Scaling up polarized RPE cell supernatant production on parylene membrane.

Age-related macular degeneration (AMD), a leading cause of vision loss, primarily arises from the degeneration of retinal pigment epithelium (RPE) and photoreceptors. Current therapeutic options for dry AMD are limited. Encouragingly, cultured RPE cells on parylene-based biomimetic Bruch's membrane demonstrate characteristics akin to the native RPE layer. In this study, we cultivated human embryonic stem cell-derived polarized RPE (hESC-PRPE) cells on parylene membranes at both small- and large-scale settings, collecting conditioned supernatant, denoted as PRPE-SF. We conducted a comprehensive analysis of the morphology of the cultured hESC-RPE cells and the secreted growth factors in PRPE-SF. To evaluate the in vivo efficacy of these products, the product was administered via intravitreal injections of PRPE-SF in immunodeficient Royal College of Surgeons (iRCS) rats, a model for retinal degeneration. Our study not only demonstrated the scalability of PRPE-SF production while maintaining RPE cell phenotype but also showed consistent protein concentrations between small- and large-scale batches. We consistently identified 10 key factors in PRPE-SF, including BMP-7, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, MANF, PEDF, PDGF-AA, TGFß1, and VEGF. Following intravitreal administration of PRPE-SF, we observed a significant increase in the thickness of the outer nuclear layer (ONL) and photoreceptor preservation in iRCS rats. Furthermore, correlation analysis revealed that IGFBP-3, IGFBP-4, MANF, PEDF, and TGFß1 displayed positive associations with in vivo bioactivity, while GDF-15 exhibited a negative correlation. Overall, this study highlights the feasibility of scaling up PRPE-SF production on parylene membranes without compromising its essential constituents. The outcomes of PRPE-SF administration in an animal model of retinal degeneration present substantial potential for photoreceptor preservation. Moreover, the identification of candidate surrogate potency markers, showing strong positive associations with in vivo bioactivity, lays a solid foundation for the development of a promising therapeutic intervention for retinal degenerative diseases.

Hematopoietic stem cell division is governed by distinct RUNX1 binding partners.

A defined number of hematopoietic stem cell (HSC) clones are born during development and expand to form the pool of adult stem cells. An intricate balance between self-renewal and differentiation of these HSCs supports hematopoiesis for life. HSC fate is determined by complex transcription factor networks that drive cell-type specific gene programs. The transcription factor RUNX1 is required for definitive hematopoiesis, and mutations in Runx1 have been shown to reduce clonal diversity. The RUNX1 cofactor, CBFý, stabilizes RUNX1 binding to DNA, and disruption of their interaction alters downstream gene expression. Chemical screening for modulators of Runx1 and HSC expansion in zebrafish led us to identify a new mechanism for the RUNX1 inhibitor, Ro5-3335. We found that Ro5-3335 increased HSC divisions in zebrafish, and animals transplanted with Ro5-3335 treated cells had enhanced chimerism compared to untreated cells. Using human CD34+ cells, we show that Ro5-3335 remodels the RUNX1 transcription complex by binding to ELF1, independent of CBFý. This allows specific expression of cell cycle and hematopoietic genes that enhance HSC self-renewal and prevent differentiation. Furthermore, we provide the first evidence to show that it is possible to pharmacologically increase the number of stem cell clones in vivo , revealing a previously unknown mechanism for enhancing clonal diversity. Our studies have revealed a mechanism by which binding partners of RUNX1 determine cell fate, with ELF transcription factors guiding cell division. This information could lead to treatments that enhance clonal diversity for blood diseases.

The LCLAT1/LYCAT acyltransferase is required for EGF-mediated phosphatidylinositol-3,4,5-trisphosphate generation and Akt signaling.

Receptor tyrosine kinases such as EGF receptor (EGFR) stimulate phosphoinositide 3 kinases to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3ß. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signaling promotes tumor progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signaling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signaling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signaling.

Sodio en panes y snacks de mayor consumo en Costa Rica: Contenido basal y verificación del etiquetado nutricional / Sodium in breads and snacks of high consumption in Costa Rica: Basal content and verification of nutrition labeling

Una categoría de alimento de amplio consumo por la población es el pan, constituyendo una de las principales fuentes de sodio en la alimentación, a pesar de ser moderada en sal. Mientras, los snacks o bocadillos tienen un elevado contenido de sodio, pero son menos consumidos por la población general. El objetivo del presente trabajo fue determinar mediante análisis directo el contenido basal de sodio en panes y snacks de mayor consumo en Costa Rica y verificar el cumplimiento del Reglamento Técnico Centroamericano de Etiquetado Nutricional. Los alimentos se clasificaron según: tipo, marca comercial, método y lugar de producción. Las muestras se recolectaron en supermercados y panaderías del Gran Área Metropolitana entre el 2011 y el 2012. La muestra primaria la constituyeron 99 panes y 84 snacks, y la analítica de 33 y 28, respectivamente. El contenido de sodio se determinó mediante espectrofotometría de emisión de llama. Los panes presentaron entre 496 y 744 mg/100g de sodio, 45% incluían etiquetado nutricional y 80% declararon mayor cantidad que el encontrado por análisis directo. Los panes industrializados, excepto el integral cumplieron con la normativa. En los snacks el contenido de sodio osciló entre 276 y 1221 mg/100g, todos presentaron etiquetado nutricional y 43% declararon menor contenido que lo analizado, incumpliendo con la normativa. El estudio provee datos basales para iniciar la reducción de sodio y confirma que el análisis directo resulta indispensable para conocer con certeza el contenido de sodio en los alimentos.

Bread is highly consumed by population, making it one of the main sources of sodium in the diet, despite being moderate in salt. Mean while, snacks have high sodium content, but are less consumed by the general population. The aim of this study was to determine by direct analysis the baseline of sodium in breads and snacks most consumed in Costa Rica and verify compliance with the Central American Technical Regulation on Nutritional Labeling. Foods samples were classified by type, trade mark, method and place of production. Samples were collected in supermarkets and bakeries in the Great Metropolitan Area between 2011and 2012. Primary sample comprised 99 breads and 84 snacks, and analytical sample 33 and 28, respectively. The sodium content was determined by flame emission spectrophotometer. Breads showed between 496 and 744mg/100g sodium, 45% included nutritional labeling and 80% reported greater amount than found by direct analysis. Industrialized breads except the whole grain varieties, complied with regulations. In snacks, sodium content ranged from 276 to 1221mg/100g, all had nutritional labeling and 43% reported less content, in breach of the regulations. The study provides baseline data to initiate sodium reduction and direct analysis confirms that it is essential to know with certainty the sodium content in foods.

Contenido y consumo humano de aflatoxinas y cáncer hepatocelular en dos cantones de Costa Rica / Aflatoxin amount and human consumption and hepatocellular cancer in two costarrican counties

Se analizó la relación entre el contenido y el consumo de aflatoxinas del maíz de la dieta y el Cáncer Hepatocelular (CHC), con un estudio de Casos y Controles tomando 13 de los 15 individuos con CHC de San Ramón y Palmares de 1979 a 1983. Ambos cantones suman un 2,01 por ciento del área total del país, 2,44 por ciento de su población media, y 8,24 por ciento de los 182 casos de CHC del país en el período 79-83. En esos cantones la Tasa de Incidencia de CHC/100.000 habitantes (Ti) quinquenal fue 5,11; 4,22 veces más que la del país (Ti=1,21). Los 39 núcleos familiares formados por 13 Casos, 13 Controles de vencindario (V1) y 13 Controles institucionales (C2), consumían regularmente maíz o sus derivados. Los 26 núcleos de la zona San Ramón-Palmares (constituidos por 13 Casos y 13 C1) consumieron más maíz en grano que corresponden a las muestras menos contaminadas por aflatoxinas (x=24,5 partes por billón (ppb) según el método de Velasco). En los 13 C2 estuvieron los núcleos no residentes enla zona y consumieron más harina de maíz industrial siendo éstas las muestras con la mayor contaminación (x= 59,7 ppb). El consumo promedio diario de aflatoxinas fue x= 2,7 ug/día para los Casos, x= 2,7 ug/día para los C1 y x= 3,01 ug/día para los C2. No hubo relación estadísticamente significativa entre CHC, el consumo diario de aflatoxinas (p,05) o el contenido mayor a 20 ppb de aflotoxinas en el maíz de la dieta (p,05), por lo que se sugiere que otros agentes etiológicos, incluyendo al virus de la hepatitis B, podrían estar relacionados con la etiología del CHC en los casos de San Ramón y Palmares.