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Autoantibodies target the RNA binding protein Ro60 in systemic lupus erythematosus (SLE) and Sjögren's syndrome. However, it is unclear whether Ro60 and its associated RNAs contribute to disease pathogenesis. We catalogued the Ro60-associated RNAs in human cell lines and found that among other RNAs, Ro60 bound an RNA motif derived from endogenous Alu retroelements. Alu transcripts were induced by type I interferon and stimulated proinflammatory cytokine secretion by human peripheral blood cells. Ro60 deletion resulted in enhanced expression of Alu RNAs and interferon-regulated genes. Anti-Ro60-positive SLE immune complexes contained Alu RNAs, and Alu transcripts were up-regulated in SLE whole blood samples relative to controls. These findings establish a link among the lupus autoantigen Ro60, Alu retroelements, and type I interferon.

We present the average pressure profile of 461 galaxy clusters from the 2500d South Pole Telescope catalogue measured in the Planck and SPT-SZ data jointly. The joint measurement is performed in Fourier space for the first time, and takes advantage of the complementarity between the high resolution of SPT (1.75 arcmin beam) and the coverage of large angular scales by Planck (> 30 arcmin). The redshift leverage of the catalogue allows the study of the evolution of the profile. We also constrain the profile shape in the outskirts.

The neocortex encodes complex and simple motor outputs in all mammalian species that have been tested. Given that changes in neocortical reorganization (and corresponding corticospinal output) have been implicated in long term motor recovery after stroke injury, there remains a need to understand this biology in order to expedite and optimize clinical care. Here, changes in the neocortical topography of complex and simple movement outputs were evaluated in mice following experimental middle cerebral artery occlusion (MCAo). Neocortical motor output was defined using long-duration parameters of intracortical microstimulation (LD-ICMS) based on area and spatial coordinates of separate motor output types to build upon our recent report in uninjured mice. LD-ICMS test sites that elicited complex (multi-joint) movement, simple (single skeletal joint) movement, as well as co-elicited FORELIMB + HINDLIMB responses were detected and recorded. Forelimb reaching behavior was assessed using the single pellet reaching (SPR) task. At 6 weeks post-surgery, behavioral deficits persisted and neocortical territories for separate movements exhibited differences in neocortical area, and spatial location, and differed between MCAo-Injured animals (i.e., the MCAo group) and Sham-Injured animals (i.e., the Control group). MCAo-Injury reduced neocortical area of complex movements while increasing area of simple movements. Limited effects of injury were detected for spatial coordinates of neocortical movements. Significant positive correlations were detected between final SPR performance and either area of complex retract or area of co-occurring FORELIMB + HINDLIMB sites., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wolsh, Brown, Brown, Pratt and Boychuk.)

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays., Highlights: DuplexSeq is an ultra-accurate NGS technology that directly quantifies mutationsENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissuesMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposureResults obtained with DuplexSeq were highly concordant between laboratoriesThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology.

Background: Decompensated hemorrhagic shock (DHS) is the leading cause of preventable death in combat casualties. "Golden hour" resuscitation effects on cerebral blood flow and perfusion following DHS in prolonged field care (PFC) are not well investigated. Using an established non-human primate model of DHS, we hypothesized noninvasive regional tissue oxygenation (rSO2) and Transcranial Doppler (TCD) would correlate to the invasive measurement of partial pressure of oxygen (PtO2) and mean arterial pressure (MAP) in guiding hypotensive resuscitation in a PFC setting., Methods: Ten rhesus macaques underwent DHS followed by a 2 h PFC phase (T0-T120), and subsequent 4 h hospital resuscitation phase (T120-T360). Invasive monitoring (PtO2, MAP) was compared against noninvasive monitoring systems (rSO2, TCD). Results were analyzed using t tests and one-way repeated measures ANOVA. Linear correlation was determined via Pearson r. Significance = P < 0.05., Results: MAP, PtO2, rSO2, and mean flow velocity (MFV) significantly decreased from baseline at T0. MAP and PtO2 were restored to baseline by T15, while rSO2 was delayed through T30. At T120, MFV returned to baseline, while the Pulsatility Index significantly elevated by T120 (1.50 ± 0.31). PtO2 versus rSO2 (R2 = 0.2099) and MAP versus MFV (R2 = 0.2891) shared very weak effect sizes, MAP versus rSO2 (R2 = 0.4636) displayed a low effect size, and PtO2 versus MFV displayed a moderate effect size (R2 = 0.5540)., Conclusions: Though noninvasive monitoring methods assessed here did not correlate strongly enough against invasive methods to warrant a surrogate in the field, they do effectively augment and direct resuscitation, while potentially serving as a substitute in the absence of invasive capabilities., Competing Interests: The authors report no conflicts of interest., (Copyright © 2020 by the Shock Society.)

The molecular functions of the majority of RNA-binding proteins (RBPs) remain unclear, highlighting a major bottleneck to a full understanding of gene expression regulation. Here, we develop a plasmid resource of 690 human RBPs that we subject to luciferase-based 3'-untranslated-region tethered function assays to pinpoint RBPs that regulate RNA stability or translation. Enhanced UV-cross-linking and immunoprecipitation of these RBPs identifies thousands of endogenous mRNA targets that respond to changes in RBP level, recapitulating effects observed in tethered function assays. Among these RBPs, the ubiquitin-associated protein 2-like (UBAP2L) protein interacts with RNA via its RGG domain and cross-links to mRNA and rRNA. Fusion of UBAP2L to RNA-targeting CRISPR-Cas9 demonstrates programmable translational enhancement. Polysome profiling indicates that UBAP2L promotes translation of target mRNAs, particularly global regulators of translation. Our tethering survey allows rapid assignment of the molecular activity of proteins, such as UBAP2L, to specific steps of mRNA metabolism.

Many proteins regulate the expression of genes by binding to specific regions encoded in the genome 1 . Here we introduce a new data set of RNA elements in the human genome that are recognized by RNA-binding proteins (RBPs), generated as part of the Encyclopedia of DNA Elements (ENCODE) project phase III. This class of regulatory elements functions only when transcribed into RNA, as they serve as the binding sites for RBPs that control post-transcriptional processes such as splicing, cleavage and polyadenylation, and the editing, localization, stability and translation of mRNAs. We describe the mapping and characterization of RNA elements recognized by a large collection of human RBPs in K562 and HepG2 cells. Integrative analyses using five assays identify RBP binding sites on RNA and chromatin in vivo, the in vitro binding preferences of RBPs, the function of RBP binding sites and the subcellular localization of RBPs, producing 1,223 replicated data sets for 356 RBPs. We describe the spectrum of RBP binding throughout the transcriptome and the connections between these interactions and various aspects of RNA biology, including RNA stability, splicing regulation and RNA localization. These data expand the catalogue of functional elements encoded in the human genome by the addition of a large set of elements that function at the RNA level by interacting with RBPs.

Background: A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types., Results: Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3' splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs., Conclusions: This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.

Alternative splicing of pre-messenger RNA transcripts enables the generation of multiple protein isoforms from the same gene locus, providing a major source of protein diversity in mammalian genomes. RNA binding proteins (RBPs) bind to RNA to control splice site choice and define which exons are included in the resulting mature RNA transcript. However, depending on where the RBPs bind relative to splice sites, they can activate or repress splice site usage. To explore this position-specific regulation, in vivo binding sites identified by methods such as cross-linking and immunoprecipitation (CLIP) are integrated with alternative splicing events identified by RNA-seq or microarray. Merging these data sets enables the generation of a "splicing map," where CLIP signal relative to a merged meta-exon provides a simple summary of the position-specific effect of binding on splicing regulation. Here, we provide RBP-Maps, a software tool to simplify generation of these maps and enable researchers to rapidly query regulatory patterns of an RBP of interest. Further, we discuss various alternative approaches to generate such splicing maps, focusing on how decisions in construction (such as the use of peak versus read density, or whole-reads versus only single-nucleotide candidate crosslink positions) can affect the interpretation of these maps using example eCLIP data from the 150 RBPs profiled by the ENCODE consortium., (© 2019 Yee et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Adenosine-to-inosine (A-to-I) editing, mediated by the ADAR enzymes, diversifies the transcriptome by altering RNA sequences. Recent studies reported global changes in RNA editing in disease and development. Such widespread editing variations necessitate an improved understanding of the regulatory mechanisms of RNA editing. Here, we study the roles of >200 RNA-binding proteins (RBPs) in mediating RNA editing in two human cell lines. Using RNA-sequencing and global protein-RNA binding data, we identify a number of RBPs as key regulators of A-to-I editing. These RBPs, such as TDP-43, DROSHA, NF45/90 and Ro60, mediate editing through various mechanisms including regulation of ADAR1 expression, interaction with ADAR1, and binding to Alu elements. We highlight that editing regulation by Ro60 is consistent with the global up-regulation of RNA editing in systemic lupus erythematosus. Additionally, most key editing regulators act in a cell type-specific manner. Together, our work provides insights for the regulatory mechanisms of RNA editing., Competing Interests: G.W.Y. is a co-founder of Locana and Eclipse Bioinnovations and member of the scientific advisory boards of Locana, Eclipse Bioinnovations and Aquinnah Pharmaceuticals. E.V.N is a co-founder and member of the scientific advisory board of Eclipse BioInnovations. The terms of these arrangements have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. The remaining authors declare no competing interests.

RNA binding proteins (RBPs) orchestrate the production, processing, and function of mRNAs. Here, we present the affinity landscapes of 78 human RBPs using an unbiased assay that determines the sequence, structure, and context preferences of these proteins in vitro by deep sequencing of bound RNAs. These data enable construction of "RNA maps" of RBP activity without requiring crosslinking-based assays. We found an unexpectedly low diversity of RNA motifs, implying frequent convergence of binding specificity toward a relatively small set of RNA motifs, many with low compositional complexity. Offsetting this trend, however, we observed extensive preferences for contextual features distinct from short linear RNA motifs, including spaced "bipartite" motifs, biased flanking nucleotide composition, and bias away from or toward RNA structure. Our results emphasize the importance of contextual features in RNA recognition, which likely enable targeting of distinct subsets of transcripts by different RBPs that recognize the same linear motif., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

HnRNPA2B1 encodes an RNA binding protein associated with neurodegeneration. However, its function in the nervous system is unclear. Transcriptome-wide crosslinking and immunoprecipitation in mouse spinal cord discover UAGG motifs enriched within ∼2,500 hnRNP A2/B1 binding sites and an unexpected role for hnRNP A2/B1 in alternative polyadenylation. HnRNP A2/B1 loss results in alternative splicing (AS), including skipping of an exon in amyotrophic lateral sclerosis (ALS)-associated D-amino acid oxidase (DAO) that reduces D-serine metabolism. ALS-associated hnRNP A2/B1 D290V mutant patient fibroblasts and motor neurons differentiated from induced pluripotent stem cells (iPSC-MNs) demonstrate abnormal splicing changes, likely due to increased nuclear-insoluble hnRNP A2/B1. Mutant iPSC-MNs display decreased survival in long-term culture and exhibit hnRNP A2/B1 localization to cytoplasmic granules as well as exacerbated changes in gene expression and splicing upon cellular stress. Our findings provide a cellular resource and reveal RNA networks relevant to neurodegeneration, regulated by normal and mutant hnRNP A2/B1. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using enhanced UV crosslinking and immunoprecipitation (eCLIP), we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region and binding site levels, IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3' UTR-enriched targets. RNA Bind-N-seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and increase in cell death. For cell adhesion, we find IMP1 maintains levels of integrin mRNA specifically regulating RNA stability of ITGB5 in hPSCs. Additionally, we show that IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcriptome-wide maps of RNA binding protein (RBP)-RNA interactions by immunoprecipitation (IP)-based methods such as RNA IP (RIP) and crosslinking and IP (CLIP) are key starting points for evaluating the molecular roles of the thousands of human RBPs. A significant bottleneck to the application of these methods in diverse cell lines, tissues, and developmental stages is the availability of validated IP-quality antibodies. Using IP followed by immunoblot assays, we have developed a validated repository of 438 commercially available antibodies that interrogate 365 unique RBPs. In parallel, 362 short-hairpin RNA (shRNA) constructs against 276 unique RBPs were also used to confirm specificity of these antibodies. These antibodies can characterize subcellular RBP localization. With the burgeoning interest in the roles of RBPs in cancer, neurobiology, and development, these resources are invaluable to the broad scientific community. Detailed information about these resources is publicly available at the ENCODE portal (https://www.encodeproject.org/)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

RNA quality-control pathways get rid of faulty RNAs and therefore must be able to discriminate these RNAs from those that are normal. Here we present evidence that the adenosine triphosphatase (ATPase) cycle of the SF1 helicase Upf1 is required for mRNA discrimination during nonsense-mediated decay (NMD). Mutations affecting the Upf1 ATPase cycle disrupt the mRNA selectivity of Upf1, leading to indiscriminate accumulation of NMD complexes on both NMD target and non-target mRNAs. In addition, two modulators of NMD-translation and termination codon-proximal poly(A) binding protein-depend on the ATPase activity of Upf1 to limit Upf1-non-target association. Preferential ATPase-dependent dissociation of Upf1 from non-target mRNAs in vitro suggests that selective release of Upf1 contributes to the ATPase dependence of Upf1 target discrimination. Given the prevalence of helicases in RNA regulation, ATP hydrolysis may be a widely used activity in target RNA discrimination., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Purpose: To assess the safety and clinical outcomes of 6-month treatment with testosterone gel 1% therapy in adolescent boys with primary hypogonadism resulting from Klinefelter syndrome (KS) or anorchia., Methods: This was a subgroup analysis of a multicenter, open-label study of adolescent boys (N = 86) with delayed puberty who received .5-5.0 g testosterone gel 1% daily for ≤6 months. Adolescent boys 12-17 years of age with KS (n = 21) or anorchia (n = 8), bone age ≥10.5 years, and baseline growth data ≥6 months were included in this analysis. Serum hormone levels (total/free testosterone, luteinizing hormone, dihydrotestosterone, follicle-stimulating hormone, and estradiol) were measured using validated assays. Safety was assessed through adverse events (AEs)., Results: At baseline, patients with KS were taller, weighed more, and had higher total testosterone levels (mean 174 vs. 19 ng/dL) than patients with anorchia. At 6 months, total and free testosterone and dihydrotestosterone levels increased 1.8- to 2.3-fold in the KS group and eight- to 10-fold in anorchia patients. Estradiol levels increased 1.9-fold in the anorchia group and 1.4-fold in the KS group after treatment. No clinically significant changes were noted for luteinizing hormone, follicle-stimulating hormone, and sex hormone-binding globulin concentrations in either group. Cough was the most common AE (eight of 29), followed by acne and headache (both four of 29). One anorchia and two KS patients discontinued prematurely., Conclusions: Once-daily testosterone gel application increased serum testosterone levels into the pubertal range and maintained pubertal testosterone levels during 6-month treatment. In this study, testosterone gel 1% raised testosterone levels and was associated with cough as the most common AE., (Copyright © 2014 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3' transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98-99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.

For both historical and technological reasons, most robots, including those meant to mimic animals or operate in natural environments,3 use actuators and control systems that have high (stiff) mechanical impedance. By contrast, most animals exhibit low (soft) impedance. While a robot's stiff joints may be programmed to closely imitate the recorded motion of an animal's soft joints, any unexpected position disturbances will generate reactive forces and torques much higher for the robot than for the animal. The dual of this is also true: while an animal will react to a force disturbance by significantly yielding position, a typical robot will greatly resist.These differences cause three deleterious effects for high impedance robots. First, the higher forces may cause damage to the robot or to its environment (which is particularly important if that environment includes people). Second, the robot must acquire very precise information about its position relative to the environment so as to minimize its velocity upon impact. Third, many of the self-stabilizing effects of natural dynamics are "shorted out"4 by the robot's high impedance, so that stabilization requires more effort from the control system.Over the past 5 yr, our laboratory has designed a series of walking robots based on "Series-Elastic Actuators" and "Virtual Model Control." Using these two techniques, we have been able to build low-impedance walking robots that are both safe and robust, that operate blindly without any model of upcoming terrain, and that add minimal control effort in parallel to their self-stabilizing passive dynamics. We have discovered that it is possible to achieve surprisingly effective ambulation from rather simple mechanisms and control systems. After describing the historical and technological motivations for our approach, this paper gives an overview of our methods and shows some of the results we have obtained.