Expression of the ectodomain-releasing protease ADAM17 is directly regulated by the osteosarcoma and bone-related transcription factor RUNX2.

Osteoblast differentiation is controlled by transcription factor RUNX2 which temporally activates or represses several bone-related genes, including those encoding extracellular matrix proteins or factors that control cell-cell, and cell-matrix interactions. Cell-cell communication in the many skeletal pericellular micro-niches is critical for bone development and involves paracrine secretion of growth factors and morphogens. This paracrine signaling is in part regulated by "A Disintegrin And Metalloproteinase" (ADAM) proteins. These cell membrane-associated metalloproteinases support proteolytic release ("shedding") of protein ectodomains residing at the cell surface. We analyzed microarray and RNA-sequencing data for Adam genes and show that Adam17, Adam10, and Adam9 are stimulated during BMP2 mediated induction of osteogenic differentiation and are robustly expressed in human osteoblastic cells. ADAM17, which was initially identified as a tumor necrosis factor alpha (TNFα) converting enzyme also called (TACE), regulates TNFα-signaling pathway, which inhibits osteoblast differentiation. We demonstrate that Adam17 expression is suppressed by RUNX2 during osteoblast differentiation through the proximal Adam17 promoter region (-0.4 kb) containing two functional RUNX2 binding motifs. Adam17 downregulation during osteoblast differentiation is paralleled by increased RUNX2 expression, cytoplasmic-nuclear translocation and enhanced binding to the Adam17 proximal promoter. Forced expression of Adam17 reduces Runx2 and Alpl expression, indicating that Adam17 may negatively modulate osteoblast differentiation. These findings suggest a novel regulatory mechanism involving a reciprocal Runx2-Adam17 negative feedback loop to regulate progression through osteoblast differentiation. Our results suggest that RUNX2 may control paracrine signaling through regulation of ectodomain shedding at the cell surface of osteoblasts by directly suppressing Adam17 expression.

Wnt/ß-Catenin Signaling Activates Expression of the Bone-Related Transcription Factor RUNX2 in Select Human Osteosarcoma Cell Types.

Osteosarcoma is the most common malignant bone tumor in children and adolescents. Metastasis and poor responsiveness to chemotherapy in osteosarcoma correlates with over-expression of the runt-related transcription factor RUNX2, which normally plays a key role in osteogenic lineage commitment, osteoblast differentiation, and bone formation. Furthermore, WNT/ß-catenin signaling is over-activated in osteosarcoma and promotes tumor progression. Importantly, the WNT/ß-catenin pathway normally activates RUNX2 gene expression during osteogenic lineage commitment. Therefore, we examined whether the WNT/ß-catenin pathway controls the tumor-related elevation of RUNX2 expression in osteosarcoma. We analyzed protein levels and nuclear localization of ß-catenin and RUNX2 in a panel of human osteosarcoma cell lines (SAOS, MG63, U2OS, HOS, G292, and 143B). In all six cell lines, ß-catenin and RUNX2 are expressed to different degrees and localized in the nucleus and/or cytoplasm. SAOS cells have the highest levels of RUNX2 protein that is localized in the nucleus, while MG63 cells have the lowest RUNX2 levels which is mostly localized in the cytoplasm. Levels of ß-catenin and RUNX2 protein are enhanced in HOS, G292, and 143B cells after treatment with the GSK3ß inhibitor SB216763. Furthermore, small interfering RNA (siRNA)-mediated depletion of ß-catenin inhibits RUNX2 expression in G292 cells. Thus, WNT/ß-catenin activation is required for RUNX2 expression in at least some osteosarcoma cell types, where RUNX2 is known to promote expression of metastasis related genes. J. Cell. Biochem. 118: 3662-3674, 2017. © 2017 Wiley Periodicals, Inc.

The cancer-related transcription factor Runx2 modulates cell proliferation in human osteosarcoma cell lines.

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G(1) phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G(1)/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G(1)/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth.

SARS-CoV-2 Genome Variations in Viral Shedding of an Immunocompromised Patient with Non-Hodgkin's Lymphoma.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) is the most transmissible ß-coronavirus in history, affecting all population groups. Immunocompromised patients, particularly cancer patients, have been highlighted as a reservoir to promote accumulation of viral mutations throughout persistent infection. CASE PRESENTATION: We aimed to describe the clinical course and SARS-CoV-2 mutation profile for 102 days in an immunocompromised patient with non-Hodgkin's lymphoma and COVID-19. We used RT-qPCR to quantify SARS-CoV-2 viral load over time and whole-virus genome sequencing to identify viral lineage and mutation profile. The patient presented with a persistent infection through 102 days while being treated with cytotoxic chemotherapy for non-Hodgkin's lymphoma and received targeted therapy for COVID-19 with remdesivir and hyperimmune plasma. All sequenced samples belonged to the BA.1.1 lineage. We detected nine amino acid substitutions in five viral genes (Nucleocapsid, ORF1a, ORF1b, ORF13a, and ORF9b), grouped in two clusters: the first cluster with amino acid substitutions only detected on days 39 and 87 of sample collection, and the second cluster with amino acid substitutions only detected on day 95 of sample collection. The Spike gene remained unchanged in all samples. Viral load was dynamic but consistent with the disease flares. CONCLUSIONS: This report shows that the multiple mutations that occur in an immunocompromised patient with persistent COVID-19 could provide information regarding viral evolution and emergence of new SARS-CoV-2 variants.

The Evolutionary Landscape of SARS-CoV-2 Variant B.1.1.519 and Its Clinical Impact in Mexico City.

The SARS-CoV-2 pandemic is one of the most concerning health problems around the globe. We reported the emergence of SARS-CoV-2 variant B.1.1.519 in Mexico City. We reported the effective reproduction number (Rt) of B.1.1.519 and presented evidence of its geographical origin based on phylogenetic analysis. We also studied its evolution via haplotype analysis and identified the most recurrent haplotypes. Finally, we studied the clinical impact of B.1.1.519. The B.1.1.519 variant was predominant between November 2020 and May 2021, reaching 90% of all cases sequenced in February 2021. It is characterized by three amino acid changes in the spike protein: T478K, P681H, and T732A. Its Rt varies between 0.5 and 2.9. Its geographical origin remain to be investigated. Patients infected with variant B.1.1.519 showed a highly significant adjusted odds ratio (aOR) increase of 1.85 over non-B.1.1.519 patients for developing a severe/critical outcome (p = 0.000296, 1.33-2.6 95% CI) and a 2.35-fold increase for hospitalization (p = 0.005, 1.32-4.34 95% CI). The continuous monitoring of this and other variants will be required to control the ongoing pandemic as it evolves.

[Drug prescription patterns in patients attended at a middle- and high-complexity level institution]. / Prescripción de medicamentos en pacientes atendidos en instituciones de mediano y alto nivel de complejidad.

OBJECTIVE: Determining prescription patterns for outpatient medication authorised for patients affiliated to an EPS and assisted by medium- and high-level complexity IPS. METHODS: This was a cross-sectional study where medication prescription was evaluated in 331 second- and third-level complexity hospitals from 27 Colombian departments during 2006. RESULTS: 38 863 prescriptions for 3 663 patients' medication were analysed. 61,4 % came from third-level complexity hospitals, mainly for patients affiliated to contribution-based regimes. Average prescribed medication per person was 2,2 (2,1-2,2 95 % CI), the percentage of antibiotics formulated by prescription was 29,2 % (28,7-29,6 95 % CI), essential prescribed medicines accounted for 64,2 % (63,7-64,6 95 % CI) and injectable medicines was 22,1 % (21,7-25,5 %). More than half the medications (62,1 %; 61,5-62,7 95 % CI) were in ATC groups such as anti-infectious agents, immunomodulating agents and medications for the alimentary and metabolic tract. DU90% consisted of 64 medications, medication consumption being 8,39 daily de-fined doses (DDD)/1 000 patients, costing 5 216 Colombian pesos per DDD. CONCLUSIONS: This analysis led to identifying medications whose formulation frequency did not correlate with an epidemiologic profile as immunomudulator and growth-hormone (somatotropin) agents. There were differences in the frequency and quantity of DDD medications authorised by type of affiliation which could thus be providing evidence of obstacles to the population having access to drugs/medicaments.

Prescripción de medicamentos en pacientes atendidos en instituciones de mediano y alto nivel de complejidad / Drug prescription patterns in patients attended at a middle- and high-complexity level institution

Objetivo Determinar los patrones de prescripción de medicamentos ambulatorios autorizados en pacientes afiliados a una EPS y atendidos por IPS de mediano y alto nivel de complejidad. Métodos Se realizo un estudio analítico transversal donde se evaluaron las prescripciones de medicamentos en 331 IPS de segundo y tercer nivel de complejidad en 27 departamentos durante el 2006. Resultados Se analizaron 38 863 prescripciones de medicamentos de 3 663 pacientes. El 61,4 por ciento de estas provienen de IPS de tercer nivel de complejidad y principalmente de pacientes afiliados al régimen contributivo. El promedio de medicamentos por formula medica fue de 2,2 (IC 95 por ciento: 2,1 a 2,2 por ciento), la proporción de antibióticos por fórmula médica del 29,2 por ciento (IC 95 por ciento: 28,7-29,6), de medicamentos POS del 64,2 por ciento (IC 95 por ciento 63,7-64,6 por ciento) y de inyectables del 22,1 por ciento (IC 95 por ciento 21,7-25,5 por ciento). Más de la mitad de los medicamentos, 62,1 por ciento (IC 95 por ciento 61,5-62,7 por ciento), corresponden a agentes anti-infecciosos, antineoplasicos y del tracto alimentario .El consumo de medicamentos fue de 8,39 Dosis Diarias Definidas (DDD) /1 000 usuarios/día y el costo por DDD fue de $ 5 216 Conclusión Este análisis permitió identificar medicamentos cuya frecuencia de formulación no se correlaciono con el perfil epidemiológico como agentes inmunomoduladores y hormona de crecimiento. Existen diferencias en la frecuencia y cantidad de DDD de medicamentos autorizados por régimen de afiliación lo cual pude ser evidencia de barreras de acceso a los medicamentos en la población.

Objective Determining prescription patterns for outpatient medication authorised for patients affiliated to an EPS and assisted by medium- and high-level complexity IPS. Methods This was a cross-sectional study where medication prescription was evaluated in 331 second- and third-level complexity hospitals from 27 Colombian departments during 2006. Results 38 863 prescriptions for 3 663 patients’ medication were analysed. 61,4 percent came from third-level complexity hospitals, mainly for patients affiliated to contribution-based regimes. Average prescribed medication per person was 2,2 (2,1-2,2 95 percent CI), the percentage of antibiotics formulated by prescription was 29,2 percent (28,7-29,6 95 percent CI), essential prescribed medicines accounted for 64,2 percent (63,7-64,6 95 percent CI) and injectable medicines was 22,1 percent (21,7-25,5 percent). More than half the medications (62,1 percent; 61,5-62,7 95 percent CI) were in ATC groups such as anti-infectious agents, immunomodulating agents and medications for the alimentary and metabolic tract. DU90 percent consisted of 64 medications, medication consumption being 8,39 daily de-fined doses (DDD)/1 000 patients, costing 5 216 Colombian pesos per DDD. Conclusions This analysis led to identifying medications whose formulation frequency did not correlate with an epidemiologic profile as immunomudulator and growth-hormone (somatotropin) agents. There were differences in the frequency and quantity of DDD medications authorised by type of affiliation which could thus be providing evidence of obstacles to the population having access to drugs/medicaments.