Lack of nuclear localization of the Creb3l1 transcription factor causes defects in caudal fin bifurcation in zebrafish Danio rerio.

The formation of normal bone and bone healing require the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after fracture. Creb3l1 undergoes regulated intra-membrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes. To expand our understanding of Creb3l1 role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish D. rerio caused a frame-shift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ doesn't regulate transcription, and the creb3l1TA+/TA+ fish animals are creb3l1 transcriptional nulls. We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased capacity for regeneration, and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates. Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway, and document a novel role of Creb3l1 in tissue patterning.

TGFß signaling is required for sclerotome resegmentation during development of the spinal column in Gallus gallus.

We previously showed the importance of TGFß signaling in development of the mouse axial skeleton. Here, we provide the first direct evidence that TGFß signaling is required for resegmentation of the sclerotome using chick embryos. Lipophilic fluorescent tracers, DiO and DiD, were microinjected into adjacent somites of embryos treated with or without TGFßRI inhibitors, SB431542, SB525334 or SD208, at developmental day E2.5 (HH16). Lineage tracing of labeled cells was observed over the course of 4 days until the completion of resegmentation at E6.5 (HH32). Vertebrae were malformed and intervertebral discs were small and misshapen in inhibitor injected embryos. Hypaxial myofibers were also increased in thickness after treatment with the inhibitor. Inhibition of TGFß signaling resulted in alterations in resegmentation that ranged between full, partial, and slanted shifts in distribution of DiO or DiD labeled cells within vertebrae. Patterning of rostro-caudal markers within sclerotome was disrupted at E3.5 after treatment with TGFßRI inhibitor with rostral domains expressing both rostral and caudal markers. We propose that TGFß signaling regulates rostro-caudal polarity and subsequent resegmentation in sclerotome during spinal column development.

Tgfbr2 in Dental Pulp Cells Guides Neurite Outgrowth in Developing Teeth.

Transforming growth factor ß (TGFß) plays an important role in tooth morphogenesis and mineralization. During postnatal development, the dental pulp (DP) mesenchyme secretes neurotrophic factors that guide trigeminal nerve fibers into and throughout the DP. This process is tightly linked with dentin formation and mineralization. Our laboratory established a mouse model in which Tgfbr2 was conditionally deleted in DP mesenchyme using an Osterix promoter-driven Cre recombinase (Tgfbr2 cko ). These mice survived postnatally with significant defects in bones and teeth, including reduced mineralization and short roots. Hematoxylin and eosin staining revealed reduced axon-like structures in the mutant mice. Reporter imaging demonstrated that Osterix-Cre activity within the tooth was active in the DP and derivatives, but not in neuronal afferents. Immunofluorescence staining for ß3 tubulin (neuronal marker) was performed on serial cryosections from control and mutant molars on postnatal days 7 and 24 (P7, P24). Confocal imaging and pixel quantification demonstrated reduced innervation in Tgfbr2 cko first molars at both stages compared to controls, indicating that signals necessary to promote neurite outgrowth were disrupted by Tgfbr2 deletion. We performed mRNA-Sequence (RNA-Seq) and gene onotology analyses using RNA from the DP of P7 control and mutant mice to investigate the pathways involved in Tgfbr2-mediated tooth development. These analyses identified downregulation of several mineralization-related and neuronal genes in the Tgfbr2 cko DP compared to controls. Select gene expression patterns were confirmed by quantitative real-time PCR and immunofluorescence imaging. Lastly, trigeminal neurons were co-cultured atop Transwell filters overlying primary Tgfbr2 f/f DP cells. Tgfbr2 in the DP was deleted via Adenovirus-expressed Cre recombinase. Confocal imaging of axons through the filter pores showed increased axonal sprouting from neurons cultured with Tgfbr2-positive DP cells compared to neurons cultured alone. Axon sprouting was reduced when Tgfbr2 was knocked down in the DP cells. Immunofluorescence of dentin sialophosphoprotein in co-cultured DP cells confirmed reduced mineralization potential in cells with Tgfbr2 deletion. Both our proteomics and RNA-Seq analyses indicate that axonal guidance cues, particularly semaphorin signaling, were disrupted by Tgfbr2 deletion. Thus, Tgfbr2 in the DP mesenchyme appears to regulate differentiation and the cells' ability to guide neurite outgrowth during tooth mineralization and innervation.

Mechanisms of TGFß in prostaglandin synthesis and sperm guidance in Caenorhabditis elegans.

BACKGROUND: The transparent epidermis of Caenorhabditis elegans makes it an attractive model to study sperm motility and migration within an intact reproductive tract. C elegans synthesize specific F-series prostaglandins (PGFs) that are important for guiding sperm toward the spermatheca. These PGFs are synthesized from polyunsaturated fatty acid (PUFA) precursors, such as arachidonic acid (AA), via a novel pathway, independent of the classical cyclooxygenases (Cox) responsible for most PG synthesis. While the enzyme(s) responsible for PG synthesis has yet to be identified, the DAF-7 TGFß pathway has been implicated in modulating PG levels and sperm guidance. RESULTS: We find that the reduced PGF levels in daf-1 type I receptor mutants are responsible for the sperm guidance defect. The lower level of PGs in daf-1 mutants is due in part to the inaccessibility of AA. Finally, lipid analysis and assessment of sperm guidance in daf-1;daf-3 double mutants suggest DAF-3 suppresses PG production and sperm accumulation at the spermatheca. Our data suggest that DAF-3 functions in the nervous system, and possibly the germline, to affect sperm guidance. CONCLUSION: The C elegans TGFß pathway regulates many pathways to modulate PG metabolism and sperm guidance. These pathways likely function in the nervous system and possibly the germline.

Canonical and noncanonical TGF-ß signaling regulate fibrous tissue differentiation in the axial skeleton.

Previously, we showed that embryonic deletion of TGF-ß type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-ß regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-ß stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-ß did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-ß-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-ß-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-ß treatment. Next, the role of several noncanonical TGF-ß pathways were tested. We found that ERK1/2 was activated by TGF-ß and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-ß regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFß-mediated fibrous differentiation in sclerotome.

Drying kinetics of Bixa orellana Labil (annatto) leaves and the influence of temperature on the physicochemical and biological properties of its essential oil / Cinética de secado de las hojas de Bixa orellana Labil (annatto) e influencia de la temperatura sobre las propiedades fisicoquímicas y biológicas de su aceite esencial

SUMMARY Bixa orellana L. is widely used as a dye, and other properties are still little studied, especially in relation to leaves. This article evaluates how drying B. orellana L. influences the properties of essential oils (EOs) extracted from their leaves. The collected plant material was submitted to convective air-drying oven at temperatures of 35, 45 and 55 °C. Chemical profiles were determined by Gas Chromatography-Mass Spectrometry (GC-MS). Mathematical models were applied to represent the drying process and statistical analysis performed using Statistica 10 software. The EO's were obtained through the hydrodistillation technique with verification of physicochemical properties and antimicrobial activity by the Disc Diffusion Method. For the toxicity test, the bioassay was applied to Artemia salina. Through the results obtained it was possible to determine that the mathematical model of Verma was the one that best fitted the experimental data. Significant differences in the properties of EO's were observed. The temperature of 45 °C allowed obtaining the best EO yield, still presenting the most efficient antimicrobial activity. This study states through the activities analyzed that the drying temperature influences the physicochemical and biological properties of the EO's, thus requiring studies such as this one that evaluate the best mathematical model to predict drying as well as the specific temperatures that influence the properties of the product obtained.

RESUMEN Bixa orellana L. es ampliamente utilizado como un tinte y tiene otras propiedades que han sido poco estudiadas, especialmente en relación con las hojas. Este artículo evalúa cómo el secado B. orellana L. influye en las propiedades de los aceites esenciales (EOs) extraídos de sus hojas. El material vegetal recogido se sometió al horno de secado por aire convectivo a temperaturas de 35, 45 y 55 °C. Los perfiles químicos fueron determinados por GC-MS. Se aplicaron modelos matemáticos para representar el proceso de secado y el análisis estadístico realizado utilizando el software Statistica 10. Las EO se obtuvieron a través de la técnica de hidrodestilación con verificación de propiedades fisicoquímicas y actividad antimicrobiana. Para el ensayo de toxicidad se aplicó el bioensayo a Artemia salina. A través de los resultados obtenidos fue posible determinar el modelo matemático que se ajustaba mejor. Se observaron diferencias significativas en las propiedades de los EO. La temperatura de 45 °C permitió obtener el mejor rendimiento de EO, porque la actividad antimicrobiana fue más eficiente. Este estudio afirma a través de las actividades analizadas que la temperatura de secado influye en las propiedades fisicoquímicas y biológicas de las EO. Estudios como este evalúan el mejor modelo matemático para predecir el secado, así como las temperaturas específicas que influyen en las propiedades del producto obtenido.

Caracterização química, atividade antimicrobiana e toxicidade dos óleos essenciais da Pimenta dioica L. (pimenta da Jamaica) e Citrus sinensis L. Osbeck (laranja doce) / Chemical characterization, antimicrobial activity, and toxicity of the essential oils of Pimenta dioica L. and Citrus sinensis L. Osbeck (laranja doce)

RESUMO Este estudo avaliou a toxicidade e a atividade antimicrobiana frente a Escherichia coli e Staphylococcus aureus dos óleos essenciais de Pimenta dioica Lindl. e Citrus sinensis L. Os óleos essenciais (OE) foram extraídos por hidrodestilação, com caracterização química através de cromatografía gasosa acoplada a espectrometria de massas (CG-EM). Os parâmetros físico-químicos foram determinados de acordo com a Farmacopeia Brasileira. O ensaio de toxicidade seguiu o bioensaio com Artemia salina Leach, os OE aprovados neste ensaio seguiram para avaliação das suas propriedades biológicas. A atividade antimicrobiana seguiu a metodologia descrita pelo Clinical and Laboratory Standards Institute utilizando o método de difusão de disco, diluição em caldo para concentração inibitória mínima (CIM) e posterior concentração bactericida mínima para avaliar a ação dos OE frente a Escherichia coli e Staphylococcus aureus. Ambos os OE apresentaram toxicidade baixa, e assim foram avaliados quanto as propriedades biológicas antimicrobianas. Ambos os OE apresentaram potenciais bactericidas frente aos microrganismos testados, exibindo resultados satisfatórios para a ação deles. Os resultados indicam que os OE avaliados são compostos por substâncias que propiciam e incentivam sua aplicação em virtude de seus potenciais para atividade biológicas moluscicida e antimicrobiana.

SUMMARY This study evaluated the toxicity and antimicrobial activity in the face of Escherichia coli and Staphylococcus aureus of essential oils of Pimenta dioica Lindl. and Citrus sinensis L. The essential oils (EO) were extracted by hydrodistillation, with chemical characterization by gas chromatography coupled and mass spectrometry (GC-MS). Physicochemical parameters were determined according to the Brazilian Pharmacopeia. The toxicity test followed the bioassay with Artemia salina Leach, the EO approved in this assay followed to evaluate its biological properties. The antimicrobial activity followed the methodology described by the Clinical and Laboratory Standards Institute using the disc diffusion method, broth dilution for minimum inhibitory concentration (MIC) and subsequent minimum bactericide concentration for to evaluate the action of EO against Escherichia coli and Staphylococcus aureus. Both OE showed low toxicity, and thus were evaluated for the biological antimicrobial properties. Both OE presented bactericidal potential against the microorganisms tested, showing satisfactory results for their action. The results indicate that the evaluated OE are composed of substances that provide and encourage their application due to their potentials for biological molluscicide and antimicrobial activity.

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

Transcriptomic Profiling of DAF-7/TGFß Pathway Mutants in C. elegans.

The transforming growth factor beta superfamily encompasses a large family of ligands that are well conserved across many organisms. They are regulators of a number of physiological and pathological processes. The model nematode Caenorhabditis elegans has been instrumental in identifying key components of the transforming growth factor beta (TGFß) pathway. In C. elegans, the TGFß homolog DAF-7 signals through the DAF-1 Type I and DAF-4 Type II receptors to phosphorylate downstream R-SMADs DAF-8 and DAF-14. These R-SMADs translocate into the nucleus to inhibit Co-SMAD DAF-3. Many of the roles of the canonical DAF-7 pathway, involving both DAF-1 and DAF-3, have been identified using targeted genetic studies. Few have assessed the global transcriptomic changes in response to these genes, especially in adult animals. In this study, we performed RNA sequencing on wild type, daf-1, and daf-1; daf-3 adult hermaphrodites. To assess the overall trends of the data, we identified differentially expressed genes (DEGs) and performed gene ontology analysis to identify the types of downstream genes that are differentially expressed. Hierarchical clustering showed that the daf-1; daf-3 double mutants are transcriptionally more similar to wild type than daf-1 mutants. Analysis of the DEGs showed a disproportionally high number of genes whose expression is increased in daf-1 mutants, suggesting that DAF-1 acts as a general repressor of gene expression in wild type animals. Gene ontology analysis of the DEGs produced many significantly enriched terms, including Molting Cycle, Response to Topologically Incorrect Protein, and Response to Biotic Stimulus. Understanding the direct and indirect targets of the DAF-7 TGFß pathway through this RNA-seq dataset can provide insight into novel roles of the multifunctional signaling pathway, as well as identify novel genes that may participate in previously reported functions of TGFß signaling.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies - Secondary Publication.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2020 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:485-502, 2020.

American Society for Bone and Mineral Research-Orthopaedic Research Society Joint Task Force Report on Cell-Based Therapies.

Cell-based therapies, defined here as the delivery of cells in vivo to treat disease, have recently gained increasing public attention as a potentially promising approach to restore structure and function to musculoskeletal tissues. Although cell-based therapy has the potential to improve the treatment of disorders of the musculoskeletal system, there is also the possibility of misuse and misrepresentation of the efficacy of such treatments. The medical literature contains anecdotal reports and research studies, along with web-based marketing and patient testimonials supporting cell-based therapy. Both the American Society for Bone and Mineral Research (ASBMR) and the Orthopaedic Research Society (ORS) are committed to ensuring that the potential of cell-based therapies is realized through rigorous, reproducible, and clinically meaningful scientific discovery. The two organizations convened a multidisciplinary and international Task Force composed of physicians, surgeons, and scientists who are recognized experts in the development and use of cell-based therapies. The Task Force was charged with defining the state-of-the art in cell-based therapies and identifying the gaps in knowledge and methodologies that should guide the research agenda. The efforts of this Task Force are designed to provide researchers and clinicians with a better understanding of the current state of the science and research needed to advance the study and use of cell-based therapies for skeletal tissues. The design and implementation of rigorous, thorough protocols will be critical to leveraging these innovative treatments and optimizing clinical and functional patient outcomes. In addition to providing specific recommendations and ethical considerations for preclinical and clinical investigations, this report concludes with an outline to address knowledge gaps in how to determine the cell autonomous and nonautonomous effects of a donor population used for bone regeneration. © 2019 American Society for Bone and Mineral Research.

Scaffoldless tissue-engineered cartilage for studying transforming growth factor beta-mediated cartilage formation.

Reduced transforming growth factor beta (TGF-ß) signaling is associated with osteoarthritis (OA). TGF-ß is thought to act as a chondroprotective agent and provide anabolic cues to cartilage, thus acting as an OA suppressor in young, healthy cartilage. A potential approach for treating OA is to identify the factors that act downstream of TGF-ß's anabolic pathway and target those factors to promote cartilage regeneration or repair. The aims of the present study were to (a) develop a scaffoldless tissue-engineered cartilage model with reduced TGF-ß signaling and disrupted cartilage formation and (b) validate the system for identifying the downstream effectors of TGF-ß that promote cartilage formation. Sox9 was used to validate the model because Sox9 is known to promote cartilage formation and TGF-ß regulates Sox9 activity. Primary bovine articular chondrocytes were grown in Transwell supports to form cartilage tissues. An Alk5/TGF-ß type I receptor inhibitor, SB431542, was used to attenuate TGF-ß signaling, and an adenovirus encoding FLAG-Sox9 was used to drive the expression of Sox9 in the in vitro-generated cartilage. SB431542-treated tissues exhibited reduced cartilage formation including reduced thicknesses and reduced proteoglycan staining compared with control tissue. Expression of FLAG-Sox9 in SB431542-treated cartilage allowed the formation of cartilage despite antagonism of the TGF-ß receptor. In summary, we developed a three-dimensional in vitro cartilage model with attenuated TGF-ß signaling. Sox9 was used to validate the model for identification of anabolic agents that counteract loss of TGF-ß signaling. This model has the potential to identify additional anabolic factors that could be used to repair or regenerate damaged cartilage.

Development of the axial skeleton and intervertebral disc.

Development of the axial skeleton is a complex, stepwise process that relies on intricate signaling and coordinated cellular differentiation. Disruptions to this process can result in a myriad of skeletal malformations that range in severity. The notochord and the sclerotome are embryonic tissues that give rise to the major components of the intervertebral discs and the vertebral bodies of the spinal column. Through a number of mouse models and characterization of congenital abnormalities in human patients, various growth factors, transcription factors, and other signaling proteins have been demonstrated to have critical roles in the development of the axial skeleton. Balance between opposing growth factors as well as other environmental cues allows for cell fate specification and divergence of tissue types during development. Furthermore, characterization of progenitor cells for specific cell lineages has furthered the understanding of specific spatiotemporal cues that cells need in order to initiate and complete development of distinct tissues. Identifying specific marker genes that can distinguish between the various embryonic and mature cell types is also of importance. Clinically, understanding developmental clues can aid in the generation of therapeutics for musculoskeletal disease through the process of developmental engineering. Studies into potential stem cell therapies are based on knowledge of the normal processes that occur in the embryo, which can then be applied to stepwise tissue engineering strategies.

Antagonism of BMP signaling is insufficient to induce fibrous differentiation in primary sclerotome.

Sclerotome is the embryonic progenitor of the axial skeleton. It was previously shown that Tgfbr2 is required in sclerotome for differentiation of fibrous skeletal tissues including the annulus fibrosus of the intervertebral disc. Alternatively, BMP signaling is required to form the vertebral body through chondrogenesis. In addition, TGFß added to sclerotome cultures induces expression of markers for fibrous tissue differentiation but not cartilage or bone. The mechanism of how TGFß signaling regulates this lineage decision in sclerotome is not known and could be due to the production of instructive or inhibitory signals or a combination of the two. Here we show that TGFß antagonizes BMP/ Smad1/5 signaling in primary sclerotome likely through regulation of Noggin, an extracellular BMP antagonist, to prevent chondrogenesis. We then tested whether inhibition of BMP signaling, and inhibition of chondrogenesis, is sufficient to push cells toward the fibrous cell fate. While Noggin inhibited BMP/ Smad1/5 signaling and the formation of chondrogenic nodules in sclerotome cultures; Noggin and inhibition of BMP signaling through Gremlin or DMH2 were insufficient to induce fibrous tissue differentiation. The results suggest inhibition of BMP signaling is not sufficient to stimulate fibrous tissue differentiation and additional signals are likely required. We propose that TGFß has a dual role in regulating sclerotome fate. First, it inhibits BMP signaling potentially through Noggin to prevent chondrogenesis and, second, it provides an unknown instructive signal to promote fibrous tissue differentiation in sclerotome. The results have implications for the design of stem cell-based therapies for skeletal diseases.

Prg4 prevents osteoarthritis induced by dominant-negative interference of TGF-ß signaling in mice.

OBJECTIVE: Prg4, also known as Lubricin, acts as a joint/boundary lubricant. Prg4 has been used to prevent surgically induced osteoarthritis (OA) in mice. Surgically induced OA serves as a good model for post-traumatic OA but is not ideal for recapitulating age-related OA. Reduced expression of the TGF-ß type II receptor (TGFßR2) is associated with age-related OA in clinical samples, so we previously characterized a mouse model that exhibits OA due to expression of a mutated dominant-negative form of TGFßR2 (DNIIR). Prg4 expression was significantly reduced in DNIIR mice. Furthermore, we showed that Prg4 was a transcriptional target of TGF-ß via activation of Smad3, the main signal transducing protein for TGF-ß. The objective of the present study was to determine whether maintenance of Prg4, a down-stream transcriptional target of TGF-ß, prevents OA associated with attenuated TGF-ß signaling in mice. DESIGN: Wild-type, DNIIR, and bitransgenic mice that express both DNIIR and Prg4, were compared. Mice were assessed with a foot misplacement behavioral test, µCT, histology, and Western blot. RESULTS: Compared to DNIIR mice, bitransgenic DNIIR+Prg4 mice missed 1.3 (0.4, 2.1) fewer steps while walking (mean difference (95% confidence interval)), exhibited a cartilage fibrillation score that was 1.8 (0.4, 3.1) points lower, exhibited cartilage that was 28.2 (0.5, 55.9) µm thicker, and exhibited an OARSI score that was 6.8 (-0.9, 14.5) points lower. However, maintenance of Prg4 expression did not restore levels of phosphorylated Smad3 in DNIIR mice, indicating Prg4 does not simply stimulate TGF-ß signaling. CONCLUSIONS: Our results indicate that maintenance of Prg4 expression prevents OA progression associated with reduced TGF-ß signaling in mice. Since there was no evidence that Prg4 acts by stimulating the TGF-ß signaling cascade, we propose that Prg4, a transcriptional target of TGF-ß, attenuates OA progression through its joint lubrication function.

IVD Development: Nucleus pulposus development and sclerotome specification.

PURPOSE OF REVIEW: Intervertebral discs (IVD) are derived from embryonic notochord and sclerotome. The nucleus pulposus is derived from notochord while other connective tissues of the spine are derived from sclerotome. This manuscript will review the past 5 years of research into IVD development. RECENT FINDINGS: Over the past several years, advances in understanding the step-wise process that govern development of the nucleus pulposus and the annulus fibrosus have been made. Generation of tissues from induced or embryonic stem cells into nucleus pulposus and paraxial mesoderm derived tissues has been accomplished in vitro using pathways identified in normal development. A balance between BMP and TGF-ß signaling as well as transcription factors including Pax1/Pax9, Mkx and Nkx3.2 appear to be very important for cell fate decisions generating tissues of the IVD. SUMMARY: Understanding how the IVD develops will provide the foundation for future repair, regeneration, and tissue engineering strategies for IVD disease.

Tgfbr2 is required in Acan-expressing cells for maintenance of the intervertebral and sternocostal joints.

BACKGROUND: Members of the transforming growth factor beta (TGF-ß) family are secreted proteins that regulate skeletal development. TGF-ß signaling is critical in embryonic development of the annulus fibrosus (AF) of the intervertebral disc (IVD). To address the question of the role of TGF-ß signaling in postnatal development and maintenance of the skeleton, we generated mice in which Tgfbr2 was deleted at 2-weeks of age in Aggrecan (Acan)-expressing cells using inducible Cre/LoxP recombination. METHODS: Localization of Cre recombination was visualized by crossing Acantm1(cre/ERT2)Crm mice to fluorescent mTmG reporter mice. Acantm1(cre/ERT2)Crm mice were mated to Tgfbr2 LoxP/LoxP mice and Cre recombinase was activated by tamoxifen injection at 2-weeks postnatally. Following tamoxifen injection, mice were aged to 3, 6, and 12-months and control mice were compared to the experimental (cKO) group. Mice were initially analyzed using X-ray and skeletal preparations. Sternocostal joints and IVD tissues were further analyzed histologically by hematoxylin and eosin (H&E), Safranin O, and Picrosirius Red staining as well as Col10 immunostaining. RESULTS: Cre recombination was observed in the IVD and sternocostal joints. X-ray analysis revealed osteophyte formation within the disc space of 12-month-old cKO mice. Skeletal preparations confirmed calcification within the IVD and the sternocostal joints in cKO mice. H&E staining of cKO IVD revealed disorganized growth plates, delay in the formation of the bony endplate, and Col10 staining in the AF indicative of ectopic endochondral bone formation. Furthermore, proteoglycan loss was observed and collagen bundles within the inner AF were thinner and less organized. Alterations in the IVD were apparent beginning at 3 months and were progressively more visible at 6 and 12 months. Similarly, histological analysis of cKO sternocostal joints revealed joint calcification, proteoglycan loss, and disorganization of the collagen architecture at 12 months of age. CONCLUSIONS: TGF-ß signaling is important for postnatal development and maintenance of fibrocartilaginous IVD and sternocostal joints.

Mechanical loading regulates organization of the actin cytoskeleton and column formation in postnatal growth plate.

Longitudinal growth of bones occurs at the growth plates where chondrocytes align into columns that allow directional growth. Little is known about the mechanisms controlling the ability of chondrocytes to form columns. We hypothesize that mechanical load and the resulting force on chondrocytes are necessary during active growth for proper growth plate development and limb length. To test this hypothesis, we created a mouse model in which a portion of the sciatic nerve from one hind limb was transected at postnatal day 8 to cause paralysis to that limb. At 6 and 12 wk postsurgery, the hind limb had significantly less bone mineral density than contralateral controls, confirming reduced load. At 8 and 14 wk postsurgery, tibiae were significantly shorter than controls. The paralyzed growth plate showed disruptions to column organization, with fewer and shorter columns. Polarized light microscopy indicated alterations in collagen fiber organization in the growth plate. Furthermore, organization of the actin cytoskeleton in growth plate chondrocytes was disrupted. We conclude that mechanical load and force on chondrocytes within the growth plate regulate postnatal development of the long bones.

Desarrollo competencial y clima organizacional para el desempeño de una práctica avanzada de los profesionales de Enfermería / Competency development and organizational environment for performance of advanced practice by Nursing professionals

Objetivo: evaluar el grado de desarrollo del nivel competencial de las enfermeras de las Illes Balears relacionado con la autopercepción de los factores individuales para el desarrollo de una práctica basada en la evidencia y cultura y factores organizacionales. Método: se llevó a cabo un estudio descriptivo transversal sobre la población de profesionales de Enfermería colegiados de las Illes Balears, basado en una participación voluntaria en un proceso de autoevaluación mediante encuesta online sobre la plataforma Limesurvey ®. Las escalas de medida fueron tres escalas de competencias generales, específicas y avanzadas de Enfermería para el análisis del desarrollo competencial, así como el Nursing Work Index para medir el clima organizacional. Resultados: 600 enfermeras y enfermeros de diversos ámbitos asistenciales completaron satisfactoriamente la encuesta. El análisis arrojó resultados globales de un clima organizacional con puntuaciones moderadas-bajas, mientras que los resultados de las competencias generales y específicas puntuaban aceptablemente frente a las competencias avanzadas que puntuaron bajo en general. Discusión y conclusiones: existe una relación directa entre años de profesión y empeoramiento de la percepción del contexto organizacional. Por otro lado, y con independencia del ámbito asistencial, las competencias generales y específicas con las que los profesionales egresan de la universidad se mantienen en niveles medios-altos a lo largo de los años de profesión. No ocurre así con las competencias avanzadas que en general puntúan bajo y se ven influencias de manera directamente proporcional por el clima organizacional (AU)

Objectives: to evaluate the degree of development of the competency level of nurses in the Balearic Islands regarding selfperception of individual factors for the development of a practice based on evidence and culture and organizational factors. Method: a descriptive transversal study on the population of the Nursing professionals registered in the Balearic Islands, based on voluntary participation in a self-assessment process through online survey with the Limesurvey® platform. The measurement scales were three scales for overall, specific and advanced competencies in Nursing for the analysis of competency development, as well as the Nursing Work Index in order to measure the organizational environment. Results: six hundred (600) female and male nurses from different healthcare settings completed successfully the survey. The analysis showed overall results of an organizational environment with moderate-low scores, while general and specific competencies showed an acceptable score vs. advanced competencies, with overall low scores. Discussion and conclusions: there is a direct relationship between years in the profession and a worse perception of the organizational setting. On the other hand, and regardless of the healthcare setting, the overall and specific competencies which professionals have when they complete their University studies are maintained in medium-high levels throughout their years in the profession. This is not the case with advanced competencies, which show an overall low score, and have a directly proportional influence by the organisational environment (AU)