Biology subjects

Lee, C. W.

Publications and source records attributed to Lee, C. W..

9 recordsLinked to original sources

bioRxiv2026

Fixative eXchange (FX)-seq: Scalable Single-nucleus RNA Sequencing Analysis of PFA-fixed or FFPE Tissue

Single-nucleus RNA sequencing (snRNA-seq) of clinical formalin-fixed, paraffin-embedded (FFPE) samples has long been a challenge due to low reverse transcription (RT) yields. Here, we present Fixative-eXchange (FX)-seq, a highly scalable snRNA-seq method for heavily paraformaldehyde (PFA)-fixed and/or FFPE samples. We employ an organocatalyst to facilitate the removal of PFA crosslinks to increase RT yield and additional regiospecific Pt(II)-based crosslinking of RNA molecules to prevent leakage. FX-seq reveals cellular heterogeneity across multiple fixed samples by analyzing 321,710 nuclei, including PFA-fixed tissue, FFPE blocks, thin FFPE and hematoxylin and eosin (H&E)-stained sections from mouse brain and human cancer specimens such as gastrointestinal stromal tumor and colorectal cancer. FX-seq enables integrated analysis with pathologist annotation to label tumor and non-tumor regions of H&E-stained sections. FX-seq can also be applied to PFA-perfusion-based animal studies, large human cohort studies, and personalized drug treatment through precision medicine.

genomics↗

bioRxiv2026

Insight into the scaffolding function of USP18 from a high resolution cryo-EM structure of STAT2-USP18-ISG15 ternary complex

USP18 is a primary negative regulator of the type I interferon (IFN-I) signaling which regulates hundreds of IFN-stimulated genes for viral protection and anti-cancer immunity. USP18 plays dual roles in the IFN-I signaling: 1) deubiquitinase enzymatic function which cleaves ISG15 from its substrates and 2) scaffolding function through forming a complex with STAT2 to suppress IFN-I signaling. Targeting the scaffolding function of USP18, instead of its enzyme activity, is crucial for reducing cancer cell fitness and boosting anti-tumor immunity. However, the molecular basis of USP18s scaffolding function remains unclear due to the lack of structural information. Here, using a fusion tag strategy, we captured the transient USP18-STAT2 complex and determined a ternary complex structure of STAT2-USP18-ISG15 at 3.05 [A] resolution by cryogenic electron microscopy (cryo-EM) that delineated detailed USP18-STAT2 interactions. Remarkably, the ternary complex impairs USP18s enzymatic function by STAT2-mediated disruption of its catalytic triad. Structural analysis and mutagenesis identify specific USP18 point mutations, facilitating further investigation into the role of USP18 in IFN-I signaling. Taken together, our findings suggest that USP18s scaffolding function could present an untapped opportunity for cancer therapy.

molecular biology↗

bioRxiv2024

Structural basis of the catalytic and allosteric mechanism of bacterial acetyltransferase PatZ

GCN5-related N-Acetyltransferases (GNATs) play a crucial role in regulating bacterial metabolism by acetylating specific target proteins. Despite their importance in bacterial physiology, the mechanisms underlying GNATs enzymatic and regulatory functions remain poorly understood. In this study, we elucidated the structures of Escherichia coli PatZ, a type I GNAT, and investigated its ligand interactions, catalytic processes, and allosterism. PatZ functions as a homotetramer, with each subunit comprising a catalytic domain and a regulatory domain. Our findings reveal that the regulatory domain is essential for acetyltransferase activity, as it not only induces cooperative conformational changes in the catalytic domain but also directly contributes to the formation of substrate binding pockets. Furthermore, a protein structure-based analysis on the evolution of bacterial GNAT types reveals a distinct pattern of the regulatory domain across phyla, underscoring the regulatory domains critical role in responding to cellular energy status. SIGNIFICANCE STATEMENTPost-translational modifications, particularly acetylation mediated by GCN5-related N-Acetyltransferases (GNATs), play a crucial role in bacterial physiology. Protein acetyltransferase Z (PatZ) is a key GNAT with diverse substrates, essential for understanding the bacterial acetylome. This study employs cryogenic electron microscopy, X-ray crystallography, and biochemical analyses to elucidate the mechanistic regulation of Escherichia coli PatZ. Our high-resolution structures reveal PatZs homo-tetrameric architecture, with each subunit comprising regulatory and GNAT domains. We characterize ligand-PatZ interactions, demonstrating ligand-induced conformational changes that facilitate allosteric regulation of the catalytic domain. Furthermore, our analyses elucidate the regulatory domains contribution to substrate binding pocket formation, potentially enhancing substrate specificity. Structure-based phylogenetic analysis provides insights into the evolution of diverse regulatory domains in the GNAT superfamily across bacterial taxonomy. This first visualization of PatZ advances our mechanistic understanding of bacterial physiology, offering novel insights into GNAT-mediated bacterial adaptations. HIGHLIGHTS- E. coli PatZ forms a homotetramer, with each subunit possessing a GNAT catalytic domain and a regulatory domain. - Cooperative binding of acetyl-CoA to the regulatory domains is a prerequisite for inducing the structural compatibility of the catalytic domain with a substrate. - Diverse regulatory domains in GNATs evolved to adapt to varied metabolic conditions across bacterial taxonomy.

biochemistry↗

bioRxiv2024

Expression study of Krabbe Disease GALC missense variants -- Insights from quantification profiles of residual enzyme activity, secretion and psychosine levels

Krabbe disease (KD) is an autosomal recessive lysosomal storage disorder caused by loss-of-function mutations in the GALC gene, which encodes for the enzyme galactosylceramidase (GALC). GALC is crucial for myelin metabolism. Functional deficiency of GALC leads to toxic accumulation of psychosine, dysfunction and death of oligodendrocytes, and eventual brain demyelination. To date, 46 clinically-relevant, pathogenic GALC missense mutations (MMs) have been identified in KD patients. These MMs are present in [~]70% of KD cases reported over 8 published studies between 1996 - 2019. However, the mechanisms by which these MMs lead to GALC functional deficiency and their correlations with clinical phenotype remain poorly understood. To address this, we generated a GALC-knockout human oligodendrocytic cell line (MO3.13/ GALC-KO) using CRISPR-Cas9 method to assess GALC function and GALC secretion. We evaluated 5 polymorphic and 31 clinically-relevant MM variants (MMVs) using transient expression assays. Our results showed that 26 MMVs, including 10 co-variants with p.I562T, reduced GALC activity by 92% - 100% compared to wild-type GALC (WT-GALC). MMVs from infantile-onset KD patients produced < 2% of WT activity, whereas those associated with juvenile- and adult-onset cases retained up to 7% of WT activity. Residual GALC activity was correlated with mature, lysosomal GALC protein levels (Pearson r = 0.93, P<0.0001). Many low-activity MMVs did not correspondingly impair GALC secretion. Twenty-one of the 26 low-activity MMVs showed a 21% - 100% reduction in sec-GALC levels, indicating varying degrees of GALC mis-trafficking among these variants. Importantly, GALC activity among MMVs strongly correlates with clinical disease severity, based on the age of symptom onset in patients with either homozygous MM (Pearson r = 0.98, P<0.0001, n = 7) or compound heterozygous (Pearson r = 0.94, P<0.0001, n = 12) MM-null mutation genotypes. Thus, our data suggests that GALC activity could serve as a prognostic disease indicator under specific experimental conditions. We further investigated the impact of pathogenic MMVs on psychosine accumulation, a key biomarker for KD. Psychosine levels were 21-fold higher in mock control cells compared to WT-GALC transfected cells (mock = 0.349 pmol/mg, WT-GALC = 0.016 pmol/mg), but negatively correlated with GALC activity among pathogenic MMVs (Pearson r = -0.63, P < 0.01, n = 15). Although psychosine levels were higher in most MMVs associated with infantile-onset KD, no significant correlations with clinical onset were detected. Overall, our study provides a comprehensive quantitative analysis of the functional deficits and mis-trafficking associated with clinically-relevant GALC MMVs, enhancing our understanding of the molecular genetics and genotype-phenotype correlations of the GALC gene in Krabbe disease.

cell biology↗

bioRxiv2023

Development and validation of a high throughput screening platform to enable target identification in skeletal muscle cells from Duchenne Muscular Dystrophy (DMD) patients

Duchenne muscular dystrophy (DMD) is a progressive and fatal muscle degenerating disease caused by dystrophin deficiency. Effective methods for drug discovery for the treatment of DMD requires systems to be physiologically relevant, scalable, and effective. To this end, the Myoscreen platform offers a scalable and physiologically relevant system for generating and characterizing patient-derived myotubes. Morphological profiling is a powerful technique involving the simultaneous measurement of hundreds of morphological parameters from fluorescence microscopy images and using machine learning to predict cellular activity. Here, we describe combining the Myoscreen platform and high dimensional morphological profiling to accurately predict a phenotype associated with the lack of Dystrophin expression in patient derived myotubes. Using this methodology, we evaluated a series of Dystrophin-associated protein complex (DAPC) candidates and identified that the combination of Utrophin and - Sarcoglycan yielded highest morphological differences between DMD and non-DMD donors. Finally, we validated this methodology by knocking down Dystrophin expression in non-DMD cells as well as introducing Dystrophin expression in DMD cells. Knocking down Dystrophin in non- DMD cells shifted their morphological profile to one that is similar to DMD cells while introducing Dystrophin in DMD cells shifted their morphological profile towards non-DMD cells. In conclusion, we have developed a platform that accurately predicts the DMD disease phenotype in a disease relevant cell type. Ultimately this platform may have wide applications in the drug development process include identification of disease modifier genes, screening of novel therapeutic moieties, and as a potency assay for future therapeutics.

cell biology↗

bioRxiv2023

Human USP18 protects diverse cancer lineages from Type I Interferon independently of its canonical catalytic function

Precise temporal regulation of Type I interferon signaling is imperative to effectively fight infections and cancerous cells without triggering autoimmunity. The key negative regulator of Type I interferon signaling is ubiquitin-specific protease 18 (USP18). USP18 cleaves interferon-inducible ubiquitin-like modifications through its canonical catalytic function and directly inhibits interferon receptor signaling through its scaffold role. USP18 loss-of-function dramatically impacts autoimmune disease, viral susceptibility, and cancer cell survival. However, the relative contribution of catalytic versus scaffold function is unresolved and must be determined to design effective therapeutics targeting USP18. To precisely delineate individual contribution, we evaluated the functional impact of single amino acid mutations that disrupt catalytic or scaffold activity. Here we demonstrate catalytic activity does not contribute to cell autonomous Type I interferon sensitivity across multiple cancer cell lineages. Furthermore, introducing a patient-derived mutation that disrupts scaffold function is sufficient to inhibit cancer growth. These findings establish a fundamental mechanistic basis for USP18 therapeutic design across diseases. OVERVIEWO_LIUSP18 is the key negative regulator of Type I interferon signaling in humans, mediating autoimmune disease, viral susceptibility, and cancer cell survival. C_LIO_LIUSP18 cleaves interferon-inducible ubiquitin-like modifications through its canonical catalytic function and attenuates interferon receptor signaling through its scaffold role. C_LIO_LIDelineating the contribution of each function is critical to resolve the mechanistic basis of interferon regulation and enable the development of therapeutics targeting USP18. C_LIO_LIWe demonstrate that cell intrinsic interferon sensitivity is not mediated by loss of catalytic activity. However, disruption of scaffold function by a patient-specific mutation inhibits cancer cell growth. C_LIO_LIFurthermore, we discovered that canonical catalytic activity is surprisingly inefficient in human cells. C_LIO_LIThese results clarify a fundamental mechanism of immune regulation and cancer cell survival in humans. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/533924v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@d0a353org.highwire.dtl.DTLVardef@192a09dorg.highwire.dtl.DTLVardef@16eb296org.highwire.dtl.DTLVardef@8e98a3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Preliminary Minimum Reporting Requirements for Reporting In-Vivo Neural Interface Research: I. Implantable Neural Interfaces

The pace of research and development in neuroscience, neurotechnology, and neurorehabilitation is rapidly accelerating, with the number of publications doubling every 4.2 years. Maintaining this progress requires technological standards and scientific reporting guidelines to provide frameworks for communication and interoperability. The present lack of such standards for neurotechnologies limits the transparency, reproducibility, and meta-analysis of this growing body of research, posing an ongoing barrier to research, clinical, and commercial objectives. Continued neurotechnological innovation requires the development of some minimal standards to promote integration between this broad spectrum of technologies and therapies. To preserve design freedom and accelerate the translation of research into safe and effective technologies with maximal user benefit, such standards must be collaboratively co-developed by a full spectrum of neuroscience and neurotechnology stakeholders. This paper summarizes the preliminary recommendations of IEEE Working Group P2794, developing a Reporting Standard for in-vivo Neural Interface Research (RSNIR). Impact StatementThis work provides a preliminary set of reporting guidelines for implantable neural interface research, developed by IEEE WG P2794 in open collaboration between a range of stakeholders to accelerate the research, development, and integration of innovative neurotechnologies.

bioengineering↗

bioRxiv2020

Respiratory and gut microbiota in commercial turkey flocks with disparate weight gain trajectories display differential compositional dynamics

Host-associated communities of bacteria (microbiota) substantially contribute to the overall poultry health and performance. Gut microbiota are known to play roles in resistance to pathogen infection and optimal weight gain in turkey flocks. However, knowledge of turkey respiratory microbiota and its link to gut microbiota is lacking. This study presents a 16S rRNA gene-based census of the turkey respiratory microbiota (nasal cavity and trachea) alongside gut microbiota (cecum and ileum) in two identical commercial Hybrid Converter turkey flocks raised in parallel under typical field commercial conditions. The flocks were housed in adjacent barns during the brood stage and in geographically separated farms during the grow-out stage. Several bacterial taxa that were acquired in the respiratory tract (RT) at the beginning of the brood stage persisted throughout the flock cycle, primarily Staphylococcus. Late-emerging predominant taxa in RT included Deinococcus and Corynebacterium. Tracheal and nasal microbiota of turkeys were identifiably distinct from one another and from gut microbiota. Nevertheless, gut and RT microbiota changed in parallel over time and appeared to share many taxa. During the brood stage, the two flocks generally acquired similar gut and RT microbiota, and their average body weights were comparable. Separating the flocks during the grow-out stage resulted in divergent microbial profiles and body weight gain trajectories. Lower weight gain corresponded with emergence of Deinococcus and Ornithobacterium in RT, and Fusobacterium and Parasutterella in gut. This study provides an overview of turkey microbiota under field conditions and suggests several hypotheses concerning the respiratory microbiome. IMPORTANCETurkey meat is an important source of animal protein, and the industry around its production contributes significantly to the agricultural economy. The nonpathogenic symbionts present in the gut of turkeys are known to impact bird health and flock performance. However, the respiratory microbiota in turkeys are entirely unexplored. This study has elucidated the microbiota of respiratory tracts of turkeys from two commercial flocks raised in parallel throughout a normal flock cycle. Further, the study suggests that bacteria originating in the gut or in poultry house environments may influence respiratory communities and consequently induce poor performance, either directly or indirectly. Future attempts to develop microbiome-based interventions for turkey health should delimit the contributions of respiratory microbiota and aim to limit disturbances to those communities.

microbiology↗

bioRxiv2018

An open source platform for analyzing and sharing worm behavior data

Animal behavior is increasingly being recorded in systematic imaging studies that generate large data sets. To maximize the usefulness of these data there is a need for improved resources for analyzing and sharing behavior data that will encourage re-analysis and method development by computational scientists1. However, unlike genomic or protein structural data, there are no widely used standards for behavior data. It is therefore desirable to make the data available in a relatively raw form so that different investigators can use their own representations and derive their own features. For computational ethology to approach the level of maturity of other areas of bioinformatics, we need to address at least three challenges: storing and accessing video files, defining flexible data formats to facilitate data sharing, and making software to read, write, browse, and analyze the data. We have developed an open resource to begin addressing these challenges using worm tracking as a model.

animal behavior and cognition↗