XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells.

X chromosome inactivation (XCI) serves as a paradigm for RNA-mediated regulation of gene expression, wherein the long non-coding RNA XIST spreads across the X chromosome in cis to mediate gene silencing chromosome-wide. In female naive human pluripotent stem cells (hPSCs), XIST is in a dispersed configuration, and XCI does not occur, raising questions about XIST's function. We found that XIST spreads across the X chromosome and induces dampening of X-linked gene expression in naive hPSCs. Surprisingly, XIST also targets specific autosomal regions, where it induces repressive chromatin changes and gene expression dampening. Thereby, XIST equalizes X-linked gene dosage between male and female cells while inducing differences in autosomes. The dispersed Xist configuration and autosomal localization also occur transiently during XCI initiation in mouse PSCs. Together, our study identifies XIST as the regulator of X chromosome dampening, uncovers an evolutionarily conserved trans-acting role of XIST/Xist, and reveals a correlation between XIST/Xist dispersal and autosomal targeting.

Mitogen-activated protein kinase signaling causes malignant melanoma cells to differentially alter extracellular matrix biosynthesis to promote cell survival.

BACKGROUND: Intrinsic and acquired resistance to drug therapies remains a challenge for malignant melanoma patients. Intratumoral heterogeneities within the tumor microenvironment contribute additional complexity to the determinants of drug efficacy and acquired resistance. METHODS: We use 3D biomimetic platforms to understand dynamics in extracellular matrix (ECM) biogenesis following pharmaceutical intervention against mitogen-activated protein kinases (MAPK) signaling. We further determined temporal evolution of secreted ECM components by isogenic melanoma cell clones. RESULTS: We found that the cell clones differentially secrete and assemble a myriad of ECM molecules into dense fibrillar and globular networks. We show that cells can modulate their ECM biosynthesis in response to external insults. Fibronectin (FN) is one of the key architectural components, modulating the efficacy of a broad spectrum of drug therapies. Stable cell lines engineered to secrete minimal levels of FN showed a concomitant increase in secretion of Tenascin-C and became sensitive to BRAF(V600E) and ERK inhibition as clonally- derived 3D tumor aggregates. These cells failed to assemble exogenous FN despite maintaining the integrin machinery to facilitate cell- ECM cross-talk. We determined that only clones that increased FN production via p38 MAPK and ß1 integrin survived drug treatment. CONCLUSIONS: These data suggest that tumor cells engineer drug resistance by altering their ECM biosynthesis. Therefore, drug treatment may induce ECM biosynthesis, contributing to de novo resistance.

Effects of linezolid on suppressing in vivo production of staphylococcal toxins and improving survival outcomes in a rabbit model of methicillin-resistant Staphylococcus aureus necrotizing pneumonia.

BACKGROUND: Linezolid is recommended for treatment of pneumonia and other invasive infections caused by methicillin-resistant Staphylococcus aureus (MRSA). The premise underlying this recommendation is that linezolid inhibits in vivo production of potent staphylococcal exotoxins, including Panton-Valentine leukocidin (PVL) and α-hemolysin (Hla), although supporting evidence is lacking. METHODS: A rabbit model of necrotizing pneumonia using MRSA clone USA300 was used to compare therapeutic effects of linezolid (50 mg/kg 3 times/day) and vancomycin (30 mg/kg 2 times/day) administered 1.5, 4, and 9 hours after infection on host survival outcomes and in vivo bacterial toxin production. RESULTS: Mortality rates were 100% for untreated rabbits and 83%-100% for vancomycin-treated rabbits. In contrast, mortality rates were 25%, 50%, and 100% for rabbits treated with linezolid 1.5, 4, and 9 hours after infection, respectively. Compared with untreated and vancomycin-treated rabbits, improved survival of rabbits treated 1.5 hours after infection with linezolid was associated with a significant decrease in bacterial counts, suppressed bacterial production of PVL and Hla, and reduced production of the neutrophil-chemoattractant interleukin 8 in the lungs. CONCLUSIONS: Across the study interval, only early treatment with linezolid resulted in significant suppression of exotoxin synthesis and improved survival outcomes in a rabbit model of MRSA necrotizing pneumonia.

Optimization of RNA Pepper Sensors for the Detection of Arbitrary RNA Targets.

The development of fluorescent light-up RNA aptamers (FLAPs) has paved the way for the creation of sensors to track RNA in live cells. A major challenge with FLAP sensors is their brightness and limited signal-to-background ratio both in vivo and in vitro. To address this, we develop sensors using the Pepper aptamer, which exhibits superior brightness and photostability when compared to other FLAPs. The sensors are designed to fold into a low fluorescence conformation and to switch to a high fluorescence conformation through toehold or loop-mediated interactions with their RNA target. Our sensors detect RNA targets as short as 20 nucleotides in length with a wide dynamic range over 300-fold in vitro, and we describe strategies for optimizing the sensor's performance for any given RNA target. To demonstrate the versatility of our design approach, we generated Pepper sensors for a range of specific, biologically relevant RNA sequences. Our design and optimization strategies are portable to other FLAPs and offer a promising foundation for future development of RNA sensors with high specificity and sensitivity for detecting RNA biomarkers with multiple applications.