Biology subjects

Le, K.

Publications and source records attributed to Le, K..

11 recordsLinked to original sources

bioRxiv2026

Single-cell foundation models identify shared and divergent transcriptomic signatures of aging across invertebrates and mammals

Cross-species aging clocks have so far stayed within mammals. We asked whether a single model can predict age from single cells of fly, worm, mouse, and human, species separated by roughly 800 million years of evolution. We fine-tuned scGPT and Geneformer on 1.3 million such cells, using one parameter set per architecture. Both models classified cells as young, middle, or old, reaching 78.6% and 80.5% accuracy. SHAP attribution then showed where the two architectures agreed and where they diverged. scGPT ranked ribosomal proteins highest in every species, with RPL12 first throughout. Geneformer did the same in mouse and human but favored signaling, chromatin, and ubiquitin-ligase genes in fly and worm. Hiding all 51 ribosomal-protein genes at inference cost 22-28 percentage points of accuracy, against 0.3 points for size-matched random gene sets. Bootstrap resampling, five-fold cross-validation, and comparison with six published aging studies left the rankings largely unchanged. Age-associated signal is therefore accessible to a pooled cross-species model, but which genes carry it depends on how the model encodes expression.

genomics↗

bioRxiv2026

A novel rat model of body first subtype of Parkinsons disease

The recent growing evidence support the existence of two subtypes of Parkinsons disease (PD), a body first and brain first subtype owing to variability in the disease site of onset as well as disease progression. Animal models which could replicate the specific differences of these subtypes are important to explore the pathophysiology as well as to evaluate novel treatment options. Here, we describe an animal model of body first PD subtype developed using repeated low dose exposure of environmental neurotoxin Paraquat (P) and Lectin (L) to characterize its PD-like manifestations. We administered P+L (P+L, p.o.) daily to rats for 90 days. These animals underwent motor and non-motor behavioral tests at various time intervals. After 21 weeks, post-mortem histopathological analysis was performed to assess neurodegeneration. Onset of motor deficits initiated unilaterally from week 4 of P+L followed by gradual progression towards bilateral symptoms that were levodopa responsive. This model also replicates non motor features including cognitive deficits in tests like Novel Object Recognition Test and Y maze as well as sleep abnormalities. The histopathology showed nigrostriatal dopaminergic degeneration and proteinase K resistant S129 alpha-synucleinopathy both in the gut and the brain. The replication of both progressive motor and non-motor features in this rat model corroborates body first subtype of PD therefore making it an attractive option for testing neuroprotective experimental therapeutics and avenue to understand pathophysiological mechanisms.

neuroscience↗

bioRxiv2026

Rigorous Validation of Paw Preference Using Three Complementary Behavioral Assays in Sprague Dawley Rats

Paw preference, or handedness, is a widely studied behavioral trait used to assess lateralization and motor function in rodents. This study aimed to determine the consistency and reliability of three commonly used behavioral tests to rigorously assess paw preference: the Collins Test, the Staircase Test, and the Pawedness Trait Test. Thirty Sprague Dawley rats (12-48 weeks; 20 females, 10 males) were subjected to all three behavioral tests. Paw uses were recorded, and the laterality index was calculated for each test. Additional cohorts of younger rats (6-9 weeks; 45 females, 45 males) and older rats (12-48 weeks; 38 females, 45 males) were tested to assess the effects of age and sex on paw preference. ANOVA, Fleiss and pairwise Cohens Kappa were used for statistical analysis. All three tests yielded comparable measures of paw preference (ANOVA, p = 0.801). Substantial inter-test agreement was demonstrated by Fleiss kappa ({kappa} = 0.761, p = 3.93 x 10{square}12). Paw preference did not significantly vary by age or sex, and the distribution of left, right, and ambidextrous preference categories aligned with existing literature. The Collins, Staircase, and Pawedness Trait Tests provide consistent, reliable assessments of paw preference in Sprague Dawley rats. These validated behavioral assays can serve as essential tools for preclinical research, including but not limited to models of motor asymmetry observed in stroke, cerebral palsy, traumatic brain injury, and language lateralization, as well as neurodegenerative diseases. HighlightsO_LIRigorously validated paw preference in Sprague Dawley rats using three commonly used behavioral tests. C_LIO_LIDemonstrated strong inter-test agreement across Collins, Staircase, and PaTRaT ({kappa} = 0.761) C_LIO_LIShowed that paw preference remains stable across age and sex in large cohorts C_LIO_LIApplied standardized LI thresholds to enable cross-test comparability C_LI

neuroscience↗

bioRxiv2024

Cell type specific suppression of hyper-recombination by human RAD18 is linked to PCNA K164 ubiquitination

Homologous recombination (HR) and translesion synthesis (TLS) promote gap-filling DNA synthesis to complete genome replication. One factor involved in both pathways is RAD18, an E3 ubiquitin ligase. Although RAD18s role in promoting TLS through the ubiquitination of PCNA at lysine 164 (K164) is well established, its requirement for HR-based mechanisms is currently less clear. To assess this, we inactivated RAD18 in three human cell lines. Our analyses found that loss of RAD18 in HCT116, but neither hTERT RPE-1 nor DLD1 cell lines, resulted in elevated sister chromatid exchange, gene conversion, and gene targeting, i.e. HCT116 mutants were hyper-recombinogenic (hyper-rec). Loss of RAD18 also impaired TLS activity in HCT116 cells, but unexpectedly, did not reduce clonogenic survival. Interestingly, these phenotypes appear linked to PCNA K164 ubiquitination, as HCT116 PCNAK164R/+ mutants were also hyper-rec and showed reduced TLS activity, consistent with previous studies in rad18-/-or pcnaK164R avian DT40 mutant cells. Importantly, knockdown of UBC9 to prevent PCNA K164 SUMOylation did not affect hyper-recombination, strengthening the link between increased recombination and RAD18-catalyzed PCNA K164 ubiquitination, but not K164 SUMOylation. Taken together, these data suggest that the roles of human RAD18 in directing distinct gap-filling DNA synthesis pathways varies depending on cell type and that these functions are linked to PCNA ubiquitination.

molecular biology↗

bioRxiv2024

CBP/P300 BRD Inhibition Reduces Neutrophil Accumulation and Activates Antitumor Immunity in TNBC

Tumor-associated neutrophils (TANs) have been shown to promote immunosuppression and tumor progression, and a high TAN frequency predicts poor prognosis in triple-negative breast cancer (TNBC). Dysregulation of CREB binding protein (CBP)/P300 function has been observed with multiple cancer types. The bromodomain (BRD) of CBP/P300 has been shown to regulate its activity. In this study, we found that IACS-70654, a novel and selective CBP/P300 BRD inhibitor, reduced TANs and inhibited the growth of neutrophil-enriched TNBC models. In the bone marrow, CBP/P300 BRD inhibition reduced the tumor-driven abnormal differentiation and proliferation of neutrophil progenitors. Inhibition of CBP/P300 BRD also stimulated the immune response by inducing an IFN response and MHCI expression in tumor cells and increasing tumor-infiltrated CTLs. Moreover, IACS-70654 improved the response of a neutrophil-enriched TNBC model to docetaxel and immune checkpoint blockade. This provides a rationale for combining a CBP/P300 BRD inhibitor with standard-of-care therapies in future clinical trials for neutrophil-enriched TNBC. SummaryIn neutrophil-enriched triple-negative breast cancer (TNBC) models, CREB binding protein (CBP)/P300 bromodomain (BRD) inhibition reduces tumor growth and systemic neutrophil accumulation while stimulating an antitumor immune response. This improves standard-of-care therapies, suggesting a potential therapeutic benefit of CBP/P300 BRD inhibitors for neutrophil-enriched TNBC.

cancer biology↗

bioRxiv2024

Diverse competitor B cell responses to a germline-targeting priming immunogen in human Ig loci transgenic mice

Broadly neutralizing antibodies (bnAbs) have promise to protect against HIV infection, but induction of bnAbs by immunization is an unsolved vaccine design challenge. Germline-targeting priming immunogens aim to initiate the induction of bnAbs by specifically activating rare bnAb-precursor B cells that can subsequently be matured using suitable heterologous boosting and shepherding immunogens. Several pre-clinical studies, and the IAVI G001 human clinical trial, have demonstrated the ability of a germline-targeting priming immunogen, eOD-GT8 60mer, to induce precursors of the VRC01 class of bnAbs. However, much less is known about B cells induced against other epitopes of the immunogen. Here, we performed unbiased analysis of B cells induced by eOD-GT8 60mers in Intelliselect Transgenic mice (Kymice) that are transgenic for the human Ig loci and produce human-like BCRs. B cells isolated with intact eOD-GT8 60mer nanoparticles showed a large diversity of non-VRC01-class B cells, with 38% unique clonotypes and only 5% of BCRs belonging to public lineages shared among all animals. We found that many competitors recognize epitopes in close proximity to or overlapping with the VRC01 epitope. These results indicate that optimal boosting of VRC01-class bnAb-precursor B cells primed by eOD-GT8 60mer might require a first-boost immunogen that minimizes recognition of competitor B cells, and such competitors isolated from Kymice could serve as valuable reagents for boost development.

immunology↗

bioRxiv2023

A scalable platform for efficient CRISPR-Cas9 chemical-genetic screens of DNA damage-inducing compounds

Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screen platform by generating a DNA damage response (DDR)-focused custom sgRNA library. We performed five proof-of-principle compound screens and found that the compounds known modes-of-action (MoA) were enriched among the compounds CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late time point interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale and produce biologically informative CGI profiles.

systems biology↗

bioRxiv2021

Deep learning for robust and flexible tracking in behavioral studies for C. elegans

Robust and accurate behavioral tracking is essential for ethological studies. Common methods for tracking and extracting behavior rely on user adjusted heuristics that can significantly vary across different individuals, environments, and experimental conditions. As a result, they are difficult to implement in large-scale behavioral studies with complex, heterogenous environmental conditions. Recently developed deep-learning methods for object recognition such as Faster R-CNN have advantages in their speed, accuracy, and robustness. Here, we show that Faster R-CNN can be employed for identification and detection of Caenorhabditis elegans in a variety of life stages in complex environments. We applied the algorithm to track animal speeds during development, fecundity rates and spatial distribution in reproductive adults, and behavioral decline in aging populations. By doing so, we demonstrate the flexibility, speed, and scalability of Faster R-CNN across a variety of experimental conditions, illustrating its generalized use for future large-scale behavioral studies.

bioengineering↗

bioRxiv2020

Population structure of eulachon Thaleichthys pacificus from Northern California to Alaska using single nucleotide polymorphisms from direct amplicon sequencing

Eulachon Thaleichthys pacificus, a culturally and ecologically important anadromous smelt (Family Osmeridae), ranges from Northern California to the southeast Bering Sea. In recent decades, some populations have experienced declines. Here we use a contig-level genome assembly combined with previously published RADseq-derived markers to construct an amplicon panel for eulachon. Using this panel, we develop a filtered genetic baseline of 521 variant loci genotyped in 1,989 individuals from 14 populations ranging from Northern California through Central Alaska. Consistent with prior genetic studies, the strongest separation occurs among three main regions: from Northern California up to and including the Fraser River; north of the Fraser River to southeast Alaska; and within the Gulf of Alaska. Separating the Fraser River from southern US populations, and refining additional substructure within the central coast may be possible in mixed-stock analysis; this will be addressed in future work. The amplicon panel outperformed the previous microsatellite panel, and thus will be used in future mixed-stock analyses of eulachon in order to provide new insights for management and conservation of eulachon.

genetics↗

bioRxiv2020

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Heterologously-expressed bacterial nitroreductase (NTR) enzymes sensitize eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrate systems. However, first-generation NTRs require confoundingly toxic prodrug treatments (e.g. 10 mM MTZ) and some cell types have proven resistant. We used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy. Toxicity tests in zebrafish showed no deleterious effects of prolonged MTZ treatments of [≤]1 mM. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and enable modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic resources to facilitate dissemination of NTR 2.0 to the research community.

cell biology↗

bioRxiv2018

Reprogramming the antigen specificity of B cells using genome-editing technologies

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody, PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved anti-HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses.

immunology↗