Biology subjects

Lee, L.

Publications and source records attributed to Lee, L..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

A robust platform for recombinant production of animal venom toxin modulators of ion channels

Background and PurposePeptide toxins isolated from animal venom are potent and selective modulators of ion channels, and promising therapeutic leads. Due to intricate disulphide bridge patterns, they are often challenging to produce in standard laboratory settings, which limits engineering approaches to manipulate their structure-function properties. Given the low cost, wide accessibility, and versatility of recombinant expression systems for protein production, we set out to establish a straightforward high-yield strategy across a broad panel of peptide toxins from snakes, spiders and scorpions. Experimental Approach13 toxin DNA sequences were genetically fused to the C-terminus of either bivalent or monovalent human IgG1 antibody fragment crystallisable (Fc) domain sequences and expressed recombinantly from mammalian Expi293F cells. Affinity-purified proteins were evaluated by SDS-PAGE and size-exclusion chromatography (SEC). Function was assessed by Ca2+ flux assays on CN21 cells, or whole-cell electrophysiology on human embryonic kidney (HEK293T) cells, Chinese hamster ovary (CHO) cells, or dorsal root ganglion (DRG) neurons. Immunocytochemistry using HEK293T cells and mouse DRG neurons assessed Fc-toxin fusion binding. Key ResultsMonovalent Fc-toxin fusions consistently yielded 1-6 mg of pure, non-proteolytically cleaved protein from 20-70 ml cultures for several toxin types, including three-finger toxins from snakes, inhibitory cystine knot (ICK) toxins from spiders, and -toxins from scorpions, substantially surpassing the performance of unfused toxins or bivalent Fc-toxin fusions which gave low or no yield. Snake toxins targeting nicotinic acetylcholine receptors retained high single digit nanomolar inhibitory potency. Spider and scorpion toxins targeting the voltage-gated Na+ channel Nav1.7 retained pharmacological function and selectivity across a panel of five Nav subtypes, albeit with reduced potencies that did not exceed [~]70 nM. Conclusions and ImplicationsWe present a strategy for straightforward robust production of pure, monodisperse, and functional animal venom-derived toxins. This lowers the barrier to toxin production in a standard laboratory setting for follow-on engineering purposes.

pharmacology and toxicology↗

bioRxiv2025

Comparative genomics reveals multipartite genomes undergoing loss in the fungal endosymbiotic genus Mycetohabitans

Endosymbiotic bacteria extensively impact phenotypes of their eukaryotic hosts, while experiencing dramatic changes to their own genome as they become more host-restricted in lifestyle. Understanding the trajectory of such a genome has largely been done through study of animal-associated bacteria, especially insect endosymbionts. Yet, endofungal bacteria provide another natural experimental model for investigating how microbial genomes change when living inside of a host cell. Mycetohabitans spp. are culturable bacterial endosymbionts of the Mucoromycota fungus Rhizopus microsporus. To investigate the genome dynamics resulting from the endohyphal nature of this emerging model genus, we long-read sequenced and assembled new complete genomes to combine with previous assemblies, creating a global dataset of 28 complete Mycetohabitans genomes. All genomes were between 3.3 and 3.9 Mbp in size and were multipartite, structured into two conserved replicons with some strains having an additional plasmid. Based on evolutionary rate and gene content analysis of the different replicons, we termed the two major ones a chromosome and chromid. The differential presence of a third, mobilome-rich plasmid in some strains and the proliferation of transposable elements provide putative mechanisms for recombination or gene loss. The conservation of intact prophage and putative toxin-antitoxin systems, and extensive enrichment of secondary metabolite clusters in the Mycetohabitans genomes highlight the dynamic nature of this reducing genome. With fungal-bacterial symbioses becoming increasingly apparent phenomena, lessons learned from this symbiosis will inform our understanding of bacterial adaptation to novel hosts, and the process of microbe-microbe coevolution. SIGNIFICANCEGenomes change when organisms become closely associated, as bacterial partners undergo less opportunities for genetic exchange and become more reliant on their hosts. Our pangenome analysis of the endofungal genus Mycetohabitans shows a snapshot of evolution where the bacteria are moving from a facultative towards an obligate relationship with their host fungi. We find that Mycetohabitans genomes are marked by transposable elements, pseudogenes, a large biosynthetic repertoire, and novel bacteriophage. We provide a genomic resource to match publicly accessible strains for future work on endosymbiotic genetics and evolution.

genomics↗

bioRxiv2025

UITOTO: a new software for molecular diagnoses in species descriptions

Millions of species remain undescribed, and each eventually will require a species description that includes a diagnosis. However, no software currently exists that fully integrates the derivation and validation of state-specific and contrastive molecular diagnoses. Here we introduce UITOTO which addresses this gap and facilitates the identification, testing, and visualization of Diagnostic Molecular Combinations (DMCs). The software uses a weighted random sampling algorithm based on the Jaccard Index for building candidate DMCs. It then selects DMCs with the highest specificity stability, meeting user-defined thresholds for exclusive character states. If multiple optimal DMCs are identified, UITOTO derives a majority-consensus DMC. To verify whether the newly generated DMCs are contrastive, UITOTO includes a validation module that tests DMCs against database sequences, efficiently handling up to hundreds of thousands of aligned or unaligned sequences. In this manuscript we propose and assess UITOTOs performance with DMCs obtained from other software (e.g., MOLD) for three large empirical datasets: i) Megaselia (Diptera: Phoridae: 69 species, 2,229 training and 30,289 testing barcodes); ii) Mycetophilidae, (Diptera: 118 species, 1,456 training and 60,349 testing barcodes); and iii) European Lepidoptera (49 species, 591 training and 21,483 testing barcodes). Based on several classification metrics (e.g., Accuracy, F1 Score), UITOTOs DMCs outcompete DMCs obtained with other software. We furthermore provide guidelines for generating molecular diagnoses and facilitate obtaining DMCs by providing a user-friendly Shiny App GUI and a module for obtaining publication-quality DMC visualizations in RStudio. Overall, our study confirms that the biggest challenge for generating molecular and morphological diagnoses is similar, i.e., balancing specificity and length. Short diagnoses often lack specificity, while excessively long DMCs are often so specific that they do not accommodate intraspecific variation.

bioinformatics↗

bioRxiv2025

Single-cell profiling of human bone marrow reveals multiple myeloma progression is accompanied by an increase in CD56bright bone marrow resident NK cells.

Natural killer (NK) cells play a key role in the innate immune response against tumour progression. While the immune microenvironment in multiple myeloma (MM) becomes increasingly dysfunctional during disease evolution, little is known about changes in the NK cell compartment. Using primary samples from clinical trial patients, we performed detailed phenotypic analyses of bone marrow mononuclear cells from MGUS, SMM, and newly diagnosed MM patients. We found that disease progression is associated with an increase in CD56bright NK cells with a dynamic positive association between this NK cell subset and local tumour burden. We generated a large single-cell RNA sequencing dataset of >100,000 NK cells from healthy donor individuals and plasma cell disorder patients and identify a bone marrow specific CD56bright-like NK cell population (BM-NK) that is enriched in the marrow of MM patients. These findings highlight the evolution of the NK-cell compartment in MM and suggest a role for BM-resident CD56bright NK cells with impaired cytotoxicity in promoting immune evasion.

cancer biology↗

bioRxiv2024

Characterizing vascular function in mouse models of Alzheimer's disease, atherosclerosis, and mixed Alzheimer's and atherosclerosis

SignificanceAlzheimers disease does not occur in isolation and there are many comorbidities associated with the disease - especially diseases of the vasculature. Atherosclerosis is a known risk factor for the subsequent development of Alzheimers disease, therefore understanding how both diseases interact will provide a greater understanding of co-morbid disease progression and aid the development of potential new treatments. AimThe current study characterizes hemodynamic responses and cognitive performance in APP/PS1 Alzheimers mice, atherosclerosis mice, and a mixed disease group (APP/PS1 & atherosclerosis) between the ages of 9 and 12 months. ApproachWhisker-evoked hemodynamic responses and recognition memory were assessed in awake mice, immunohistochemistry to assess amyloid pathology, and histology to characterize atherosclerotic plaque load. ResultsWe observed hemodynamic deficits in atherosclerosis mice (vs Alzheimers, mixed disease or wild-type mice), with reduced short-duration stimulus-evoked hemodynamic responses occurring when there was no concurrent locomotion during the stimulation period. Mixed Alzheimers and atherosclerosis models did not show differences in amyloid beta coverage in the cortex or hippocampus or atherosclerotic plaque burden in the aortic arch vs relevant Alzheimers or atherosclerosis controls. Consistent with the subtle vascular deficits and no pathology differences, we also observed no difference in performance on the novel object recognition task across groups. ConclusionsThese results emphasize the importance of experimental design for characterizing vascular function across disease groups, as locomotion and stimulus duration impacted the ability to detect differences between groups. Whilst atherosclerosis did reduce hemodynamic responses, these were recovered in the presence of co-occurring Alzheimers disease which may provide targets for future studies to explore the potentially contrasting vasodilatory mechanisms these diseases impact.

neuroscience↗

bioRxiv2024

RESOLVING BIOLOGY'S DARK MATTER: SPECIES RICHNESS, SPATIOTEMPORAL DISTRIBUTION, AND COMMUNITY COMPOSITION OF A DARK TAXON

BackgroundZoologys dark matter comprises hyperdiverse, poorly known taxa that are numerically dominant but largely unstudied, even in temperate regions where charismatic taxa are well understood. It is everywhere, but high diversity, abundance, and small size have historically stymied its study. We demonstrate how entomological dark matter can be elucidated using high-throughput DNA barcoding ("megabarcoding"). We reveal the high abundance and diversity of scuttle flies (Diptera: Phoridae) in Sweden using 31,800 specimens from 37 sites across four seasonal periods. We investigate the number of scuttle fly species in Sweden and the environmental factors driving community changes across time and space. ResultsSwedish scuttle fly diversity is much higher than previously known, with 549 mOTUs (putative species) detected, compared to 374 previously recorded species. Hierarchical Modelling of Species Communities reveals that scuttle fly communities are highly structured by latitude and strongly driven by climatic factors. Large dissimilarities between sites and seasons are driven by turnover rather than nestedness. Climate changes are predicted to significantly affect the 47% of species that show significant responses to mean annual temperature. Results were robust whether using haplotype diversity or species-proxies (mOTUs) as response variables. Additionally, species-level models of common taxa adequately predict overall species richness. ConclusionsUnderstanding the bulk of the diversity around us is imperative during an era of biodiversity loss. We show that dark insect taxa can be efficiently characterized and surveyed with megabarcoding. Undersampling of rare taxa and choice of operational taxonomic units do not alter the main ecological inferences, making it an opportune time to tackle zoologys dark matter.

ecology↗

bioRxiv2024

Rapid in situ mutation detection in extracellular vesicle-DNA

A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-minute DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.

bioengineering↗

bioRxiv2023

c-Maf and Mafb function combinatorially to establish SGN subtype-specific synaptic properties

Neurons develop diverse synapses that vary in content, morphology, and size. Although transcriptional regulators of neurotransmitter identity have been identified, it remains unclear how other synaptic features are patterned among neuronal subtypes. In the auditory system, glutamatergic synaptic properties vary across three subtypes of spiral ganglion neurons (SGNs) that collectively encode sound information. Here, we show that a combinatorial Maf transcription factor code establishes SGN subtype identity and shapes both shared and subtype-appropriate synaptic properties. We find that c-Maf and Mafb have independent and opposing effects on synaptic morphology and auditory function, while also acting redundantly to impart subtype identities and drive synaptic differentiation needed for normal auditory responses. Additionally, c-Maf and Mafb are expressed at different levels across subtypes and regulate subtype-appropriate gene expression in a dose-dependent manner. Thus, functional diversity of Maf family members enables flexible and robust control of gene expression needed to generate synaptic heterogeneity across neuronal subtypes.

neuroscience↗

bioRxiv2023

Epigenetic coordination of transcriptional and translational programs in hypoxia

Adaptation to cellular stresses entails an incompletely understood coordination of transcriptional and post-transcriptional gene expression programs. Here, we quantified hypoxia-dependent transcriptomes, epigenomes, and translatomes in T47D breast cancer cells and H9 human embryonic stem cells. This revealed pervasive changes in transcription start site (TSS) selection associated with nucleosome repositioning and alterations in H3K4me3 distribution. Importantly, hypoxia-associated TSS switching was induced or reversed via pharmacological modulation of H3K4me3 in the absence of hypoxia, defining a role for H3K4me3 in TSS selection independent of HIF1-transcriptional programs. By remodelling 5UTRs, TSS switching selectively alters protein synthesis, including enhanced translation of mRNAs encoding pyruvate dehydrogenase kinase 1 (PDK1) that is essential for the metabolic adaptation to hypoxia. These results demonstrate a previously unappreciated mechanism of translational regulation during hypoxia driven by epigenetic reprogramming of the 5UTRome.

cancer biology↗

bioRxiv2023

Do working forest easements work for conservation?

Conservation easements are voluntary legal agreements designed to achieve conservation goals on private land. Extensive public and private funding has been used to establish "working forest" conservation easements (WFCE) that aim to protect conservation values while maintaining commercial timber production. We use variation in the timing and location of easements to estimate the impacts of WFCEs in Maine from a 33-year time-series of forest loss and harvesting. Compared to matched controls, WFCEs reduced forest loss rates by just 0.0004% yr-1, the equivalent of 3ha yr-1, and increased the rate of harvesting by 0.37% yr-1, or 2935ha yr-1 within the 839,000 ha enrolled. More recent easements contained stricter restrictions on harvest practices and these easements reduced harvest by 0.62% yr-1. Overall, WFCEs supported continued harvests, but did not appear to provide substantial ecological benefits. Future easements could be more effective if they include additional provisions for harvest restrictions and monitoring.

ecology↗

bioRxiv2023

Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

bioengineering↗

bioRxiv2022

SnapFISH: a computational pipeline to identify chromatin loops from multiplexed DNA FISH data

Multiplexed DNA fluorescence in situ hybridization (FISH) imaging technologies have been developed to map the folding of chromatin fibers at tens of nanometer and tens of kilobase resolution in single cells. However, computational methods to reliably identify chromatin loops from such imaging datasets are still lacking. Here we present a Single-Nucleus Analysis Pipeline for multiplexed DNA FISH (SnapFISH), to process the multiplexed DNA FISH data and identify chromatin loops. SnapFISH can identify known chromatin loops from mouse embryonic stem cells with high sensitivity and accuracy. In addition, SnapFISH obtained comparable results of chromatin loops across datasets generated from diverse imaging technologies.

bioinformatics↗

bioRxiv2022

Improving Protein Subcellular Localization Prediction with Structural Prediction & Graph Neural Networks

The majority of biological functions are carried out by proteins. Proteins perform their roles only upon arrival to their target location in the cell, hence elucidating protein subcellular localization is essential for better understanding their function. The exponential growth in genomic information and the high cost of experimental validation of protein localization call for the development of predictive methods. We present a method that improves subcellular localization prediction for proteins based on their sequence by leveraging structure prediction and Graph Neural Networks. We demonstrate how Language Models, trained on protein sequences, and Graph Neural Networks, trained on proteins 3D structures, are both efficient approaches for this task. They both learn meaningful, yet different representations of proteins; hence, ensembling them outperforms the reigning state of the art method. Our architecture improves the localization prediction performance while being lighter and more cost-effective.

bioinformatics↗

bioRxiv2022

Network analysis with either Illumina or MinION reveals that detecting vertebrate species requires metabarcoding of iDNA from a diverse fly community

Metabarcoding of vertebrate DNA obtained from invertebrates (iDNA) has been used to survey vertebrate communities, but we here show that it can also be used to study species interactions between invertebrates and vertebrates in a spatial context. We sampled the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310m from a road). Approximately, 60% of the baited 407 flies yield 294 vertebrate identifications based on two COI fragments and 16S sequenced with Illumina and/or MinION. A bipartite network analysis finds no specialization in the interaction between flies and vertebrate species, but a spatial analysis revealed that surprisingly 18 of the 20 vertebrate species can be detected within 150m of the road. We show that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families) and carried DNA for mammals, birds, and reptiles. They included common and rare ground-dwelling (e.g., wild boar, Sunda pangolin), and arboreal species (e.g., long-tailed macaque, Raffles banded langur) as well as small bodied vertebrates (skinks, rats). All of our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving feces and regurgitates from individual flies with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. Overall, flies from several families (calliphorids, muscids and sarcophagids) should be used in iDNA surveys because we show that uncommon fly species carry the signal for several vertebrate species that are otherwise difficult to detect with iDNA.

ecology↗

bioRxiv2022

SnapHiC-D: a computational pipeline to identify differential chromatin contacts from single cell Hi-C data

Single cell Hi-C (scHi-C) has been used to map genome organization in complex tissues. However, computational tools to detect dynamic chromatin contacts from scHi-C datasets in development and through disease pathogenesis are still lacking. Here, we present SnapHiC-D, a computational pipeline to identify differential chromatin contacts (DCCs) between two scHi-C datasets. Compared to methods designed for bulk Hi-C data, SnapHiC-D detects DCCs with high sensitivity and accuracy. We used SnapHiC-D to identify celltype-specific chromatin contacts at 10 kilobase resolution in mouse hippocampal and human prefrontal cortical tissues, and demonstrated that DCCs detected in the cortical and hippocampal cell types are generally correlated with cell-type-specific gene expression patterns and epigenomic features.

bioinformatics↗

bioRxiv2022

Cost-efficient whole genome-sequencing using novel mostly natural sequencing-by-synthesis chemistry and open fluidics platform

We introduce a massively parallel novel sequencing platform that combines an open flow cell design on a circular wafer with a large surface area and mostly natural nucleotides that allow optical end-point detection without reversible terminators. This platform enables sequencing billions of reads with longer read length ([~]300bp) and fast runs times (<20hrs) with high base accuracy (Q30 > 85%), at a low cost of $1/Gb. We establish system performance by whole-genome sequencing of the Genome-In-A-Bottle reference samples HG001-7, demonstrating high accuracy for SNPs (99.6%) and Indels in homopolymers up to length 10 (96.4%) across the vast majority (>98%) of the defined high-confidence regions of these samples. We demonstrate scalability of the whole-genome sequencing workflow by sequencing an additional 224 selected samples from the 1000 Genomes project achieving high concordance with reference data.

genomics↗

bioRxiv2022

Nitric oxide is not responsible for the initial sensory-induced neurovascular coupling response in mouse cortex.

Neurovascular coupling ensures that changes in neural activity are accompanied by localised changes in cerebral blood flow. While much is known about the involvement of excitatory neurons in neurovascular coupling, the role of inhibitory interneurons is unresolved. While nNOS-expressing interneurons have been shown to be capable of eliciting vasodilation, the role of nitric oxide in functional hyperemia remains a matter of debate. Therefore in the present study we applied a combination of optogenetic and pharmacological approaches, 2-dimensional optical imaging spectroscopy, and electrophysiology to investigate the role of nitric oxide in neurovascular coupling responses evoked by nNOS-expressing interneurons and whisker stimulation in mouse sensory cortex. The haemodynamic response evoked by nNOS-expressing interneurons was significantly altered in the presence of the NOS inhibitor LNAME, revealing a large initial 20-HETE-dependent vasoconstriction. In contrast, the haemodynamic response induced by sensory stimulation was largely unchanged by LNAME. Our results suggest that while nitric oxide plays a key role in neurovascular responses evoked by nNOS-expressing interneurons it does not mediate the initial sensory-induced neurovascular coupling response in mouse cortex. Thus, our results call into question the involvement of nNOS-expressing interneurons and nitric oxide in sensory-evoked functional hyperemia.

neuroscience↗

bioRxiv2022

Bidirectional alterations in brain temperature profoundly modulate spatiotemporal neurovascular responses in-vivo: Implications for theragnostics

Neurovascular coupling (NVC) is a mechanism that, amongst other known and latent critical functions, ensures activated brain regions are adequately supplied with oxygen and glucose. This biological phenomenon underpins non-invasive perfusion-related neuroimaging techniques and recent reports have implicated NVC impairment in several neurodegenerative disorders. Yet, much remains unknown regarding NVC in health and disease, and only recently has there been burgeoning recognition of a close interplay with brain thermodynamics. Accordingly, we developed a novel multi-modal approach to systematically modulate cortical temperature and interrogate the spatiotemporal dynamics of sensory-evoked NVC. We show that changes in cortical temperature profoundly and intricately modulate NVC, with low temperatures associated with diminished oxygen delivery, and high temperatures inducing a distinct vascular oscillation. These observations provide novel insights into the relationship between NVC and brain thermodynamics, with important implications for brain-temperature related therapies, functional biomarkers of elevated brain temperature, and in-vivo methods to study neurovascular coupling.

neuroscience↗