Protocol for chromatin accessibility profiling of human endothelial cells cultured under fluid shear stress using ATAC-seq.

Chromatin accessibility influences gene regulation and can be quantified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Recapitulating in vivo fluid shear stress (FSS) mechano-regimes in vitro allows the study of atheroprone and atheroprotective mechanisms. In this protocol, we show how to culture and harvest endothelial cells from microfluidic channels for the preparation of ATAC-seq, highlighting optional growth factor stimulation and different FSS rates. This extends the application of ATAC-seq to the analysis of in vitro mechanically stimulated cells. For complete details on the use and execution of this protocol, please refer to Jatzlau et al.1.

Fluid shear stress-modulated chromatin accessibility reveals the mechano-dependency of endothelial SMAD1/5-mediated gene transcription.

Bone morphogenetic protein (BMP) signaling and fluid shear stress (FSS) mediate complementary functions in vascular homeostasis and disease development. It remains to be shown whether altered chromatin accessibility downstream of BMP and FSS offers a crosstalk level to explain changes in SMAD-dependent transcription. Here, we employed ATAC-seq to analyze arterial endothelial cells stimulated with BMP9 and/or FSS. We found that BMP9-sensitive regions harbor non-palindromic GC-rich SMAD-binding elements (GGCTCC) and 69.7% of these regions become BMP-insensitive in the presence of FSS. While GATA and KLF transcription factor (TF) motifs are unique to BMP9- and FSS-sensitive regions, respectively, SOX motifs are common to both. Finally, we show that both SOX(13/18) and GATA(2/3/6) family members are directly upregulated by SMAD1/5. These findings highlight the mechano-dependency of SMAD-signaling by a sequential mechanism of first elevated pioneer TF expression, allowing subsequent chromatin opening to eventually providing accessibility to novel SMAD binding sites.

The Florida Clinical Skills Collaborative: A New Regional Consortium for the Assessment of Clinical Skills.

Discontinuation of the United States Medical Licensing Examination (USMLE) Step 2 Clinical Skills (CS) exam and Comprehensive Osteopathic Medical Licensing Examination (COMLEX) Level 2 Performance Evaluation (2-PE) raised questions about the ability of medical schools to ensure the clinical skills competence of graduating students. In February 2021, representatives from all Florida, United States, allopathic and osteopathic schools initiated a collaboration to address this critically important issue in the evolving landscape of medical education. A 5-point Likert scale survey of all members (n=18/20 individuals representing 10/10 institutions) reveals that initial interest in joining the collaboration was high among both individuals (mean 4.78, SD 0.43) and institutions (mean 4.69, SD 0.48). Most individuals (mean 4.78, SD 0.55) and institutions (mean 4.53, SD 0.72) are highly satisfied with their decision to join. Members most commonly cited a "desire to establish a shared assessment in place of Step 2 CS/2-PE" as their most important reason for joining. Experienced benefits of membership were ranked as the following: 1) Networking, 2) Shared resources for curriculum implementation, 3) Scholarship, and 4) Work towards a shared assessment in place of Step 2 CS/2-PE. Challenges of membership were ranked as the following: 1) Logistics such as scheduling and technology, 2) Agreement on common goals, 3) Total time commitment, and 4) Large group size. Members cited the "administration of a joint assessment pilot" as the highest priority for the coming year. Florida has successfully launched a regional consortium for the assessment of clinical skills competency with high levels of member satisfaction which may serve as a model for future regional consortia.

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes.

BACKGROUND: Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions. RESULTS: We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress. CONCLUSIONS: We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions.

Developing Awareness and Allies: Simulating Difficult Conversations about Microaggressions for Faculty and Students.

OBJECTIVES: Discrimination and bias in clinical training often take the form of microaggressions, which, albeit unintentional, are detrimental to the learning environment and well-being of students. Although there are a few reports of medical schools training students to respond to microaggressions, none have included a complementery student-led faculty training module. The aim of this study was to develop and evaluate a case-based approach to improving student resilience and increasing faculty awareness of microaggressions in the clinical setting. METHODS: We created four realistic cases of microaggressions and uncomfortable conversations, based on students' experiences on the wards, to implement training for incoming third-year students and their core faculty. Standardized patients were trained to effectively portray discriminatory faculty, residents, and patients. Institutional review board-approved surveys were administered and statistically analyzed to evaluate for efficacy. RESULTS: Students had greater mean confidence scores for responding to microaggressions immediately and at 6 months after the sessions (P < 0.05). Faculty showed improved mean confidence and understanding of the definition of a microaggression (P < 0.05). CONCLUSIONS: This approach had results similar to other studies, with the additional benefit of training faculty with the same scenarios. We believe that this method helped bridge the gap between students' notions of discrimination and faculty understanding of microaggressions.

Visualization and Quantification of TGFß/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay.

Transforming Growth Factor ß (TGFß)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGFß/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGFß/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA). Active TGFß/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGFß and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy. The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGFß/BMP signaling in ECs, both quantitatively and spatially.

Differential Impact of Fluid Shear Stress and YAP/TAZ on BMP/TGF-ß Induced Osteogenic Target Genes.

Bone is a remarkable dynamic structure, which integrates mechanical and biochemical signaling inputs. Interstitial fluid in the intramedullary space transmits signals derived from compression-induced fluid shear stress (FSS) to stimulate osteoblasts for bone formation. Using a flow system and human osteoblasts, this study demonstrates how BMP/TGF-ß  signaling integrates stimuli derived from FSS and YAP/TAZ and confirms these findings by transcriptome analyses. Here, FSS positively affects the phosphorylation of both SMAD1/5 and SMAD2/3, the respective BMP- and TGFß-R-SMADs. Increase in phosphorylated SMAD1/5 levels affects distinct target genes, which are susceptible to low levels of phosphorylated SMADs (such as ID1-3) or dependent on high levels of phosphorylated SMAD1/5 (NOG, noggin). Thus, FSS lowers the threshold for genes dependent on high levels of phosphorylated SMAD1/5 when less BMP is available. While the impact of FSS on direct BMP target genes is independent of YAP/TAZ, FSS acts cooperatively with YAP/TAZ on TGF-ß  target genes, which are shared by both pathways (such as CTGF). As mechanical stimuli are key in bone regeneration, their crosstalk to biochemical signaling pathways such as BMP and TGF-ß and YAP/TAZ acts on different levels, which allows now to think about new and more specified intervention strategies for age-related bone loss.

It Takes Two to Tango: Endothelial TGFß/BMP Signaling Crosstalk with Mechanobiology.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGFß) superfamily of cytokines. While some ligand members are potent inducers of angiogenesis, others promote vascular homeostasis. However, the precise understanding of the molecular mechanisms underlying these functions is still a growing research field. In bone, the tissue in which BMPs were first discovered, crosstalk of TGFß/BMP signaling with mechanobiology is well understood. Likewise, the endothelium represents a tissue that is constantly exposed to multiple mechanical triggers, such as wall shear stress, elicited by blood flow or strain, and tension from the surrounding cells and to the extracellular matrix. To integrate mechanical stimuli, the cytoskeleton plays a pivotal role in the transduction of these forces in endothelial cells. Importantly, mechanical forces integrate on several levels of the TGFß/BMP pathway, such as receptors and SMADs, but also global cell-architecture and nuclear chromatin re-organization. Here, we summarize the current literature on crosstalk mechanisms between biochemical cues elicited by TGFß/BMP growth factors and mechanical cues, as shear stress or matrix stiffness that collectively orchestrate endothelial function. We focus on the different subcellular compartments in which the forces are sensed and integrated into the TGFß/BMP growth factor signaling.

Multifocal myopathy in a patient with polyarteritis nodosa; usefulness of magnetic nuclear resonance as a diagnostic test / Miopatia multifocal en paciente con poliarteritis nodosa: utilidad de la resonancia nuclear magnética como prueba diagnóstica

ABSTRACT Polyarteritis nodosa is part of the primary systemic vasculitis that specifically compromises vessels of medium caliber, and can affect virtually any organ. The diagnosis of this disease is based on clinical criteria, such as pain and weakness in the lower limbs, as well as laboratory results and the histology report that shows necrotizing, segmental and focal inflammation of the vessels involved. The case is presented of a 42 year-old woman with a previous diagnosis of polyarteritis nodosa, who, 12 years later, presented with an episode of activation of the disease associated with venous thrombosis and multifocal myopathy in the lower limbs. This is a rare presentation that should be suspected in this type of patients.

RESUMEN La poliarteritis nodosa hace parte de las vasculitis sistémicas primarias, específicamente compromete vasos de mediano calibre pudiendo afectar virtualmente a cualquier órgano. El diagnóstico de esta enfermedad se basa en criterios clínicos, como el dolor y la debilidad en los miembros inferiores, además de reportes paraclínicos y el compromiso histológico, que evidencia inflamación necrosante, segmentaria y focal de los vasos involucrados. A continuación, presentamos el caso clínico de una mujer de 42 arios, con diagnóstico previo de poliarteritis nodosa, que presenta, 12 años después, un episodio de activación de la enfermedad asociado a trombosis venosa y miopatía multifocal en miembros inferiores, una presentación poco frecuente pero que debe sospecharse en este tipo de pacientes.

Step Up-Not On-The Step 2 Clinical Skills Exam: Directors of Clinical Skills Courses (DOCS) Oppose Ending Step 2 CS.

Recently, a student-initiated movement to end the United States Medical Licensing Examination Step 2 Clinical Skills and the Comprehensive Osteopathic Medical Licensing Examination Level 2-Performance Evaluation has gained momentum. These are the only national licensing examinations designed to assess clinical skills competence in the stepwise process through which physicians gain licensure and certification. Therefore, the movement to end these examinations and the ensuing debate merit careful consideration. The authors, elected representatives of the Directors of Clinical Skills Courses, an organization comprising clinical skills educators in the United States and beyond, believe abolishing the national clinical skills examinations would have a major negative impact on the clinical skills training of medical students, and that forfeiting a national clinical skills competency standard has the potential to diminish the quality of care provided to patients. In this Perspective, the authors offer important additional background information, outline key concerns regarding the consequences of ending these national clinical skills examinations, and provide recommendations for moving forward: reducing the costs for students, exploring alternatives, increasing the value and transparency of the current examinations, recognizing and enhancing the strengths of the current examinations, and engaging in a national dialogue about the issue.

[An adolescent with lumbosacral instability due to the presence of a hydatid cyst: a case report]. / Adolescente con inestabilidad lumbosacra por presencia de quiste hidatídico: reporte de caso.

Hydatidosis, caused by Echinococcus granulosus, affects the spinal column in 0.5-2% of all cases, with the sacral column being a rare location typically observed only in adults. The case is reported of a 14-year-old boy from Juliaca (Puno, Peru) who presented with chronic lower back pain and cauda equina syndrome. He was initially attended to in Juliaca, and then transferred to the Hospital Edgardo Rebagliati Martins (Lima, Peru) with a diagnosis of sacral tumor. In magnetic resonance imaging, a multicystic tumor was seen with L5 and S1 lysis and instability. The surgical treatment was an L5- S2 laminectomy and extraction of multiple hydatid cysts followed by lumbopelvic stabilization with transpedicular screws. Post-operatively, he was prescribed albendazol (15 mg/kg/d) for six months. Hydatid cysts should be considered within the differential diagnosis when there are cystic lesions in the spinal column, particularly in endemic areas such as Peru.

Adolescente con inestabilidad lumbosacra por presencia de quiste hidatídico: reporte de caso / An adolescent with lumbosacral instability due to the presence of a hydatid cyst: a case report

RESUMEN La hidatidosis, causada por el Echinococcus granulosus, afecta a la columna vertebral en 0,5-2% del total de casos, siendo la columna sacra una ubicación rara, típica de personas en edad adulta. Se reporta el caso de un varón de 14 años oriundo de Juliaca (Puno, Perú) quien presentó lumbalgia crónica y síndrome de cauda equina. Atendido inicialmente en Juliaca, fue trasladado al Hospital Edgardo Rebagliati Martins (Lima, Perú) con el diagnóstico de tumoración sacra. En la resonancia magnética se mostró una tumoración multiquística con lisis del L5 y S1 e inestabilidad. El tratamiento quirúrgico fue una laminectomía L5-S2 y extracción de múltiples quistes de hidátide más estabilización lumbopélvica con tornillos transpediculares. En el posoperatorio se recetó albendazol (15 mg/kg/d) por el periodo de seis meses. Se debe considerar al quiste hidatídico dentro del diagnóstico diferencial cuando existan lesiones quísticas en la columna vertebral, sobre todo en regiones endémicas como el Perú.

ABSTRACT Hydatidosis, caused by Echinococcus granulosus, affects the spinal column in 0.5-2% of all cases, with the sacral column being a rare location typically observed only in adults. The case is reported of a 14-year-old boy from Juliaca (Puno, Peru) who presented with chronic lower back pain and cauda equina syndrome. He was initially attended to in Juliaca, and then transferred to the Hospital Edgardo Rebagliati Martins (Lima, Peru) with a diagnosis of sacral tumor. In magnetic resonance imaging, a multicystic tumor was seen with L5 and S1 lysis and instability. The surgical treatment was an L5- S2 laminectomy and extraction of multiple hydatid cysts followed by lumbopelvic stabilization with transpedicular screws. Post-operatively, he was prescribed albendazol (15 mg/kg/d) for six months. Hydatid cysts should be considered within the differential diagnosis when there are cystic lesions in the spinal column, particularly in endemic areas such as Peru.

Síndrome de macrofago activado y lupus eritematoso sistemico. Presentación de un caso / Macrophage activation syndrome and systemic lupus erythematosus: one case

El síndrome del macrófago activado representa una entidad rara, presentándose principalmente en la infancia y caracterizado por la proliferación de macrófagos persistentemente activados y linfocitos. El síndrome se ha descrito principalmente como una entidad secundaria en niños que tienen ciertas enfermedades sistémicas, la mayoría de casos informados asociados a la artritis reumatoide juvenil, y algunos pocos casos asociados al lupus eritematoso sistémico (LES). Las manifestaciones clínicas del síndrome están asociadas a la producción de citoquinas , y en muchos casos ponen en peligro la vida. Describimos un caso de una niña con LES que tenía muchas manifestaciones clínicas, ninguna asociada a actividad de la enfermedad de base y habiendo descartado infección sistémica, presentó mejoría luego de los estudios confirmatorios del síndrome de macrófago activado y luego de recibir tratamiento con ciclosporina. Este artículo describe una corta revisión de la literatura sobre este síndrome y la asociación con LES, se discute su fisiopatología y la diferenciación con la actividad de enfermedad de base, infección agregada o el síndrome de macrófago activado, lo cual representa un problema frecuente

Aplicacion clinica de los anticuerpos en lupus eritematoso sistemico / Clinical aplication of antibodies in systemic lupus erythematosus

En la práctica diaria, el médico tiene como uno de los retos frente a un paciente el establecer un diagnóstico, y quisiera tener una prueba o ayuda diagnóstica 100 por ciento sensible y específica para corroborar su diagnóstico de trabajo. Sin embargo, esto es infrecuente en la consulta reumatológica, es por esto que presentamos en este artículo una visión detallada sobre el uso y significado clínico de los anticuerpos más utilizados en la valoración inicial y seguimiento de los pacientes con lupus eritematoso sistémico.

Historia de los anticuerpos antinucleares / History of antinuclear antibodies

Durante la práctica médica en reumatología y en otras áreas de la medicina con frecuencia se busca identificar anticuerpos dirigidos contra algunos componentes antigénicos nucleares. Estos anticuerpos se han relacionado con algunas enfermedades y con el tipo de alteraciones que ellas presentan. Las técnicas empleadas para identificar estos anticuerpos han ido variando y mejorando a medida que se amplía el conocimiento en ellos. Presentamos los aspectos históricos más importantes que se dieron en el desarrollo de las técnicas para identificar estos anticuerpos

Osteoporosis por esteroides: A proposito de unc aso ilustrativo: revision de la literatura y recuento historico / Osteoporosis for steroids

Presentamos el caso de una paciente con neumonitis lúpica, a quien se le hizo tratamiento con altas dosis de glucocorticoides, presentando desmineralización marcada de hueso a nivel de columna vertebral, con fracturas severas, de aparición rápida, el cual ilustra de manera clara los efectos secundarios de estos medicamentos sobre el hueso y la importancia de conocerlos, prevenirlos e implementar una terapia adecuada tempranamente. Hacemos una corta revisión histórica desde el inicio del descubrimiento de los glucocorticoides hasta llegar a las primeras descripciones sobre efectos secundarios de estos medicamentos, en especial la osteoporosis; analizamos la epidemiología, la etiopatogénesis y las formas de prevención y tratamiento de la osteoporosis inducida por glucocorticoides

Tolerancia inmunologica: ¿por que convivimos con nuestros tejidos? / Inmunologic tolerance

En este artículo se resumen los aspectos generales de la tolerancia inmunológica con énfasis en la distinción entre tolerancia e ignorancia. Este último concepto está relacionado con la incapacidad del sistema inmunitario para reconocer todos los auto-antígenos o con la ausencia de linfocitos vírgenes recirculantes en los tejidos periféricos

Enfermedad quistica pulmonar y sindrome de Sjogren: Informe del primer caso en Colombia y revision de la literatura / Cystic lung disease and Sjorgren's syndrome: firts case in Colombia

En este artículo presentamos el caso de una paciente con síndrome de Sjögren primario, hipotiroidismo autoinmune y compromiso pulmonar severo dado por enfermedad quística difusa, revisamos la literatura y reseñamos las principales características del compromiso pulmonar quístico y bulloso en el síndrome de Sjögren. Es el primer caso informado en Colombia con esta patología

Calcinosis distrofica pulmonar asociada a esclerosis sistemica: Informe del primer caso en Colombia / Dystrophic calcinosis associated with systemic sclerosis: first case in Colombia

La asociación de calcinosis pulmonar con esclerosis sistémica no ha sido descrita en la literatura médica. Las clases de calcificación pulmonar se restringen a los tipos metastásico y distrófico; en las enfermedades de tejido conectivo el tipo de calcinosis predominante es el distrófico, asociándose principalmente a dermatopolimiositis y esclerosis sistémica. Describimos el primer caso de calcinosis pulmonar asociada a esclerosis sistémica