Biology subjects

Lee, D. J.

Publications and source records attributed to Lee, D. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Distributed brain-spinal cord dynamics during micturition revealed by functional ultrasound imaging

Micturition relies on coordinated activity across distributed neural circuits spanning the brain and spinal cord, yet the spatiotemporal dynamics of these processes remain poorly understood. Disruption of these pathways in conditions such as spinal cord injury, Parkinson's disease, multiple sclerosis, and aging can lead to neurogenic bladder dysfunction. Here, we used functional ultrasound imaging (fUSI), a modality with high spatiotemporal resolution and single-trial sensitivity, to investigate central nervous system activity during micturition. In anesthetized rats undergoing controlled bladder filling and volitional voiding, we recorded power Doppler signals from either the brain or the lumbosacral spinal cord. We observed distinct, region-specific hemodynamic responses associated with micturition across multiple brain regions, including the thalamus, hypothalamus, midbrain, and neocortex - particularly sensorimotor and retrosplenial areas - as well as dorsal and ventral spinal cord circuits. Furthermore, population-level activity in these regions enabled accurate decoding of bladder pressure dynamics. These results provide a systems-level characterization of central nervous system activity during micturition and establish fUSI as a platform for developing neural interfaces to restore bladder control.

bioengineering↗

bioRxiv2026

Revisiting Analog Electrical Stimulation with Current Focusing in a Guinea Pig Model of Cochlear Implants.

Modern cochlear implants (CIs) use pulsatile stimulation to restore hearing for individuals with severe hearing loss. CIs provide robust speech recognition in quiet but poorly represent temporal fine structure (TFS), needed for challenging listening situations. Analog stimulation preserves the acoustic waveform and may better encode TFS, yet it has not been evaluated combined with modern current-focusing strategies. We compared neural responses in the inferior colliculus (IC) evoked by CI stimuli consisting of 100 pulses/s biphasic pulse trains and 100 cycles/s sinusoidal analog stimulation with monopolar, bipolar, and tripolar electrode configurations in urethane-anesthetized guinea pigs. Following cochlear implantation, multiunit activity was recorded from the tonotopic axis of the central nucleus of the IC using 16-channel silicon probes. Detection thresholds, spread of excitation, vector strength, sustained response percentage, and temporal response properties were quantified. Analog stimulation consistently evoked significantly lower activation thresholds than pulsatile stimulation while maintaining comparable or sometimes narrower spatial selectivity across stimulation modes. In contrast, analog stimulation generated lower vector strength, larger tonic response components, and a pronounced level-dependent polarity effect. At low stimulus levels, responses were dominated by the cathodic phase of the sinusoidal waveform, whereas increasing stimulus level responses were elicited by both phases, producing synchronization at twice the stimulus frequency. These findings demonstrate that stimulation waveform strongly influences temporal coding while having relatively little effect on the spatial distribution of neural activation. These results provide a physiological basis for reexamining analog stimulation as an alternative strategy for cochlear implant sound coding.

neuroscience↗

bioRxiv2026

Stereoselective methyl-swapping demonstrates target specificity of cognitive enhancer

The Integrated Stress Response (ISR) couples cellular stress sensing to translational control, playing a critical role in the homeostatic regulation of cell health. However, prolonged and unmitigated ISR activation becomes maladaptive and drives the progression of a wide range of pathologies, including cognitive decline. Pharmacological inhibition of the ISR with the small, drug-like molecule ISRIB has proven remarkably effective in reversing cognitive deficits and pathology in animal models, highlighting its potential for therapeutic intervention in humans. We engineered an allele-specific ISRIB analog (mISRIB) that selectively targets a mutant form of eIF2B, the molecular target of ISRIB, without affecting wild-type eIF2B. Notably, mISRIB treatment in mice homozygous for the eIF2B mutant allele enhances synaptic plasticity and long-term memory, confirming the on-target mechanism underlying ISRIBs cognitive benefits. Our results provide a framework for dissecting the ISRs contributions within complex cellular networks, such as those governing brain function, with precise temporal and spatial resolution.

biochemistry↗

bioRxiv2025

Cereblon Promotes Influenza Virus Replication through AMPK Ubiquitination

Influenza A and B viruses (IAV and IBV) continually threaten global health, with IAV posing a risk of emerging pandemics. Rapid viral evolution makes current treatments less effective, highlighting the urgent need for broad-spectrum antivirals. Targeting host factors essential for viral replication may offer a highly promising broad-spectrum antiviral strategy. In this context, cereblon (CRBN), a substrate adaptor of the CRL4 E3 ubiquitin ligase complex, is found to promote both IAV and IBV replication as a key pro-viral host factor. Mechanistically, CRBN targets and degrades AMP-activated protein kinase (AMPK) via the proteasome. This CRBN-driven degradation shifts the metabolism of the infected cells toward anabolism, promoting lipid droplet (LD) formation and creating a microenvironment favorable for viral replication. Genetic depletion or inhibition of CRBN stabilizes AMPK, significantly reduces LD formation, and effectively suppresses the replication of various IAV and IBV strains in vivo, demonstrating its broad-spectrum potential. Notably, Crbn knockout mice show marked resistance to lethal IAV infection. CRBN inhibition with immunomodulatory imide drugs--known CRBN inhibitors--significantly decreases IAV replication in vivo. This research underscores CRBN as a crucial regulator of host metabolism during viral infection, revealing the CRBN-AMPK axis as a promising target for host-directed pan-influenza antiviral development. Author SummaryInfluenza A and B viruses are response for recurring seasonal epidemics and occasional pandemics that pose serious global health challenges. Current antiviral drugs often lose effectiveness as influenza viruses rapidly develop resistance through genetic mutations. To overcome this limitation, our study focused on a host factor targeted by influenza viruses-the ubiquitin E3 ligase substrate adaptor cereblon (CRBN). We discovered that influenza viruses exploit CRBN to enhance their replication by altering host cell metabolism. Specifically, CRBN binds to a key energy sensor - AMPK and induces the ubiquitination and degradation of AMPK{gamma}. Consequently, the AMPK suppression shifts the cellular environment from catabolism to anabolism, activating lipogenic enzymes and promoting lipid droplets formation which provide a favorable environment for viral growth. Our study highlight that blocking CRBN either genetically or chemical inhibition (immunomodulatory imide drugs) of CRBN reduces LD formation and strongly suppresses diverse influenza strains replication in vitro and in vivo. These findings reveal not only how viruses hijack host metabolism via the CRBN-AMPK pathway but also present a potential therapeutic for novel broad-spectrum anti-influenza.

microbiology↗

bioRxiv2025

Product-stabilized filamentation by human glutamine synthetase allosterically tunes metabolic activity

To maintain metabolic homeostasis, enzymes must adapt to fluctuating nutrient levels through mechanisms beyond gene expression. Here, we demonstrate that human glutamine synthetase (GS) can reversibly polymerize into filaments aided by a composite binding site formed at the filament interface by the product, glutamine. Time-resolved cryo-electron microscopy (cryo-EM) confirms that glutamine binding stabilizes these filaments, which in turn exhibit reduced catalytic specificity for ammonia at physiological concentrations. This inhibition appears induced by a conformational change that remodulates the active site loop ensemble gating substrate entry. Metadynamics ensemble refinement revealed >10 [A] conformational range for the active site loop and that the loop is stabilized by transient contacts. This disorder is significant, as we show that the transient contacts which stabilize this loop in a closed conformation are essential for catalysis both in vitro and in cells. We propose that GS filament formation constitutes a negative-feedback mechanism, directly linking product concentration to the structural and functional remodeling of the enzyme.

bioinformatics↗

bioRxiv2025

Harnessing the Evolution of Proteostasis Networks to Reverse Cognitive Dysfunction

The integrated stress response (ISR) is a highly conserved network essential for maintaining cellular homeostasis and cognitive function. Here, we investigated how persistent ISR activation impacts cognitive performance, primarily focusing on a PPP1R15BR658C genetic variant associated with intellectual disability. By generating a novel mouse model that mimics this human condition, we revealed that this variant destabilizes the PPP1R15B*PP1 phosphatase complex, resulting in chronic ISR activation, impaired protein synthesis, and deficits in long-term memory. Importantly, we found that the cognitive and synaptic deficits in Ppp1r15bR658C mice are directly due to ISR activation. Leveraging insights from evolutionary biology, we characterized DP71L, a viral orthologue of PPP1R15B, through detailed molecular and structural analyses, uncovering its mechanism of action as a potent pan-ISR inhibitor. Remarkably, we found that DP71L not only buffers cognitive decline associated with a wide array of conditions--including Down syndrome, Alzheimers disease and aging--but also enhances long-term synaptic plasticity and memory in healthy mice. These findings highlight the promise of utilizing evolutionary insight to inform innovative therapeutic strategies.

neuroscience↗

bioRxiv2024

Mechanisms of sustaining oxygen extraction efficiency in dragonfly nymphs during aquatic hypoxia

Despite breathing water using their tidally ventilated rectal gills, dragonfly nymphs show a surprising ability to maintain oxygen (O2) extraction from the water during hypoxia. However, an increase in convective O2 transfer is insufficient to sustain aerobic demands by itself, which suggests that diffusive mechanisms must also be involved. This study examines the contributions of changing the O2 partial pressure gradient (PO2) and/or O2 conductance across the rectal gill in maintaining O2 extraction efficiency (OEE) of dragonfly nymphs during hypoxia. Data were collected using the same custom-designed respiro-spirometer described in a previous study with the addition of an implanted O2 sensor to measure hemolymph PO2. Results show that the implantation of the O2 sensor does not affect the respiratory and ventilatory response of nymphs to hypoxia. Hemolymph PO2 fell from 6.3 {+/-} 1.6 kPa at normoxia to 2.5 {+/-} 0.6 kPa at 16.0 kPa, which resulted in the PO2 diffusion gradient remaining statistically constant at these two water PO2s (17.5 {+/-} 1.7 and 15.4 {+/-} 0.7 kPa during normoxia and 16.0 kPa respectively). Beyond 16.0 kPa, a progressive reduction in hemolymph PO2 was unable to sustain the diffusion gradient. Mathematical modeling revealed that while the addition of hemolymph PO2 in tandem with ventilation frequency was able to elevate OEE during 16.0 kPa to that of normoxia, both were still insufficient during severe hypoxia and required an increase in O2 conductance. Estimating the change in whole-gill conductance showed that nymphs are indeed increasing their conductance as the water becomes hypoxic, demonstrating a reliance on both diffusion gradient and O2 conductance to enhance diffusive O2 transfer in conjunction with convective mechanisms to maintain O2 extraction during hypoxia.

zoology↗

bioRxiv2024

Human spinal cord activation during filling and emptying of the bladder

Recording neural activity from the spinal cord is crucial for gaining insights into how it functions. However, the neural activity of the human spinal cord is notoriously difficult to measure. The bony and fascial enclosures combined with the relatively small anatomic size of the spinal cord make it an unfavorable target for traditional functional neuroimaging techniques. Functional ultrasound imaging (fUSI) is an emerging neuroimaging technology that represents a new platform for studying large-scale neural dynamics with high sensitivity, spatial coverage and spatiotemporal resolution. Although it was originally developed for studying brain function, fUSI was recently extended for imaging the spinal cord in animals and humans. While these studies are significant, their primary focus is on the neuroactivation of the spinal cord in response to external sensory stimulations. Here, we combined fUSI with urodynamically-controlled bladder filling and emptying to characterize the hemodynamic response of the human spinal cord during the micturition cycle. Our findings provide the first practical evidence of the existence of bladder pressure-responsive regions, whose hemodynamic signal is strongly correlated with the bladder pressure.

neuroscience↗

bioRxiv2023

Immediate and Long-Term Electrophysiological Biomarkers of Antidepressant Behavioral Effects After Subanesthetic Ketamine and Medial Prefrontal Cortex Deep Brain Stimulation Treatment

1Ketamine and medial prefrontal cortex (mPFC) deep brain stimulation (DBS) are emerging depression treatments, however their mechanisms and efficacy biomarkers have not been established. This study investigates immediate and long-term mPFC electrophysiological correlates of their antidepressant effects, and explores the effects of combinatorial treatment. Local field potential electrodes were implanted into the mPFC of male Sprague Dawley rats, then corticosterone was administered for 21 days, except in the control group (n = 8). In the last week of depression induction, the remaining groups received mPFC DBS (n = 8), ketamine (n = 9), both (n = 9), or neither (n = 8). All groups then performed a behavioral assay. We found that DBS treatment immediately suppressed relative low gamma power and improved cognition in the long-term, which correlated with rescued apathy-like behavior in the Groom Test (GT). In contrast, ketamine treatment suppressed sample entropy in the long-term, which correlated with its immediate decrease in sample entropy as well as rescued GT behavior. In the combinatorial group these changes were inhibited, which correlated with a failure to fully rescue GT behavior. Despair-like behavior in the Forced Swim Test was rescued by all three treatments, which correlated with increased relative high frequency oscillation (HFO) power immediately after ketamine and combinatorial treatment. Our findings support the utility of low gamma and HFO power, sample entropy, and cognitive tests in the search for mechanisms and biomarkers of depression and novel treatments. 2 Significance StatementThere is growing interest in psychedelic pharmacological agents, such as ketamine, and direct neurostimulation techniques, like deep brain stimulation, as treatments for depression. However, their mechanisms are poorly understood, particularly those underlying their sustained benefits. Our study addresses this by correlating behavioral rescue with immediate and long-term changes in the function of the medial prefrontal cortex. These antidepressant correlates may represent therapeutically relevant functional changes, and therefore also have potential as biomarkers. Clinically, such biomarkers enable predicting and monitoring of treatment efficacy, and thereby contribute to the personalization of care. In research, utilizing these translatable biomarkers enhances the etiological validity of disease models, and enables the development of more targeted, next-generation treatment strategies.

neuroscience↗

bioRxiv2023

Comparison of image registration techniques in functional ultrasound imaging

Functional Ultrasound Imaging (fUSI) is an emerging hemodynamic-based functional neuroimaging technique that combines high spatiotemporal resolution and sensitivity, as well as extensive brain coverage, enabling a range of applications in both control and disease animal models. Based on power Doppler (pD) imaging, fUSI measures changes in cerebral blood volume (CBV) by detecting the back-scattered echoes from red blood cells moving within its field of view (FOV). However, the expansion of fUSI technology is partly limited by the challenge to co-register pD vascular maps acquired across different sessions or animals to one reference; an approach that could widen the scope of experimental paradigms and enable advanced data analysis tools. In this study, we seek to address this critical limitation. We evaluate six image registration techniques, predominantly used in other neuroimaging studies, using 2D sagittal whole-brain fUSI data from 82 anesthetized mice, and tested the quality of registration using multiple metrics. Our findings indicate a substantial enhancement in the alignment of fUSI images post registration. Among the tested techniques, the non-rigid registration algorithm Imregdeform yielded superior performance. We offer the first comparative study of image registration techniques for a 2D fUSI brain dataset, paving a way for improved utilization of fUSI in future pre-clinical research applications.

neuroscience↗

bioRxiv2023

Microfluidic Quaking-Induced Conversion (Micro-QuIC) for Rapid On-Site Amplification and Detection of Misfolded Proteins

Protein misfolding diseases, such as prion diseases, Alzheimers, and Parkinsons, share a common molecular mechanism involving the misfolding and aggregation of specific proteins. There is an urgent need for point-of-care (POC) diagnostic technologies that can accurately detect these misfolded proteins, facilitating early diagnosis and intervention. Here, we introduce the Microfluidic Quaking Induced Conversion (Micro-QuIC), a novel acoustofluidic platform for the rapid and sensitive detection of protein misfolding diseases. We demonstrate the utility of our technology using chronic wasting disease (CWD) as a model system, as samples from wild white-tailed deer are readily accessible, and CWD shares similarities with human protein misfolding diseases. Acoustofluidic mixing enables homogeneous mixing of reagents in a high-Reynolds-number regime, significantly accelerating the turnaround time for CWD diagnosis. Our Micro-QuIC assay amplifies prions by an order of magnitude faster than the current gold standard, real-time quaking-induced conversion (RT-QuIC). Furthermore, we integrated Micro-QuIC with a gold nanoparticle-based, naked-eye detection method, which enables visual discrimination between CWD positive and negative samples without the need for a bulky fluorescence detection module. This integration creates a rapid, POC testing platform capable of detecting misfolded proteins associated with a variety of protein misfolding diseases. TOC graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/549283v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@885eeaorg.highwire.dtl.DTLVardef@10f820eorg.highwire.dtl.DTLVardef@12396eaorg.highwire.dtl.DTLVardef@188fe0f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2023

Signatures of Jamming in the Cellular Potts Model

We explore the jamming transition in the Cellular Potts Model (CPM) as a function of confinement, cell adhesion, and cell shape. To accurately characterize jamming, we compare Potts simulations of unconfined single cells, cellular aggregates, and confluent monolayers as a function of cell adhesion energies and target cell shape. We consider metrics that may identify signatures of the jamming transition, including diffusion coefficients, anomalous diffusion exponents, cell shape, cell-cell rearrangements, and velocity correlations. We find that the onset of jamming coincides with an abrupt drop in cell mobility, rapid transition to sub-diffusive behavior, and cessation of rearrangements between neighboring cells that is unique to confluent monolayers. Velocity correlations reveal collective migration as a natural consequence of high energy barriers to neighbor rearrangements for certain cell types. Cell shapes across the jamming transition in the Potts model are found to be generally consistent with predictions of vertex-type simulations and trends from experiment. Finally, we demonstrate that changes in cell shape can fluidize cellular monolayers at cellular interaction energies where jamming otherwise occurs.

biophysics↗

bioRxiv2022

A mutation rate model at the basepair resolution identifies the mutagenic effect of Polymerase III transcription

De novo mutations occur with substantially different rates depending on genomic location, sequence context and DNA strand1-4. The success of many human genetics techniques, especially when applied to large population sequencing datasets with numerous recurrent mutations5-7, depends strongly on assumptions about the local mutation rate. Such techniques include estimation of selection intensity8, inference of demographic history9, and mapping of rare disease genes10. Here, we present Roulette, a genome-wide mutation rate model at the basepair resolution that incorporates known determinants of local mutation rate (http://genetics.bwh.harvard.edu/downloads/Vova/Roulette/). Roulette is shown to be more accurate than existing models1,6. Roulette has sufficient resolution at high mutation rate sites to model allele frequencies under recurrent mutation. We use Roulette to refine estimates of population growth within Europe by incorporating the full range of human mutation rates. The analysis of significant deviations from the model predictions revealed a 10-fold increase in mutation rate in nearly all genes transcribed by Polymerase III, suggesting a new mutagenic mechanism. We also detected an elevated mutation rate within transcription factor binding sites restricted to sites actively utilized in testis and residing in promoters.

genetics↗

bioRxiv2022

Functional Ultrasound Imaging of the Human Spinal Cord

The integration of functional responses in the human spinal cord into the nervous system is not well understood. Herein we demonstrate the first in-human functional ultrasound imaging (fUSI) of spinal cord response to epidural electrical stimulation. fUSI is a minimally invasive neuroimaging technique that can record blood flow at a level of spatial and temporal precision not previously achieved in the human spinal cord. By leveraging fUSI and epidural electrical spinal cord stimulation in patients who underwent surgery, we recorded and characterized for the first-time hemodynamic responses of the human spinal cord to an electrical neuromodulatory intervention commonly used for treating pain, and increasingly used for sensory-motor and autonomic functions. We found that the hemodynamic response to epidural stimulation reflects a spatiotemporal modulation of the spinal cord circuitry not previously recognized. The impact of this analytical capability is significant for several reasons. It offers a mechanism to assess blood flow changes with a new level of precision which can be obtained in real time under in vivo conditions. Additionally, we demonstrate that fUSI can successfully decode the spinal cord state in a single trial, which is of fundamental importance for developing real-time closed-loop neuromodulation systems. Also, we show that spinal cord hemodynamic changes due to epidural electrical stimulation occur primarily at the level of small vessels. Overall, our work is a critical step towards developing a vital technique to study spinal cord function and understand the potential effects of clinical neuromodulation for spinal cord and other neurological disorders. One Sentence SummaryThe first in-human quantitative evaluation of spinal cord hemodynamics using functional ultrasound imaging (fUSI).

bioengineering↗

bioRxiv2022

Genetic architecture of creativity and extensive genetic overlap with psychiatric disorders revealed from genome-wide association analyses of 241,736 individuals

Creativity is heritable and exhibits familial aggregation with psychiatric disorders, but its genomic basis and genetic relationship with psychiatric disorders remain largely unknown. Here, we conducted a genome-wide association study (GWAS) using an expanded, machine learning-based definition of creativity in individuals of European ancestry from the UK Biobank (n = 241,736) and identified 25 creativity-associated loci. Extensive genetic overlap with psychiatric disorders, including schizophrenia, major depression, bipolar I disorder, attention deficit/hyperactivity disorder, and anorexia nervosa, was demonstrated by the genetic correlation, polygenic risk score, and MiXeR analyses. The condFDR and conjFDR analyses identified additional loci for creativity and psychiatric disorders, as well as shared genetic loci between creativity and psychiatric disorders. This GWAS showed significant correlations with GWASs using traditional definitions of creativity and GWASs adjusted for educational attainment. Our findings contribute to the understanding of the genetic architecture of creativity and reveal its polygenic relationships with psychiatric disorders.

genomics↗

bioRxiv2022

Magnetic Stimulation Allows Focal Activation of the Mouse Cochlea

Cochlear implants (CIs) strive to restore hearing to those with severe to profound hearing loss by artificially stimulating the auditory nerve. While most CI users can understand speech in a quiet environment, hearing that utilizes complex neural coding (e.g., appreciating music) has proved elusive, probably because of the inability of CIs to create narrow regions of spectral activation. Several novel approaches have recently shown promise for improving spatial selectivity, but substantial design differences from conventional CIs will necessitate much additional safety testing before clinical viability is established. Outside the cochlea, magnetic stimulation from small coils (micro-coils) has been shown to confine activation more narrowly than that from conventional micro-electrodes, raising the possibility that coil-based stimulation of the cochlea could improve the spectral resolution of CIs. To explore this, we delivered magnetic stimulation from micro-coils to multiple locations of the cochlea and measured the spread of activation utilizing a multi-electrode array inserted into the inferior colliculus; responses to magnetic stimulation were compared to analogous experiments with conventional micro-electrodes as well as to the responses to auditory monotones. Encouragingly, the extent of activation with micro-coils was [~]60% narrower than that from electric stimulation and largely similar to the spread arising from acoustic stimulation. The dynamic range of coils was more than three times larger than that of electrodes, further supporting a smaller spread of activation. While much additional testing is required, these results support the notion that coil-based CIs can produce a larger number of independent spectral channels and may therefore improve functional performance. Further, because coil-based devices are structurally similar to existing CIs, fewer impediments to clinical translational are likely to arise.

neuroscience↗

bioRxiv2021

NGSpop: A desktop software that supports population studies by identifying sequence variations from next-generation sequencing data

Next-generation sequencing (NGS) is widely used in all areas of genetic research, such as for genetic disease diagnosis and breeding, and it can produce massive amounts of data. The identification of sequence variants is an important step when processing large NGS datasets; however, currently, the process is complicated, repetitive, and requires concentration, which can be taxing on the researcher. Therefore, to support researchers who are not familiar with bioinformatics in identifying sequence variations regularly from large datasets, we have developed a fully automated desktop software, NGSpop. NGSpop includes functionalities for all the variant calling and visualization procedures used when processing NGS data, such as quality control, mapping, filtering details, and variant calling. In the variant calling step, the user can select the GATK or DeepVariant algorithm for variant calling. These algorithms can be executed using pre-set pipelines and options or customized with the user-specified options. NGSpop is implemented using JavaFX (version 1.8) and can thus be run on Unix like operating systems such as Ubuntu Linux (version 16.04, 18.0.4). Although there are several pipelines and visualization tools available for NGS data analysis, most integrated environments do not support batch processes; thus, variant detection cannot be automated for population-level studies. The NGSpop software, developed in this study, has an easy-to-use interface and helps in rapid analysis of multiple NGS data from population studies.

bioinformatics↗

bioRxiv2021

Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.

biophysics↗