Fluorescent ubiquitination-based cell cycle indicator (FUCCI) embryonic stem cells (ESCs), which fluoresce green during the S-G2-M phases, generate an S-shaped curve for the accumulation of cells during normal stemness (NS) culture with leukemia-inhibitory factor (LIF). Since it was hypothesized that a culture of ESCs was heterogeneous in the cell cycle, it was expected that increased S-G2-M-phases of the cell cycle would make an S-shaped curve parallel to the accumulation curve. Unexpectedly, it was observed that the fraction of FUCCI ESCs in green decreases over time to a nadir at ∼24 h after previous feeding and then rapidly enters S-G2-M-phases after medium change. G1 delay by infrequent medium change is a mild stress, as it does not affect growth significantly when frequency is increased to 12 h. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) were used as examples of members of the per- and polyfluoroalkyl substances (PFAS) and phthalate families of chemicals, respectively. Two adverse outcomes were used to compare dose- and time-dependent effects of PFOA and DEP. The first was cell accumulation assay by time-lapse confluence measurements, largely at Tfinal/T74 h. The second was by quantifying dominant toxicant stress shown by the suppression of mild stress that creates a green fed/unfed peak. In terms of speed, PFOA is 26 times faster than DEP for producing a time-dependent LOAEL dose at 100 uM (that is, 2 h for PFOA and 52 h for DEP). PFOA has 1000-fold more sensitive LOAEL doses than DEP for suppressing ESC accumulation (confluence) at day 3 and day 2. There were two means to compare the magnitude of the growth suppression of PFOA and DEP. For the suppression of the accumulation of cells measured by confluence at Tfinal/T74h, there was a 13-fold suppression at the highest dose of PFOA > the highest dose of DEP. For the suppression of entry into the cell cycle after the G1 phase by stress on day 1 and 2, there is 10-fold more suppression by PFOA than DEP. The data presented here suggest that FUCCI ESCs can assay the suppression of accumulated growth or predict the suppression of future growth by the suppression of fed/unfed green fluorescence peaks and that PFOA’s adverse effects are faster and larger and can occur at more sensitive lower doses than DEP.
An efficient immune defense against pathogens requires sufficient basal sensing mechanisms that can deliver prompt responses. Type I IFNs are protective against acute viral infections and respond to viral and bacterial infections, but their efficacy depends on constitutive basal activity that promotes the expression of downstream genes known as IFN-stimulated genes (ISGs). Type I IFNs and ISGs are constitutively produced at low quantities and yet exert profound effects essential for numerous physiological processes beyond antiviral and antimicrobial defense, including immunomodulation, cell cycle regulation, cell survival, and cell differentiation. Although the canonical response pathway for type I IFNs has been extensively characterized, less is known regarding the transcriptional regulation of constitutive ISG expression. Zika virus (ZIKV) infection is a major risk for human pregnancy complications and fetal development and depends on an appropriate IFN-β response. However, it is poorly understood how ZIKV, despite an IFN-β response, causes miscarriages. We have uncovered a mechanism for this function specifically in the context of the early antiviral response. Our results demonstrate that IFN regulatory factor (IRF9) is critical in the early response to ZIKV infection in human trophoblast. This function is contingent on IRF9 binding to Twist1. In this signaling cascade, Twist1 was not only a required partner that promotes IRF9 binding to the IFN-stimulated response element but also an upstream regulator that controls basal levels of IRF9. The absence of Twist1 renders human trophoblast cells susceptible to ZIKV infection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.