The rockefeller university

Purpose: Many cancer treatments suffer from dose-limiting toxicities to vital organs due to poor therapeutic indices. To overcome these challenges we developed a novel multimerization platform that rapidly removes tumor-targeting proteins from the blood to substantially improve therapeutic index. Experimental Design: The platform was designed as a fusion of a self-assembling and disassembling (SADA) domain to a tandem single-chain bispecific antibody (BsAb, anti-gangliosideGD2 x anti-DOTA). SADA-BsAbs were assessed with multiple in vivo tumor models using two-step pretargeted radioimmunotherapy (PRIT) to evaluate tumor uptake, dosimetry, and antitumor responses. Results: SADA-BsAbs self-assembled into stable tetramers (220 kDa), but could also disassemble into dimers or monomers (55 kDa) that rapidly cleared via renal filtration and substantially reduced immunogenicity in mice. When used with rapidly clearing DOTA-caged PET isotopes, SADA-BsAbs demonstrated accurate tumor localization, dosimetry, and improved imaging contrast by PET/CT. When combined with therapeutic isotopes, two-step SADA-PRIT safely delivered massive doses of alpha-emitting (Ac-225, 1.48 MBq/kg) or beta-emitting (Lu-177, 6,660 MBq/kg) S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (DOTA) payloads to tumors, ablating them without any short-term or long-term toxicities to the bone marrow, kidneys, or liver. Conclusions: The SADA-BsAb platform safely delivered large doses of radioisotopes to tumors and demonstrated no toxicities to the bone marrow, kidneys, or liver. Because of its modularity, SADA-BsAbs can be easily adapted to most tumor antigens, tumor types, or drug delivery approaches to improve therapeutic index and maximize the delivered dose.

Precision tools for spatiotemporal control of cytoskeletal motor function are needed to dissect fundamental biological processes ranging from intracellular transport to cell migration and division. Direct optical control of motor speed and direction is one promising approach, but it remains a challenge to engineer controllable motors with desirable properties such as the speed and processivity required for transport applications in living cells. Here, we develop engineered myosin motors that combine large optical modulation depths with high velocities, and create processive myosin motors with optically controllable directionality. We characterize the performance of the motors using in vitro motility assays, single-molecule tracking and live-cell imaging. Bidirectional processive motors move efficiently toward the tips of cellular protrusions in the presence of blue light, and can transport molecular cargo in cells. Robust gearshifting myosins will further enable programmable transport in contexts ranging from in vitro active matter reconstitutions to microfabricated systems that harness molecular propulsion.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides with little or no protein coding potential. The expanding list of lncRNAs and accumulating evidence of their functions in plants have necessitated the creation of a comprehensive database for lncRNA research. However, currently available plant lncRNA databases have some deficiencies, including the lack of lncRNA data from some model plants, uneven annotation standards, a lack of visualization for expression patterns, and the absence of epigenetic information. To overcome these problems, we upgraded our Plant Long noncoding RNA Database (PLncDB, http://plncdb. tobaccodb.org/), which was based on a uniform annotation pipeline. PLncDB V2.0 currently contains 1 246 372 lncRNAs for 80 plant species based on 13 834 RNA-Seq datasets, integrating lncRNA information from four other resources including EVLncRNAs, RNAcentral and etc. Expression patterns and epigenetic signals can be visualized using multiple tools (JBrowse, eFP Browser and EPexplorer). Targets and regulatory networks for lncRNAs are also provided for function exploration. In addition, PLncDB V2.0 is hierarchical and user-friendly and has five builtin search engines. We believe PLncDB V2.0 is useful for the plant lncRNA community and data mining studies and provides a comprehensive resource for data-driven lncRNA research in plants.

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing similar to 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

Despite the strong genetic basis of psychiatric disorders, the underlying molecular mechanisms are largely unmapped. RNA-binding proteins (RBPs) are responsible for most post-transcriptional regulation, from splicing to translation to localization. RBPs thus act as key gatekeepers of cellular homeostasis, especially in the brain. However, quantifying the pathogenic contribution of noncoding variants impacting RBP target sites is challenging. Here, we leverage a deep learning approach that can accurately predict the RBP target site dysregulation effects of mutations and discover that RBP dysregulation is a principal contributor to psychiatric disorder risk. RBP dysregulation explains a substantial amount of heritability not captured by large-scale molecular quantitative trait loci studies and has a stronger impact than common coding region variants. We share the genome-wide profiles of RBP dysregulation, which we use to identify DDHD2 as a candidate schizophrenia risk gene. This resource provides a new analytical framework to connect the full range of RNA regulation to complex disease.

The widely abused prescription opioid oxycodone is a mu-opioid receptor (MOP-r) agonist and addiction to such opioids is a relapsing disorder. The human MOP-r gene (OPRM1) has an important functional single nucleotide polymorphism (SNP), A118G, which affects risk of severe opioid use disorders. A112G (G/G) knock-in mice are models of human A118G carriers. We examined oxycodone self-administration (SA) in male and female G/G versus wild type (A/A) mice in SA sessions and in relapse-like behavior. Adult male and female G/G and A/A mice self-administered oxycodone (0.25 mg/kg/infusion, FR1) for 10 consecutive days. Following 10-day home cage drug free withdrawal, the mice were re-exposed to oxycodone SA for a further 10 days. MOP-r receptor mRNA in various brain regions were examined immediately after the last re-exposure session. We found that G/G mice had greater oxycodone SA than A/A mice in the initial and in re-exposure sessions. Mice of both genotypes had greater oxycodone intake during the re-exposure period than during the initial exposure. We also detected differences in MOP-r gene expression due to genotype, sex and oxycodone SA history in the dorsal striatum, hippocampus, and prefrontal cortex. These studies may improve our understanding of MOP-r-agonist self-exposure and relapse in human carriers of the A118G SNP.

The 14 authors of the first review article on hidradenitis suppurativa (HS) pathogenesis published 2008 in EXPERIMENTAL DERMATOLOGY cumulating from the 1st International Hidradenitis Suppurativa Research Symposium held March 30-April 2, 2006 in Dessau, Germany with 33 participants were prophetic when they wrote "Hopefully, this heralds a welcome new tradition: to get to the molecular heart of HS pathogenesis, which can only be achieved by a renaissance of solid basic HS research, as the key to developing more effective HS therapy." (Kurzen et al. What causes hidradenitis suppurativa? Exp Dermatol 2008;17:455). Fifteen years later, there is no doubt that the desired renaissance of solid basic HS research is progressing with rapid steps and that HS has developed deep roots among inflammatory diseases in Dermatology and beyond, recognized as "the only inflammatory skin disease than can be healed". This anniversary article of 43 research-performing authors from all around the globe in the official journal of the European Hidradenitis Suppurativa Foundation e.V. (EHSF e.V.) and the Hidradenitis Suppurativa Foundation, Inc (HSF USA) summarizes the evidence of the intense HS clinical and experimental research during the last 15 years in all aspects of the disease and provides information of the developments to come in the near future.

Limitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.

Cancer cells rewire their metabolism and rely on endogenous antioxidants to mitigate lethal oxidative damage to lipids. However, the metabolic processes that modulate the response to lipid peroxidation are poorly defined. Using genetic screens, we compared metabolic genes essential for proliferation upon inhibition of cystine uptake or glutathione peroxidase-4 (GPX4). Interestingly, very few genes were commonly required under both conditions, suggesting that cystine limitation and GPX4 inhibition may impair proliferation via distinct mechanisms. Our screens also identify tetrahydrobiopterin (BH4) biosynthesis as an essential metabolic pathway upon GPX4 inhibition. Mechanistically, BH4 is a potent radical-trapping antioxidant that protects lipid membranes from autoxidation, alone and in synergy with vitamin E. Dihydrofolate reductase catalyzes the regeneration of BH4, and its inhibition by methotrexate synergizes with GPX4 inhibition. Altogether, our work identifies the mechanism by which BH4 acts as an endogenous antioxidant and provides a compendium of metabolic modifiers of lipid peroxidation. Genetic screens reveal a compendium of metabolic modifiers of lipid peroxidation. Tetrahydrobiopterin is essential under GPX4 inhibition, acting as a radical-trapping antioxidant that inhibits lipid peroxidation and is regenerated by DHFR.

Through recurrent bouts synchronous with the hair cycle, quiescent melanocyte stem cells (McSCs) become activated to generate proliferative progeny that differentiate into pigment-producing melanocytes. The signaling factors orchestrating these events remain incompletely understood. Here, we use single-cell RNA sequencing with comparative gene expression analysis to elucidate the transcriptional dynamics of McSCs through quiescence, activation, and melanocyte maturation. Unearthing converging signs of increased WNT and BMP signaling along this progression, we endeavored to understand how these pathways are integrated. Employing conditional lineage-specific genetic ablation studies in mice, we found that loss of BMP signaling in the lineage leads to hair graying due to a block in melanocyte maturation. We show that interestingly, BMP signaling functions downstream from activated McSCs and maintains WNT effector, transcription factor LEF1. Employing pseudotime analysis, genetics, and chromatin landscaping, we show that following WNT-mediated activation of McSCs, BMP and WNT pathways collaborate to trigger the commitment of proliferative progeny by fueling LEF1and MITF-dependent differentiation. Our findings shed light upon the signaling interplay and timing of cues that orchestrate melanocyte lineage progression in the hair follicle and underscore a key role for BMP signaling in driving complete differentiation.