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Technologies capable of programmable translation activation offer strategies to develop therapeutics for diseases caused by insufficient gene expression. Here, we present “translation-activating RNAs” (taRNAs), a bifunctional RNA-based molecular technology that binds to a specific mRNA of interest and directly upregulates its translation. taRNAs are constructed from a variety of viral or mammalian RNA internal ribosome entry sites (IRESs) and upregulate translation for a suite of target mRNAs. We minimize the taRNA scaffold to 94 nucleotides, identify two translation initiation factor proteins responsible for taRNA activity, and validate the technology by amplifying SYNGAP1 expression, a haploinsufficiency disease target, in patient-derived cells. Finally, taRNAs are suitable for delivery as RNA molecules by lipid nanoparticles (LNPs) to cell lines, primary neurons, and mouse liver in vivo. taRNAs provide a general and compact nucleic acid-based technology to upregulate protein production from endogenous mRNAs, and may open up possibilities for therapeutic RNA research. Many diseases are driven by the insufficient expression of critical genes, but few technologies are capable of rescuing these endogenous protein levels. Here, Cao et al. present an RNA-based technology that boosts protein production from endogenous mRNAs by upregulating their translation.

De binder discovery is unpredictable and inefficient due to a lack of quantitative understanding of protein-protein interaction (PPI) sequence-function landscapes. Here, we use our PANCS-Binder technology to perform >1,300 independent selections of various library sizes and compositions of a randomized small protein to identify binders to a panel of 96 distinct target proteins. For successful selections, we discovered reproducible fitness landscapes that group into a few, target-specific, clusters. Each cluster defines a minimal binding motif whose frequency is inversely proportional to the number of specified amino acids (∼2-8) and determines selection success, which is quantifiable by the density of binders to the target within a theoretical sequence space. We leverage these data to develop a supervised contrastive learning approach that discriminates binders from non-binders and demonstrates generalization beyond a threshold amount of data. Together, this framework renders PPI landscapes measurable and predictive, accelerating binder discovery and optimization.

Proteins that selectively bind to a target of interest are foundational components of research pipelines , diagnostics , and therapeutics . Current immunization-based , display-based , and computational approaches for discovering binders are laborious and time-consuming - taking months or more, suffer from high false positives - necessitating extensive secondary screening, and have a high failure rate, especially for disordered proteins and other challenging target classes. Here we establish Phage-Assisted Non-Continuous Selection of Protein Binders (PANCS-binders), an selection platform that links the life cycle of M13 phage to target protein binding though customized proximity-dependent split RNA polymerase biosensors, allowing for complete and comprehensive high-throughput screening of billion-plus member protein variant libraries with high signal-to-noise. We showcase the utility of PANCS-Binders by screening multiple protein libraries each against a panel of 95 separate therapeutically relevant targets, thereby individually assessing over 10 protein-protein interaction pairs, completed in two days. These selections yielded large, high-quality datasets and hundreds of novel binders, which we showed can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. PANCS-Binders dramatically accelerates and simplifies the binder discovery process, the democratization of which will help unlock new creative potential in proteome-targeting with engineered binder-based biotechnologies.

Fe-containing transition-metal (oxy)hydroxides are highly active oxygen-evolution reaction (OER) electrocatalysts in alkaline media and ubiquitously form across many materials systems. The complexity and dynamics of the Fe sites within the (oxy)hydroxide have slowed understanding of how and where the Fe-based active sites form—information critical for designing catalysts and electrolytes with higher activity and stability. We show that where/how Fe species in the electrolyte incorporate into host Ni or Co (oxy)hydroxides depends on the electrochemical history and structural properties of the host material. Substantially less Fe is incorporated from Fe-spiked electrolyte into Ni (oxy)hydroxide at anodic potentials, past the nominally Ni 2+/3+ redox wave, compared to during potential cycling. The Fe adsorbed under constant anodic potentials leads to impressively high per-Fe OER turn-over frequency (TOF Fe ) of ~40 s −1 at 350 mV overpotential which we attribute to under-coordinated “surface” Fe. By systematically controlling the concentration of surface Fe, we find TOF Fe increases linearly with the Fe concentration. This suggests a changing OER mechanism with increased Fe concentration, consistent with a mechanism involving cooperative Fe sites in FeO x clusters. Fe-based oxyhydroxides are promising catalysts for oxygen evolution reactions. However, the dynamics of Fe sites require further investigation. Here, the authors report Fe oxyhydroxide clusters on nickel or cobalt oxyhydroxides as key active sites for water oxidation and reveal how these clusters are selectively formed under controlled electrochemical conditions.

Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with epithelial identity is obscure. Here, we use single cell transcriptomics to generate a high-resolution cell state map of the embryonic mouse foregut. This identifies a diversity of SM cell types that develop in close register with the organ-specific epithelium. We infer a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. We validate key predictions with mouse genetics, showing the importance of endoderm-derived signals in mesoderm patterning. Finally, leveraging these signaling interactions, we generate different SM subtypes from human pluripotent stem cells (hPSCs), which previously have been elusive. The single cell data can be explored at: https://research.cchmc.org/ZornLab-singlecell . The fetal murine foregut develops into visceral organs via interactions between the mesoderm and endoderm, but how is unclear. Here, the authors use single cell RNAseq to show a diversity in organ specific splanchnic mesoderm cell-types, infer a signalling network governing organogenesis and use this to differentiate human pluripotent stem cells.

The cultivated [Glycine max (L) Merr.] and wild [Glycine soja Siebold & Zucc.] soybean species comprise wide variation in seed composition traits. Compared to wild soybean, cultivated soybean contains low protein, high oil and high sucrose. In this study, an inter-specific population was derived from a cross between G. max (Williams 82) and G. soja (PI 483460B). This recombinant inbred line (RIL) population of 188 lines was sequenced at 0.3x depth. Based on 91,342 single nucleotide polymorphisms (SNPs), recombination events in RILs were defined, and a high-resolution bin map was developed (4,070 bins). In addition to bin mapping, QTL analysis for protein, oil and sucrose was performed using 3,343 polymorphic SNPs (3K-SNP), derived from Illumina Infinium BeadChip sequencing platform. The QTL regions from both platforms were compared and a significant concordance was observed between bin and 3K-SNP markers. Importantly, the bin map derived from next generation sequencing technology enhanced mapping resolution (from 1325 Kb to 50 Kb). A total of 5, 9 and 4 QTLs were identified for protein, oil and sucrose content, respectively and some of the QTLs coincided with soybean domestication related genomic loci. The major QTL for protein and oil was mapped on Chr. 20 (qPro_20) and suggested negative correlation between oil and protein. In terms of sucrose content, a novel and major QTL was identified on Chr. 8 (qSuc_08) and harbors putative genes involved in sugar transport. In addition, genome-wide association (GWAS) using 91,342 SNPs confirmed the genomic loci derived from QTL mapping. A QTL based haplotype using whole genome resequencing of 106 diverse soybean lines identified unique allelic variation in wild soybean that could be utilized to widen the genetic base in cultivated soybean.

Immune checkpoint inhibitors are associated with immune-related adverse events (irAEs), including arthritis (arthritis-irAE). Management of arthritis-irAE is challenging because immunomodulatory therapy for arthritis should not impede antitumor immunity. Understanding of the mechanisms of arthritis-irAE is critical to overcome this challenge, but the pathophysiology remains unknown. Here, we comprehensively analyze peripheral blood and/or synovial fluid samples from 20 patients with arthritis-irAE, and unmask a prominent Th1-CD8 + T cell axis in both blood and inflamed joints. CX3CR1 hi CD8 + T cells in blood and CXCR3 hi CD8 + T cells in synovial fluid, the most clonally expanded T cells, significantly share TCR repertoires. The migration of blood CX3CR1 hi CD8 + T cells into joints is possibly mediated by CXCL9/10/11/16 expressed by myeloid cells. Furthermore, arthritis after combined CTLA-4 and PD-1 inhibitor therapy preferentially has enhanced Th17 and transient Th1/Th17 cell signatures. Our data provide insights into the mechanisms, predictive biomarkers, and therapeutic targets for arthritis-irAE. Arthritis is the most common rheumatic immune-related adverse event (irAE) occurring in cancer patients receiving immune checkpoint inhibitors. Here the authors study the immune landscape of blood and synovial fluid samples from patients with arthritis-irAE, reporting immunological differences and similarities with classic autoimmune arthritis.

Background Health-related behaviours (HRBs) cluster within individuals. Evidence for the association between HRB clustering and cognitive functioning is limited. We aimed to examine and compare the associations between three HRB clusters: “multi-HRB cluster”, “inactive cluster” and “(ex-)smoking cluster” (identified in previous work based on HRBs including smoking, alcohol consumption, physical activity and social activity) and episodic memory trajectories among men and women, separately, in the United States of America (USA) and England. Methods Data were from the waves 10–14 (2010–2018) of the Health and Retirement Study in the USA and the waves 5–9 (2010–2018) of the English Longitudinal Study of Ageing in England. We included 17,750 US and 8,491 English participants aged 50 years and over. The gender-specific HRB clustering was identified at the baseline wave in 2010, including the multi-HRB (multiple positive behaviours), inactive and ex-smoking clusters in both US and English women, the multi-HRB, inactive and smoking clusters in US men, and only the multi-HRB and inactive clusters in English men. Episodic memory was measured by a sum score of immediate and delayed word recall tests across waves. For within country associations, a quadratic growth curve model (age-cohort model, allowing for random intercepts and slopes) was applied to assess the gender-stratified associations between HRB clustering and episodic memory trajectories, considering a range of confounding factors. For between country comparisons, we combined country-specific data into one pooled dataset and generated a country variable (0 = USA and 1 = England), which allowed us to quantify between-country inequalities in the trajectories of episodic memory over age across the HRB clusters. This hypothesis was formally tested by examining a quadratic growth curve model with the inclusion of a three-way interaction term (age × HRB clustering × country). Results We found that within countries, US and English participants within the multi-HRB cluster had higher scores of episodic memory than their counterparts within the inactive and (ex-)smoking clusters. Between countries, among both men and women within each HRB cluster, faster declines in episodic memory were observed in England than in the USA (e.g., b England versus the USA for men: multi-HRB cluster  = -0.05, 95%CI: -0.06, -0.03, b England versus the USA for women: ex-smoking cluster  = -0.06, 95%CI: -0.07, -0.04). Additionally, the range of mean memory scores was larger in England than in the USA when comparing means between two cluster groups, including the range of means between inactive and multi-HRB cluster for men (b England versus the USA  = -0.56, 95%CI: -0.85, -0.27), and between ex-smoking and multi-HRB cluster for women (b England versus the USA  = -1.73, 95%CI: -1.97, -1.49). Conclusions HRB clustering was associated with trajectories of episodic memory in both the USA and England. The effect of HRB clustering on episodic memory seemed larger in England than in the USA. Our study highlighted the importance of being aware of the interconnections between health behaviours for a better understanding of how these behaviours affect cognitive health. Governments, particularly in England, could pay more attention to the adverse effects of health behaviours on cognitive health in the ageing population.

In patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study. We prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient's tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients' tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/- adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing. We confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM. ACTRN12612000345886.

Background Neuroinflammation associated with advanced human immunodeficiency virus (HIV)-1 infection is often exacerbated by chronic cocaine abuse. Cocaine exposure has been demonstrated to mediate up-regulation of inflammatory mediators in in vitro cultures of microglia. The molecular mechanisms involved in this process, however, remain poorly understood. In this study, we sought to explore the underlying signaling pathways involved in cocaine-mediated activation of microglial cells. Methods BV2 microglial cells were exposed to cocaine and assessed for toll-like receptor (TLR2) expression by quantitative polymerase chain reaction (qPCR), western blot, flow cytometry, and immunofluorescence staining. The mRNA and protein levels of cytokines (TNFα, IL-6, MCP-1) were detected by qPCR and ELISA, respectively; level of reactive oxygen species (ROS) production was examined by the Image-iT LIVE Green ROS detection kit; activation of endoplasmic reticulum (ER)-stress pathways were detected by western blot. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of activating transcription factor 4 (ATF4) with the TLR2 promoter. Immunoprecipitation followed by western blotting with tyrosine antibody was used to determine phosphorylation of TLR2. Cocaine-mediated up-regulation of TLR2 expression and microglial activation was validated in cocaine-injected mice. Results Exposure of microglial cells to cocaine resulted in increased expression of TLR2 with a concomitant induction of microglial activation. Furthermore, this effect was mediated by NADPH oxidase-mediated rapid accumulation of ROS with downstream activation of the ER-stress pathways as evidenced by the fact that cocaine exposure led to up-regulation of pPERK/peIF2α/ATF4 and TLR2. The novel role of ATF4 in the regulation of TLR2 expression was confirmed using genetic and pharmacological approaches. Conclusions xThe current study demonstrates that cocaine-mediated activation of microglia involves up-regulation of TLR2 through the ROS-ER stress-ATF4-TLR2 axis. Understanding the mechanism(s) involved in cocaine-mediated up-regulation of ROS-ER stress/TLR2 expression and microglial activation could have implications for the development of potential therapeutic targets aimed at resolving neuroinflammation in cocaine abusers.