Biology subjects

Lee, G. M.

Publications and source records attributed to Lee, G. M..

16 recordsLinked to original sources

bioRxiv2026

Curvature tuning in areas V2 and V4 of the developing macaque

Visual areas V2 and V4 are critical for the perception of visual forms in primates. Neurons in area V4 of macaque monkeys are often sensitive to the curvature of specific boundary segments within shapes, but it is unknown how curvature tuning is represented in the developing brain. To address this, we recorded multiunit neural activity from areas V2 and V4 of two macaque monkeys, at both 30 and 58 weeks of age, in response to shape stimuli which primarily varied in curvature along a single segment. Observable curvature tuning was adult-like from 30 weeks of age in both V2 and V4. We compared the tuning of sites to shapes presented at multiple positions. We saw evidence of position-invariant tuning in V4, but not in V2. Position invariance in V4 was stable from 30 weeks of age. Finally, we fit two models - a stimulus-centric model of boundary curvature tuning, and a simple linear model based on the spike-triggered average response to all stimuli. We found many sites in both V2 and V4 whose responses could be captured by one or both models, but no evidence of age-related changes in curvature tuning within the space of either model. Our results suggest that the neural representation of curvature in both areas reaches maturity soon after birth, and that object-centric representations of curvature first emerge in V4, not V2.

neuroscience↗

bioRxiv2026

Gardos potassium channel amplifies PIEZO1-TMEM16F coupling in red blood cells

In red blood cells (RBCs), the mechanosensitive channel PIEZO1 provides an upstream Ca2+ signal that activates TMEM16F, a Ca2+-activated phospholipid scramblase (CaPLSase) responsible for phosphatidylserine (PS) externalization. However, limited PIEZO1-mediated Ca{superscript 2} entry and the relatively low Ca2+ sensitivity of TMEM16F suggest the need for signal amplification. Here, we identify the Gardos (KCNN4) Ca2+-activated K channel as a critical amplifier of the PIEZO1-TMEM16F axis. Gardos activation induces membrane hyperpolarization, thereby increasing the driving force for Ca2+ entry and enhancing TMEM16F activation and phospholipid scrambling. Gardos-mediated amplification also contributes to excessive PS externalization in sickle cell disease (SCD) and hereditary xerocytosis (HX) RBCs, and functional disruption of Gardos-mediated K+ efflux attenuates this response. These findings demonstrate Gardos as a critical amplifier of RBC mechanotransduction and highlight Gardos as a potential therapeutic target for mitigating pathogenic PS exposure. HIGHLIGHTSO_LIGardos amplifies PIEZO1-mediated Ca{superscript 2} influx to promote TMEM16F-dependent phosphatidylserine exposure in healthy and diseased RBCs. C_LIO_LIUnexpected effects of some Gardos inhibitors may complicate the use in hematologic diseases. C_LI

physiology↗

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

Improving Recombinant Antibody Production Using FcBAR: An In Situ Approach to Detect and Amplify Protein-Protein Interactions

Recombinant proteins, in particular monoclonal antibodies and related molecules, have become dominant therapeutics. As they are produced in mammalian cells, they require the concerted function of hundreds of host cell proteins in the protein secretion pathway. However, the comprehensive set of host cell machinery involved remains unclear. Thus, it is often unknown why some recombinant proteins fail to express well. Here we present and deploy an approach called Fc-targeting Biotinylation by Antibody Recognition (FcBAR), which allows for the in situ detection of protein-protein interactions for any recombinant protein with Fc domain. Briefly, cells are permeabilized and incubated with an anti-Fc antibody, conjugated with horseradish peroxidase. All proteins interacting with Fc-bearing proteins are then biotinylated, pulled down and identified via mass spectrometry. We applied this method on a panel of rituximab-producing CHO-S clones with a range of productivity levels. Through analysis of FcBAR protein-protein interactions and RNA-Seq, we identified protein interactions positively correlated with rituximab secretion, and tested 7 of these targets. We found overexpression of AGPAT4, EPHX1, and NSDHL significantly increased rituximab production. Thus, FcBAR provides an unbiased approach to measure PPIs supporting recombinant antibody production in situ, and can guide efforts to boost production of biotherapeutics and biosimilars by addressing production bottlenecks.

systems biology↗

bioRxiv2025

Mass spectrometry-based quantification of proteins and post-translational modifications in dried blood: longitudinal sampling of patients with sepsis in Tanzania

The proteomic analysis of blood is routine for disease phenotyping and biomarker development. Whole blood is commonly separated into soluble and cellular fractions. However, this can introduce pre-analytical variability; and analysis of a single component (which is common) may ignore important pathophysiology. We have recently developed methods for the facile processing of dried blood for mass spectrometry-based quantification of the proteome, N-glycoproteome and phosphoproteome. Here, we applied this approach to 38 patients in Tanzania who presented to the hospital with sepsis. Blood was collected on Mitra devices at presentation and 1, 3 and 28-42 days post-enrollment. Processing of 96 total samples was performed in plate-based formats and completed within two days. Approximately 2,000 protein groups and 8,000 post-translational modifications were quantified in 3 LC-MS/MS runs at [~]1.5 hours per sample. Analysis of differential abundance revealed blood proteome signatures of acute phase response and neutrophilic inflammation that partially resolved at the 28-42 day timepoint. Numerous analytes correlated with clinical laboratory values for c-reactive protein and white blood cell counts, as well as the Universal Vital Assessment illness severity score. These datasets serve as proof-of-concept for large scale MS-based (sub)phenotyping of disease using dried blood and are available via the ProteomeXchange consortium (PXD060377).

systems biology↗

bioRxiv2025

Evaluation of distal facial nerve branches contribution to facial nerve paralysis in rodents

Introduction/AimsFacial nerve paralysis is a complex and devastating condition. Translational research of facial paralysis recovery remains largely limited to animal studies, for which there are many potential models employed. When studying facial nerve regeneration in rodents, it is important to understand the converging contributions of the motor supply into the whisker pad. A consensus surgical approach and animal model has yet to be defined. Of particular interest for movement of the nose and whiskers are the buccal and marginal mandibular nerves. This study aims to evaluate how these distal nerve branches contribute to facial nerve paralysis and identify key morphological changes at the neuromuscular junctions (NMJs) in the whisker pad of rodents. MethodsAdult rats underwent isolated transection of the buccal branch of the facial nerve, both the buccal and marginal mandibular branches of the facial nerve, or control sham surgery. ResultsHistological, electrophysiological, and behavior assessments confirmed that the transection of the buccal branch alone did not cease whisker movement in rats, but when combined with a transection of the marginal mandibular branch, it resulted in full paralysis of the whisker and nose movement. DiscussionThese results are indicative of the distinct roles of these nerves branches in facial paralysis repair following a transection injury. Further, our results suggest additional targets for facial nerve repair treatments.

neuroscience↗

bioRxiv2025

Emergence of a contrast-invariant representation of naturalistic texture in macaque visual cortex

Sensory stimuli vary across a variety of dimensions, like contrast, orientation, or texture. The brain must rely on population representations to distinguish changes in one dimension from changes in another. To understand how the visual system might extract separable stimulus representations, we recorded multiunit neuronal responses to texture images varying along two dimensions: contrast, a property represented as early as the retina, and naturalistic statistical structure, a property that modulates neuronal responses in V2 and V4, but not in V1. We measured how sites in these 3 cortical areas responded to variation in both dimensions. Contrast modulated responses in all areas. In V2 and V4, the presence of naturalistic structure both modulated responses and increased contrast sensitivity. Tuning for naturalistic structure was both strongest and most dispersed in V4. We measured how well populations in each area could support the linear readout of both dimensions. Populations in V2 and V4 could support the linear readout of naturalistic structure, but in V4, this readout was more robust to variations in contrast. Significance StatementTo support flexible behavior, the brain must simultaneously represent different stimulus dimensions. Single neurons are typically modulated by multiple dimensions, and so cannot distinguish them - they must be extracted by decoding neural populations. We studied neuronal responses in three cortical visual areas - V1, V2, V4 - using texture images varying in both contrast and naturalistic image structure. We used population decoders to read out each dimension. In all areas, contrast was well decoded independently of image structure. On the other hand, V1 could not decode image structure independent of contrast, while V2 and V4 could. V4 decoding was greatly superior, because the selectivity of individual sites for texture was more diverse than in V1 or V2.

neuroscience↗

bioRxiv2024

Multiplex genome editing eliminates the Warburg Effect without impacting growth rate in mammalian cells

The Warburg effect is ubiquitous in proliferative mammalian cells, including cancer cells, but poses challenges for biopharmaceutical production, as lactate accumulation inhibits cell growth and protein production. Previous efforts to eliminate lactate production via knockout have failed in mammalian bioprocessing since lactate dehydrogenase has proven essential. However, here we eliminated the Warburg effect in Chinese hamster ovary (CHO) and HEK293 cells by simultaneously knocking out lactate dehydrogenase and regulators involved in a negative feedback loop that typically inhibits pyruvate conversion to acetyl-CoA. In contrast to long-standing assumptions about the role of aerobic glycolysis, Warburg-null cells maintain wildtype growth rate while producing negligible lactate. Further characterization of Warburg-null CHO cells showed a compensatory increase in oxygen consumption, a near total reliance on oxidative metabolism, and higher cell densities in fed-batch cell culture. These cells remained amenable for production of diverse biotherapeutic proteins, reaching industrially relevant titers and maintaining product glycosylation. Thus, the ability to eliminate the Warburg effect is an important development for biotherapeutic production and provides a tool for investigating a near-universal metabolic phenomenon.

bioengineering↗

bioRxiv2024

Developmentally stable representations of naturalistic image structure in macaque visual cortex

We studied visual development in macaque monkeys using texture stimuli, matched in local spectral content but varying in "naturalistic" structure. In adult monkeys, naturalistic textures preferentially drive neurons in areas V2 and V4, but not V1. We paired behavioral measurements of naturalness sensitivity with separately-obtained neuronal population recordings from neurons in areas V1, V2, V4, and inferotemporal cortex (IT). We made behavioral measurements from 16 weeks of age and physiological measurements as early as 20 weeks, and continued through 56 weeks. Behavioral sensitivity reached half of maximum at roughly 25 weeks of age. Neural sensitivities remained stable from the earliest ages tested. As in adults, neural sensitivity to naturalistic structure increased from V1 to V2 to V4. While sensitivities in V2 and IT were similar, the dimensionality of the IT representation was more similar to V4s than to V2s.

neuroscience↗

bioRxiv2022

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

bioengineering↗

bioRxiv2022

Sensitivity to naturalistic texture relies primarily on high spatial frequencies

Natural images contain information at multiple spatial scales. Although we understand how early visual mechanisms split multi-scale images into distinct spatial frequency channels, we do not know how the outputs of these channels are processed further by mid-level visual mechanisms. We have recently developed a naturalness discrimination task that uses synthesized, multi-scale textures to isolate these mid-level mechanisms (Freeman et. al. 2013). Here, we use three experimental manipulations (image blur, image rescaling, and eccentric viewing) to show that naturalness sensitivity is strongly dependent on image features at high object spatial frequencies (measured in cycles/image). As a result, sensitivity depends on a texture acuity limit, a property of the visual system that sets the highest retinal spatial frequency (measured in cycles/degree) that can be used to solve the task. A model observer analysis shows that high object spatial frequencies carry more task-relevant information than low object spatial frequencies. Comparing the outcome of this analysis with human performance reveals that human observers efficiency is similar for all object spatial frequencies. We conclude that the mid-level mechanisms that underlie naturalness sensitivity effectively extract information from all image features below the texture acuity limit, regardless of their retinal and object spatial frequency.

neuroscience↗

bioRxiv2020

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells

Chinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase gene Mgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

genomics↗

bioRxiv2020

Using targeted genome integration for virus-free genome-wide mammalian CRISPR screen

Pooled CRISPR screens have been widely applied in mammalian cells to discover genes regulating various phenotypes of interest. In such screens, CRISPR components are generally delivered with a lentivirus. However, lentiviral CRISPR screens are limited by unpredictable genome insertion, the requirement of biosafety level II lab facilities and personnel trained to work with viruses. Here we established a virus-free (VF) genome-wide CRISPR screening platform for Chinese hamster ovary (CHO) cells with 74,617 gRNAs targeting 18,353 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad thus reducing the clonal variation. Using this VF CRISPR screening platform, 338 genes are identified as essential for CHO cell growth and 76 genes were found to be involved in the unfolded protein response (UPR) induced endoplasmic reticulum (ER) stress. Extensive validation of the candidate genes further demonstrated the robustness of this novel non-viral CRISPR screen method.

genomics↗

bioRxiv2020

A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes

Over the past decades, optimization of media formulation and feeding strategies have fueled a many-fold improvement in CHO-based biopharmaceutical production. While Design of Experiments (DOE) and media screens have led to many advances, genome editing offers another avenue for enhancing cell metabolism and bioproduction. However the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we developed a comprehensive pooled CRISPR screen for CHO cell metabolism, including [~]16,000 gRNAs against [~]2500 metabolic enzymes and regulators. We demonstrated the value of this screen by identifying a glutamine response network in CHO cells. Glutamine is particularly important since it is often substantially over-fed to drive increased TCA cycle flux but can lead to accumulation of toxic ammonia. Within the glutamine-response network, the deletion of a novel and poorly characterized lipase, Abhd11, was found to substantially increase growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait.

bioengineering↗

bioRxiv2019

Multiplex secretome engineering enhances recombinant protein production and purity

Host cell proteins (HCPs) are process-related impurities generated during biotherapeutic protein production. HCPs can be problematic if they pose a significant metabolic demand, degrade product quality, or contaminate the final product. Here, we present an effort to create a \"clean\" Chinese hamster ovary (CHO) cell by disrupting multiple genes to eliminate HCPs. Using a model of CHO cell protein secretion, we predicted the elimination of unnecessary HCPs could have a non-negligible impact on protein production. We analyzed the total HCP content of 6-protein, 11-protein, and 14-protein knockout clones and characterized their growth in shake flasks and bioreactors. These cell lines exhibited a substantial reduction in total HCP content (40%-70%). We also observed higher productivity and improved growth characteristics, in specific clones. With the reduced HCP content, protein A and ion exchange chromatography more efficiently purified a monoclonal antibody (mAb) produced in these cells during a three-step purification process. Thus, substantial improvements can be made in protein titer and purity through large-scale HCP deletion, providing an avenue to increased quality and affordability of high-value biopharmaceuticals.

bioengineering↗

bioRxiv2018

Genome-scale reconstructions of the mammalian secretory pathway predict metabolic costs and limitations of protein secretion

In mammalian cells, >25% of synthesized proteins are exported through the secretory pathway. The pathway complexity, however, obfuscates its impact on the secretion of different proteins. Unraveling its impact on diverse proteins is particularly important for biopharmaceutical production. Here we delineate the core secretory pathway functions and integrate them with genome-scale metabolic reconstructions of human, mouse, and Chinese hamster cells. The resulting reconstructions enable the computation of energetic costs and machinery demands of each secreted protein. By integrating additional omics data, we find that highly secretory cells have adapted to reduce expression and secretion of other expensive host cell proteins. Furthermore, we predict metabolic costs and maximum productivities of biotherapeutic proteins and identify protein features that most significantly impact protein secretion. Finally, the model successfully predicts the increase in secretion of a monoclonal antibody after silencing a highly expressed selection marker. This work represents a knowledgebase of the mammalian secretory pathway that serves as a novel tool for systems biotechnology.

systems biology↗