Detección canina de anaplasma platys mediante PCR en tiempo real

Anaplasma platys is an obligate gram-negative, intracellular pleomorphic bacterium that especially affects dog platelets, transmitted mainly by ticks. Pathogen of relevant importance in veterinary medicine and recognized worldwide for its zoonotic potential. The objective of the work was to determine by real-time PCR the presence of A. platys in a canine from Asunción. We highlight that it is the first molecular evidence of A. platys in the country using the qPCR method, whose sequencing of the obtained product confirmed the infection by CP046391.1 Anaplasma Platys with 100% homology. The development of the qPCR method contributes to the advancement of research with A. platys, constituting a more sensitive and specific tool for situations that indicate a possible clinical disease, but with traditional diagnostic methods such as blood cytology or negative serology.Anaplasma platys es una bacteria pleomórfica intracelular, gramnegativa obligada que afecta especialmente a las plaquetas del perro, transmitida principalmente por garrapatas. Patógeno de importancia relevante en medicina veterinaria y reconocido mundialmente por su potencial zoonótico. El objetivo del trabajo fue determinar por PCR a tiempo real la presencia de A. platys en un canino de Asunción. Destacamos que es la primera evidencia molecular de A. platys en el país utilizando el método de qPCR, cuya secuenciación del producto obtenido confirmó la infección por CP046391.1 Anaplasma Platys con homología 100%. El desarrollo del método qPCR contribuye al avance de la investigación con A. platys, constituyendo una herramienta más sensible y específica para situaciones que indiquen una posible enfermedad clínica, pero con métodos diagnósticos tradicionales como citología sanguínea o serología negativa

Detección canina de anaplasma platys mediante PCR en tiempo real

Anaplasma platys es una bacteria pleomórfica intracelular, gramnegativa obligada que afecta especialmente a las plaquetas del perro, transmitida principalmente por garrapatas. Patógeno de importancia relevante en medicina veterinaria y reconocido mundialmente por su potencial zoonótico. El objetivo del trabajo fue determinar por PCR a tiempo real la presencia de A. platys en un canino de Asunción. Destacamos que es la primera evidencia molecular de A. platys en el país utilizando el método de qPCR, cuya secuenciación del producto obtenido confirmó la infección por CP046391.1 Anaplasma Platys con homología 100%. El desarrollo del método qPCR contribuye al avance de la investigación con A. platys, constituyendo una herramienta más sensible y específica para situaciones que indiquen una posible enfermedad clínica, pero con métodos diagnósticos tradicionales como citología sanguínea o serología negativa.</jats:p

EWS/FLI mediated reprogramming of 3D chromatin promotes an altered transcriptional state in Ewing sarcoma

ABSTRACTEwing sarcoma is a prototypical fusion transcription factor-associated pediatric cancer that expresses EWS/FLI or highly related fusions. EWS/FLI dysregulates transcription to induce and maintain sarcomagenesis, but the mechanisms utilized are not fully understood. We therefore sought to define the global effects of EWS/FLI on chromatin conformation and transcription in Ewing sarcoma. We found that EWS/FLI (and EWS/ERG) genomic localization is largely conserved across multiple patient-derived Ewing sarcoma cell lines. EWS/FLI binding is primarily associated with compartment activation, establishment of topologically-associated domain (TAD) boundaries, enhancer-promoter looping that involve both intra- and inter-TAD interactions, and gene activation. Importantly, local chromatin features provide the basis for transcriptional heterogeneity in regulation of direct EWS/FLI target genes across different Ewing sarcoma cell lines. These data demonstrate a key role of EWS/FLI in mediating genome-wide changes in chromatin configuration and support the notion that fusion transcription factors serve as master regulators through three-dimensional reprogramming of chromatin.</jats:p

The FLI portion of EWS/FLI contributes a transcriptional regulatory function that is distinct and separable from its DNA-binding function in Ewing sarcoma

AbstractBackgroundEwing sarcoma is an aggressive bone cancer in children and young adults that contains a pathognomonic chromosomal translocation: t(11;22)(q24;q12). The encoded protein, EWS/FLI, fuses the low-complexity amino-terminal portion of EWS to the carboxyl-terminus of FLI. The FLI portion contains an ETS DNA-binding domain and adjacent amino- and carboxyl-regions. Early studies using non-Ewing sarcoma cellular models provided conflicting information on the role of these adjacent regions in the oncogenic function of EWS/FLI. We therefore sought to define the specific contributions of each FLI region to EWS/FLI activity in an appropriate Ewing model, and in doing so, to better understand Ewing sarcoma development mediated by the fusion protein.MethodsWe used a “knock-down/rescue” system to replace endogenous EWS/FLI expression with mutant forms of the protein in Ewing sarcoma cells and tested these for oncogenic transformation using soft-agar colony forming assays. These data were complemented by DNA-binding assays using fluorescence anisotropy, genomic localization assays using CUT&amp;RUN, transcriptional regulation studies using luciferase reporter assays and RNA-sequencing, as well as chromatin accessibility assays using ATAC-sequencing.ResultsWe found that the DNA-binding domain and short flanking regions of FLI were required for oncogenic transformation, gene expression, genomic localization and chromatin accessibility when fused to the amino-terminal EWS-portion from EWS/FLI, but that the remaining regions of FLI were dispensable for these functions. Removal of a carboxyl-terminal alpha-helix from the short flanking regions of the DNA-binding domain of FLI created a hypomorphic EWS/FLI that retained normal DNA binding, genomic localization, and chromatin accessibility, but had significantly restricted transcriptional activity and a near total loss of oncogenic transformational capacity.ConclusionsThe DNA-binding domain and carboxyl-terminal short flanking region of FLI are the only portions of FLI required for EWS/FLI-mediated oncogenic transformation in a Ewing sarcoma cellular context. In addition to the well-defined DNA-binding function of FLI, this additional alpha-helix immediately downstream of the DNA-binding domain contributes a previously-undescribed function in gene regulation and oncogenic transformation. Understanding the function of this critical region could provide new therapeutic opportunities to target EWS/FLI in Ewing sarcoma.</jats:sec

Identification of a novel FUS/ETV4 fusion and comparative analysis with other Ewing sarcoma fusion proteins

AbstractEwing sarcoma is an aggressive pediatric bone cancer defined by a chromosomal translocation fusing one of the FET family members to a member of the ETS transcription factor family. To date, there have been seven reported translocations, with the most recent translocation reported over a decade ago. We now report the first identification of a novel translocation occurring between the FUS gene and ETS family member ETV4 detected in a neonatal patient with Ewing sarcoma. Given its apparent rarity, we conducted an initial characterization of FUS/ETV4 function by performing genomic localization and transcriptional regulatory studies. We knocked down endogenous EWS/FLI in the A673 cell line, and expressed FUS/ETV4 in its stead, and performed CUT&amp;Tag and RNA-sequencing analyses. We compared these data to similar “knock-down/rescue” analyses of other rare (non-EWS/FLI) Ewing sarcoma-associated translocation products. Through this comparative analysis in the same genetic background, we demonstrate significant similarities across these fusions, and in doing so, validate this novel FUS/ETV4 translocation as a bona fide Ewing sarcoma translocation. This study presents the first genomic comparisons of the rare Ewing sarcoma-associated translocation products, and reveals that the FET/ETS fusions share highly similar, but not identical, genomic localization and transcriptional regulation patterns. These data provide insights into the roles of both the FET and ETS sides of these fusions, and provide a generic strategy to provide further strength to the notion that FET/ETS fusions are key drivers of, and thus pathognomonic for, Ewing sarcoma.SignificanceIdentification and initial characterization of the novel Ewing sarcoma fusion, FUS/ETV4, expands the family of Ewing-fusions and extends the diagnostic possibilities for this aggressive tumor of adolescents and young adults.</jats:sec

Human Teratoma-Derived Hematopoiesis Is a Highly Polyclonal Process Supported by Human Umbilical Vein Endothelial Cells

Summary: Hematopoietic stem cells (HSCs) ensure a life-long regeneration of the blood system and are therefore an important source for transplantation and gene therapy. The teratoma environment supports the complex development of functional HSCs from human pluripotent stem cells, which is difficult to recapitulate in culture. This model mimics various aspects of early hematopoiesis, but is restricted by the low spontaneous hematopoiesis rate. In this study, a feasible protocol for robust hematopoiesis has been elaborated. We achieved a significant increase of the teratoma-derived hematopoietic population when teratomas were generated in the NSGS mouse, which provides human cytokines, together with co-injection of human umbilical vein endothelial cells. Since little is known about hematopoiesis in teratomas, we addressed localization and clonality of the hematopoietic lineage. Our results indicate that early human hematopoiesis is closely reflected in teratoma formation, and thus highlight the value of this model. : Schambach and colleagues show evidence that hematopoiesis in hiPSC-derived teratomas occurs by endothelial-to-hematopoietic transition in a highly polyclonal manner. This process was supported by the presence of HUVECs in the NSGS mouse, which provides human SCF, IL-3, and GM-CSF. The established hematopoiesis model can potentially be used for disease modeling and improvement of hematopoietic differentiation protocols. Keywords: hematopoiesis, teratoma, hiPSC, EHT, genetic barcoding, HUVECs, embryogenesi

MCR-21-0354_SF4 from Identification of a Novel <i>FUS/ETV4</i> Fusion and Comparative Analysis with Other Ewing Sarcoma Fusion Proteins

Supp Figure 4 depicts the overlap analysis of EWS/ETS and FUS/ETS proteins at both the genomic localization and transcriptional regulatory levels.</p

MCR-21-0354_SF2 from Identification of a Novel <i>FUS/ETV4</i> Fusion and Comparative Analysis with Other Ewing Sarcoma Fusion Proteins

Supp Figure 2 shows knockdown of endogenous EWS/FLI and expression of alternative FET/ETS fusions in the A673 Ewing sarcoma cell line.</p

MCR-21-0354_SF5 from Identification of a Novel <i>FUS/ETV4</i> Fusion and Comparative Analysis with Other Ewing Sarcoma Fusion Proteins

Supp Figure 5 demonstrates similar DNA-binding patterns between FET/ETS proteins at EWS/FLI-specific regulatory elements.</p

MCR-21-0354_SF6 from Identification of a Novel <i>FUS/ETV4</i> Fusion and Comparative Analysis with Other Ewing Sarcoma Fusion Proteins

Supp Figure 6 demonstrates similar transcriptional regulatory patterns between FET/ETS proteins at Ewing sarcoma-specific genes.</p