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Thrombosis is a common complication of advanced cancer, yet the cellular mechanisms linking malignancy to thrombosis are poorly understood. The unfolded protein response (UPR) is an ER stress response associated with advanced cancers. A proteomic evaluation of plasma from patients with gastric and non–small cell lung cancer who were monitored prospectively for venous thromboembolism demonstrated increased levels of UPR-related markers in plasma of patients who developed clots compared with those who did not. Release of procoagulant activity into supernatants of gastric, lung, and pancreatic cancer cells was enhanced by UPR induction and blocked by antagonists of the UPR receptors inositol-requiring enzyme 1α (IRE1α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK). Release of extracellular vesicles bearing tissue factor (EVTFs) from pancreatic cancer cells was inhibited by siRNA-mediated knockdown of IRE1α/XBP1 or PERK pathways. Induction of UPR did not increase tissue factor (TF) synthesis, but rather stimulated localization of TF to the cell surface. UPR-induced TF delivery to EVTFs was inhibited by ADP-ribosylation factor 1 knockdown or GBF1 antagonism, verifying the role of vesicular trafficking. Our findings show that UPR activation resulted in increased vesicular trafficking leading to release of prothrombotic EVTFs, thus providing a mechanistic link between ER stress and cancer-associated thrombosis.

Endothelial cells (ECs) normally form an anticoagulant surface under physiological conditions, but switch to support coagulation following pathogenic stimuli. This switch promotes thrombotic cardiovascular disease. To generate thrombin at physiologic rates, coagulation proteins assemble on a membrane containing anionic phospholipid, most notably phosphatidylserine (PS). PS can be rapidly externalized to the outer cell membrane leaflet by phospholipid “scramblases,” such as TMEM16F. TMEM16F-dependent PS externalization is well characterized in platelets. In contrast, how ECs externalize phospholipids to support coagulation is not understood. We employed a focused genetic screen to evaluate the contribution of transmembrane phospholipid transport on EC procoagulant activity. We identified 2 TMEM16 family members, TMEM16F and its closest paralog, TMEM16E, which were both required to support coagulation on ECs via PS externalization. Applying an intravital laser-injury model of thrombosis, we observed, unexpectedly, that PS externalization was concentrated at the vessel wall, not on platelets. TMEM16E-null mice demonstrated reduced vessel-wall–dependent fibrin formation. The TMEM16 inhibitor benzbromarone prevented PS externalization and EC procoagulant activity and protected mice from thrombosis without increasing bleeding following tail transection. These findings indicate the activated endothelial surface is a source of procoagulant phospholipid contributing to thrombus formation. TMEM16 phospholipid scramblases may be a therapeutic target for thrombotic cardiovascular disease.

Type 2 diabetes is considered one of the top ten life-threatening diseases worldwide. Following economic growth, obesity and metabolic syndrome became the most common risk factor for type 2 diabetes. In this regard, high-fat diet-fed C57BL/6J mouse model is widely used for type 2 diabetes pathogenesis and novel therapeutics development. However, criteria for classifying type 2 diabetes progressive stages in this mouse model are yet to be determined, led to the difficulty in experimental end-point decision. In this study, we fed C57BL/6J male mice with 45% high-fat diet, which is physiologically close to human high-fat consumption, and evaluated the progression of type 2 diabetes. After consuming high-fat diet for 4 weeks, mice developed metabolic syndrome, including obesity, significant increase of fasting plasma cholesterol level, elevation of both C-peptide and fasting blood glucose levels. By combining both fasting blood glucose test and 2-hour-oral glucose tolerance test, our results illustrated clear progressive stages from metabolic syndrome into pre-diabetes before onset of type 2 diabetes in C57BL/6J mice given a 45% high-fat diet. Besides, among metabolic measurements, accumulating body weight gain > 16.23 g for 12 weeks could be utilized as a potential parameter to predict type 2 diabetes development in C57BL/6J mice. Thus, these results might support future investigations in term of selecting appropriate disease stage in high-fat diet-fed C57BL/6J mouse model for studying early prevention and treatment of type 2 diabetes.

The identification of genes affecting gonad development is essential to understand the mechanisms causing Variations/Differences in Sex Development (DSD). Recently, a DLC3 mutation was associated with male gonadal dysgenesis in 46,XY DSD patients. We have studied the requirement of Cv-c, the Drosophila ortholog of DLC3, in Drosophila gonad development, as well as the functional capacity of DLC3 human variants to rescue cv-c gonad defects. We show that Cv-c is required to maintain testis integrity during fly development. We find that Cv-c and human DLC3 can perform the same function in fly embryos, as flies carrying wild type but not patient DLC3 variations can rescue gonadal dysgenesis, suggesting functional conservation. We also demonstrate that the StART domain mediates Cv-c's function in the male gonad independently from the GAP domain's activity. This work demonstrates a role for DLC3/Cv-c in male gonadogenesis and highlights a novel StART domain mediated function required to organize the gonadal mesoderm and maintain its interaction with the germ cells during testis development.

La valoración negativa de la vejez por parte de las personas cuidadoras produce efectos adversos sobre las personas mayores cuidadas. Este estudio se planteó identificar los estereotipos negativos sobre la vejez en personas cuidadoras formales de personas adultas mayores de Montevideo, Uruguay. Además, explorar la posibilidad de modificarlos, mediante intervenciones formativas en cuidados, con perspectiva psicogerontológica. La muestra fue no probabilística. Se utilizó el Cuestionario de Estereotipos Negativos hacia la Vejez (CENVE). El análisis fue descriptivo y comparativo, siendo central el análisis de diferencias de medias. En la escala global, se evidenció un importante descenso de los estereotipos negativos altos y muy altos (de 27,3 en preintervención a 6,8 en posintervención) y la desaparición de los niveles muy altos. De los tres factores del CENVE, los estereotipos más altos fueron registrados en carácter-personalidad. Los resultados permiten subrayar la importancia de una formación gerontológica integral de las personas cuidadoras formales de personas adultas mayores.

Background: Relatively little is known about social inequality in human papillomavirus (HPV) vaccination among teenagers in the United States. This study aims to investigate whether there is a social disparity in HPV vaccination among teenagers and if so, whether it can differ according to the source of teen vaccination information (parental reports and provider records). Methods: We used the data from the 2019 National Immunization Survey-Teen (NIS-Teen; 42,668 teenagers, aged 13–17) including parent-reported vaccination status. Among them, 18,877 teenagers had adequate provider-reported vaccination records. Two socioeconomic status (SES) measures were used: mother’s education and annual family income. Multivariate logistic analyses were conducted. Results: False negatives of parental reports against provider records were more than two times higher (p < 0.001) in low-SES teens than in high-SES teens. In both SES measures, the proportion of HPV-unvaccinated teenagers was lowest at the highest SES level in analyses with parental reports. However, it was the opposite in analyses with provider records. Interestingly, regardless of the vaccination information source, the HPV unvaccinated rate was highest in the middle-SES teens (>12 years, non-college graduates; above poverty level, but not >USD 75 K). Conclusions: Significant social inequality in HPV vaccination among teenagers exists in the United States. The pattern of social inequality in HPV vaccination can be distorted when only parent-reported vaccination information is used.

Objetivos. Caracterizar a nivel molecular las bacterias patógenas de las vías respiratorias de pacientes peruanos con fibrosis quística (FQ). Materiales y métodos. Se caracterizaron las comunidades bacterianas cultivables a partir de muestras de esputo de pacientes pediátricos y adultos con FQ registrados en el Hospital Nacional Edgardo Rebagliati Martins y el Instituto Nacional de Salud del Niño (INSN). Para el cultivo bacteriano se utilizaron técnicas microbiológicas estándares, y para la caracterización molecular la secuenciación del gen ARNr 16S y espectrometría de masas de tipo desorción/ionización con láser asistido por matriz con tiempo de vuelo (MALDI TOF) y MALDI TOF/TOF. Resultados. Por secuenciación del ARNr 16S se identificaron 127 cepas, encontrando las bacterias patógenas Pseudomonas aeruginosa (31,5%), Staphylococcus aureus (12,6%), Pseudomonas spp. (11,8%), Klebsiella oxytoca (3,1%), otras especies mostraron baja prevalencia. El análisis por MALDI TOF permitió obtener una serie de espectros representativos de cada especie aislada, mientras que el análisis por MALDI TOF/TOF reveló péptidos y proteínas de las especies más comunes con informaciones complementarias que revelarían datos sobre su patogenicidad o sensibilidad a antibióticos. Conclusiones. Los principales microorganismos patógenos encontrados en las vías respiratorias son similares a los reportados en otros países. Estos son los primeros hallazgos en Perú que muestran la caracterización bacteriana por secuenciación del ARNr 16S, por MALDI TOF y MALDI TOF TOF. Los hallazgos permiten la identificación bacteriana de microorganismos nativos relacionados con la FQ basada en el análisis de su proteoma.

OBJECTIVES/GOALS: Klotho is a protein linked to improved cognition in aging adults. A specific KLOTHO gene variant, KL-VS, increases circulating levels of Klotho. The current study aims to identify if the KL-VS haplotype and Klotho levels are associated with improved neurocognition in pediatric brain tumor survivors. METHODS/STUDY POPULATION: We are actively accruing pediatric brain tumor patients at UCSF alongside an existing multi-institutional cohort study investigating radiation-induced vasculopathies and cognitive outcomes in this population. Normal controls are being enrolled in parallel. Each patient undergoes: 1) single nucleotide polymorphism genotyping to identify KL-VS haplotype status, 2) enzyme-linked immunosorbent assays to measure circulating Klotho, 3) neurocognitive assessments with a computer-based, validated Cogstate battery, and 4) brain volume and white matter lesion segmentation analyses using MRI sequences obtained as part of routine care. RESULTS/ANTICIPATED RESULTS: Genotyping has been performed on 99 enrolled patients. KL-VS heterozygosity was seen in 22.7% of patients. To date, KL-VS status is not associated with neurocognitive outcomes at baseline or Year 1 testing. Association between KL-VS status, circulating Klotho levels, neurocognitive outcomes, brain volume and white matter lesion segmentation analyses is ongoing. We hypothesize that elevated Klotho levels will be associated with improved neurocognition, increased brain volumes in regions of interest and decreased white matter lesion volumes. DISCUSSION/SIGNIFICANCE OF IMPACT: If circulating Klotho leads to improved neurocognition in pediatric brain tumor survivors, Klotho levels might serve as a prognostic biomarker. Furthermore, as Klotho is being investigated for therapeutic indications, it may represent an intervention to prevent cognitive deficits in these patients.