RESUMEN
Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation.
Asunto(s)
Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Factor Nuclear Tiroideo 1/metabolismo , Células Epiteliales Alveolares , Diferenciación Celular , Humanos , Pulmón , OrganoidesRESUMEN
Electrode materials play an important role on the electrocatalytic properties of immobilized biocatalysts. In this regard, achieving direct electronic communication between the electrode and redox sites of biocatalysts eliminates the need for additional electron transfer mediators for biocatalytic applications in fuel cells and other electrochemical energy devices. In order to increase electrocatalytic currents and power in fuel cells and metal-air batteries, conductive carbon-nanostructure-modified large surface area electrodes are quite useful. Among various electrode materials, freestanding buckypapers made from carbon nanotubes have gained significance as they do not require a solid support material and thus facilitate miniaturization. In this article, we present the effect of buckypaper (BP) thickness on the electrocatalytic properties of a bilirubin oxidase (BOD) enzyme. In this study, we prepared BPs of varying thicknesses ranging from 87 µm, the minimum thickness for suitable handling with a good stability in aqueous experiments, to 380 µm. BOD was adsorbed overnight onto the BPs, mostly via hydrophobic and π-π interactions since the nanotubes used were not chemically functionalized. Furthermore, intercalation of the BOD molecules onto the nanotubes' multicylindrical network is feasible. We determined that the lower range BP thickness (<220 µm) exhibited better sigmoidal shaped electrocatalytic currents than the higher BP-thickness-based BOD biofilms with larger capacitive currents. An oxygen reduction current density of up to 3 mA cm-2 is achieved without the use of any redox mediators or tedious electrode modifications. Using the 87 µm thick BP as the representative case, we were able to obtain distinguishable peaks for all Cu sites of BOD and assign their types, T1, T2, and T3, based on the peak-width at half-maximum in anaerobic cyclic voltammograms. Our peak assignment is further supported by the appearance of dual electrocatalytic oxygen reduction waves at a higher scan rate region (>10 mV s-1) in oxygen-saturated buffer, which is identified to be driven by an â¼3.5 times faster electron transfer rate from the buckypaper to the T2/T3 center than the T1 Cu site. Findings from this study are significant for designing enzyme electrocatalytic systems and biosensors in general and fuel cells and aerobic energy storage devices in particular, where the cathodic oxygen reduction current is often inadequate.
RESUMEN
A rapid optical microarray imaging approach for anticancer drug screening at specific cancer protein-protein interface targets with binding kinetics and validation by a mass sensor is reported for the first time. Surface plasmon resonance imager (SPRi) demonstrated a 3.5-fold greater specificity for interactions between murine double minute 2 protein (MDM2) and wild-type p53 over a nonspecific p53 mutant in a real-time microfluidic analysis. Significant percentage reflectivity changes (Δ%R) in the SPRi signals and molecular-level mass changes were detected for both the MDM2-p53 interaction and its inhibition by a small-molecule Nutlin-3 drug analogue known for its anticancer property. We additionally demonstrate that synthetic, inexpensive binding domains of interacting cancer proteins are sufficient to screen anticancer drugs by an array-based SPRi technique with excellent specificity and sensitivity. This imaging array, combined with a mass sensor, can be used to study quantitatively any protein-protein interaction and screen for small molecules with binding and potency evaluations.