neural development and identify mechanisms by which this is disrupted in intellectual disability.

Runt-Related Transcription Factor 2 Induction During Differentiation of Wharton's Jelly Mesenchymal Stem Cells to Osteoblasts Is Regulated by Jumonji AT-Rich Interactive Domain 1B Histone Demethylase.

Novel bone regeneration approaches aim to obtain immature osteoblasts from somatic stem cells. Umbilical cord Wharton's jelly mesenchymal stem cells (WJ-MSCs) are an ideal source for cell therapy. Hence, the study of mechanisms involved in WJ-MSC osteoblastic differentiation is crucial to exploit their developmental capacity. Here, we have assessed epigenetic control of the Runt-related transcription factor 2 (RUNX2) osteogenic master regulator gene in WJ-MSC. We present evidence indicating that modulation of RUNX2 expression through preventing Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase activity is relevant to enhance WJ-MSC osteoblastic potential. Hence, JARID1B loss of function in WJ-MSC results in increased RUNX2/p57 expression. Our data highlight JARID1B activity as a novel target to modulate WJ-MSC osteoblastic differentiation with potential applications in bone tissue engineering. Stem Cells 2017;35:2430-2441.

Protein Kinases in Pluripotency-Beyond the Usual Suspects.

Post-translational modification of proteins by phosphorylation plays a key role in regulating all aspects of eukaryotic biology. Embryonic stem cell (ESC) pluripotency, defined as the ability to differentiate into all cell types in the adult body, is no exception. Maintenance and dissolution of pluripotency are tightly controlled by phosphorylation. As a result, key signalling pathways that regulate pluripotency have been identified and their functions well characterised. Amongst the best studied are the fibroblast growth factor (FGF)-ERK1/2 pathway, PI3K-AKT, the leukemia inhibitory factor (LIF)-JAK-STAT3 axis, Wnt-GSK3 signalling, and the transforming growth factor (TGF)ß family. However, these kinase pathways constitute only a small proportion of the protein kinase complement of pluripotent cells, and there is accumulating evidence that diverse phosphorylation systems modulate ESC pluripotency. Here, we review recent progress in understanding the overarching role of phosphorylation in mediating communication from the cellular environment, metabolism, and cell cycle to the core pluripotency machinery.

Epigenetic Signatures at the RUNX2-P1 and Sp7 Gene Promoters Control Osteogenic Lineage Commitment of Umbilical Cord-Derived Mesenchymal Stem Cells.

Wharton's Jelly mesenchymal stem cells (WJ-MSCs) are an attractive potential source of multipotent stem cells for bone tissue replacement therapies. However, the molecular mechanisms involved in their osteogenic conversion are poorly understood. Particularly, epigenetic control operating at the promoter regions of the two master regulators of the osteogenic program, RUNX2/P57 and SP7 has not yet been described in WJ-MSCs. Via quantitative PCR profiling and chromatin immunoprecipitation (ChIP) studies, here we analyze the ability of WJ-MSCs to engage osteoblast lineage. In undifferentiated WJ-MSCs, RUNX2/P57 P1, and SP7 promoters are found deprived of significant levels of the histone post-translational marks that are normally associated with transcriptionally active genes (H3ac, H3K27ac, and H3K4me3). Moreover, the RUNX2 P1 promoter lacks two relevant histone repressive marks (H3K9me3 and H3K27me3). Importantly, RUNX2 P1 promoter is found highly enriched in the H3K4me1 mark, which has been shown recently to mediate gene repression of key regulatory genes. Upon induction of WJ-MSCs osteogenic differentiation, we found that RUNX2/P57, but not SP7 gene expression is strongly activated, in a process that is accompanied by enrichment of activating histone marks (H3K4me3, H3ac, and H3K27ac) at the P1 promoter region. Histone mark analysis showed that SP7 gene promoter is robustly enriched in epigenetic repressive marks that may explain its poor transcriptional response to osteoblast differentiating media. Together, these results point to critical regulatory steps during epigenetic control of WJ-MSCs osteogenic lineage commitment that are relevant for future applications in regenerative medicine. J. Cell. Physiol. 232: 2519-2527, 2017. © 2016 Wiley Periodicals, Inc.

Ck2-Dependent Phosphorylation Is Required to Maintain Pax7 Protein Levels in Proliferating Muscle Progenitors.

Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate.

Bromfenac 0.09% bioavailability in aqueous humor, prophylactic effect on cystoid macular edema, and clinical signs of ocular inflammation after phacoemulsification in a Mexican population.

PURPOSE: The purpose of this study was to evaluate the aqueous humor bioavailability and clinical efficacy of bromfenac 0.09% vs nepafenac on the presence of cystoid macular edema (CME) after phacoemulsification. MATERIAL AND METHODS: A Phase II, double-blind, masked, active-controlled, multicenter, clinical trial of 139 subjects, randomized to either a bromfenac 0.09% ophthalmic solution (n=69) or nepafenac 0.1% (n=70). Subjects instilled a drop three times a day for a period of 30 days. Follow-up visits were on days 2, 7, 15, 30, and 60. Biomicroscopy, clinical ocular signs, and assessment of posterior segment were performed. The primary efficacy endpoints included the presence of CME evaluated by optical coherence tomography. Safety evaluation included intraocular pressure, transaminase enzymes, lissamine green, and fluorescein stain. RESULTS: The demographic and efficacy variables were similar between groups at baseline. The presence of pain, photophobia, conjunctival hyperemia, chemosis, cellularity, and corneal edema disappeared by day 30 in both groups. The central retinal thickness did not show significant changes after treatment when compared to baseline as follows: in the bromfenac group (247.2±32.9 vs 252.0±24.9 µm; P=0.958) and in nepafenac group (250.8±34 vs 264.0±34.1 µm; P=0.137), respectively. A statistically significant difference was observed between bromfenac and nepafenac group: (252.0±24.9 vs 264.0±34.1 µm; P=0.022), at day 30, respectively; even though there was no clinical relevance in the presentation of CME. There were no significant alterations in intraocular pressure, either lissamine green or fluorescein stains. The adverse events were not related to the interventions. CONCLUSION: Bromfenac 0.09% ophthalmic solution showed similar clinical efficacy to reduce the presentation of CME after phacoemulsification compared to nepafenac 0.01%.

NEDD4 Regulates PAX7 Levels Promoting Activation of the Differentiation Program in Skeletal Muscle Precursors.

The transcription factor Pax7 regulates skeletal muscle stem cell (satellite cells) specification and maintenance through various mechanisms, including repressing the activity of the muscle regulatory factor MyoD. Hence, Pax7-to-MyoD protein ratios can determine maintenance of the committed-undifferentiated state or activation of the differentiation program. Pax7 expression decreases sharply in differentiating myoblasts but is maintained in cells (re)acquiring quiescence, yet the mechanisms regulating Pax7 levels based on differentiation status are not well understood. Here we show that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4. Our results indicate that Nedd4 is expressed in quiescent and activated satellite cells, that Nedd4 and Pax7 physically interact during early muscle differentiation-correlating with Pax7 ubiquitination and decline-and that Nedd4 loss of function prevented this effect. Furthermore, even transient nuclear accumulation of Nedd4 induced a drop in Pax7 levels and precocious muscle differentiation. Consequently, we propose that Nedd4 functions as a novel Pax7 regulator, which activity is temporally and spatially controlled to modulate the Pax7 protein levels and therefore satellite cell fate.