Biology subjects

Luo, L.

Publications and source records attributed to Luo, L..

At least 73 records · Page 4Linked to original sources

bioRxiv2024

Reduction of DNA Topoisomerase Top2 reprograms the epigenetic landscape and extends health and life span across species

DNA topoisomerases are essential molecular machines that manage DNA topology in the cell and play important roles in DNA replication and transcription. We find that knocking down the enzyme Topoisomerase Top2 or its mammalian homolog Top2b increases the life span of S. cerevisiae, C. elegans, and mice. Top2b reduction also extends the health span of mice and alleviates the pathologies of aging in multiple tissues. At the cellular/molecular level, Top2b reduction attenuates the major hallmarks of aging, such as cellular senescence, de-regulated nutrient sensing, epigenetic alteration, and lysosomal biogenesis. We observed that Top2b reduction significantly changes the epigenetic landscape in various mouse tissues toward those of the young animals, and differentially down-regulates genes with active promoter and high expression. Our observations suggest that Top2 reduction confers longevity effect across species via a conserved mechanism, and may be used as a novel therapeutic strategy for countering aging.

biochemistry↗

bioRxiv2024

Improving Accuracy of Somatic Mutation Profiling in Large Epidemiologic Studies: Addressing Cases without Matched Normal Samples

Ideally, detection of somatic mutations in a tumor is accomplished using a patient-matched sample of normal cells as the benchmark. In this way somatic mutations can be distinguished from rare germline mutations. In large retrospective studies, archival tissue collection can pose challenges in obtaining samples of normal DNA. In this article we propose a protocol that improves somatic mutation analysis in the absence of a matched normal sample. The method was motivated by the InterMEL study, a large-scale epidemiologic investigation involving multiomic, multi-institutional genomic profiling of 1000 primary melanoma samples. The key insight for accomplishing improved mutation calling is the fact that germline mutations should produce a variant allele frequency (VAF) of around 50%. While a similar VAF of 50% would also be expected for somatic mutations in pure tumor samples, typically the tumor purity is much less than 50%, resulting in a considerably lower VAF. Making use of a technique that can simultaneously estimate both tumor purity and VAF from tumor-only samples we have developed a method for better distinguishing somatic versus germline variants. Based on 137 melanomas from the InterMEL Study with matched normal tissue to provide a gold standard we show that the conventional pipeline using a panel of (unmatched) normal samples has a false positive rate of 15.6% and a false negative rate of 3.5%. Our new technique improves these error rates to 6.4% and 2.1%, respectively.

genomics↗

bioRxiv2024

Directed evolution of the multicopper oxidase laccase for cell surface proximity labeling and electron microscopy

Enzymes that oxidize aromatic substrates have shown utility in a range of cell-based technologies including live cell proximity labeling (PL) and electron microscopy (EM), but are associated with drawbacks such as the need for toxic H2O2. Here, we explore laccases as a novel enzyme class for PL and EM in mammalian cells. LaccID, generated via 11 rounds of directed evolution from an ancestral fungal laccase, catalyzes the one-electron oxidation of diverse aromatic substrates using O2 instead of toxic H2O2, and exhibits activity selective to the surface plasma membrane of both living and fixed cells. We show that LaccID can be used with mass spectrometry-based proteomics to map the changing surface composition of T cells that engage with tumor cells via antigen-specific T cell receptors. In addition, we use LaccID as a genetically-encodable tag for EM visualization of cell surface features in mammalian cell culture and in the fly brain. Our study paves the way for future cell-based applications of LaccID.

biochemistry↗

bioRxiv2024

Heterogeneity in chromatin structure drives core regulatory pathways in B-cell Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common pediatric malignancy. Based on gene expression profiling, B-ALL can be classified into distinct transcriptional subtypes with differing disease outcomes. Many of these transcriptional subtypes are defined by mutations in transcription factors and chromatin-modifying enzymes, but how such diverse mutations lead to distinct transcriptional subtypes remains unclear. To illuminate the chromatin regulatory landscape in B-ALL, we analyzed 3D genome organization, open chromatin, and gene expression in 53 primary patient samples. At the level of 3D genome organization, we identified chromatin interactions that vary across transcriptional subtypes. These sites of variable 3D chromatin interactions correlate with local gene expression changes and are enriched for core drivers of B-ALL observed in genome-wide CRISPR knock-out screens. Sites of variable 3D genome interactions are frequently shared across multiple transcriptional subtypes and are enriched for open chromatin sites found in normal B-cell development but repressed in mature B-cells. Within an individual patient sample, the chromatin landscape can resemble progenitor chromatin states at some loci and mature B-cell chromatin at others, suggesting that the chromatin in B-ALL patient tumor cells is in a partially arrested immature state. By analyzing transcriptomic data from large cohorts of B-ALL patients, we identify gene expression programs that are shared across transcriptional subtypes, associated with B-cell developmental stages, and predictive of patient survival. In combination, these results show that the 3D genome organization of B-ALL reflects B-cell developmental stages and helps illustrate how B-cell developmental arrest interacts with transcriptional subtypes to drive B-ALL.

cancer biology↗

bioRxiv2024

Distinct Neural Representations of Hunger and Thirst in Neonatal Mice before the Emergence of Food- and Water-seeking Behaviors

Hunger and thirst are two fundamental drives for maintaining homeostasis and elicit distinct food- and water-seeking behaviors essential for survival. For neonatal mammals, however, both hunger and thirst are sated by consuming milk from their mother. While distinct neural circuits underlying hunger and thirst drives in the adult brain have been characterized, it is unclear when these distinctions emerge in neonates and what processes may affect their development. Here we show that hypothalamic hunger and thirst regions already exhibit specific responses to starvation and dehydration well before a neonatal mouse can seek food and water separately. At this early age, hunger neurons drive feeding behaviors more than do thirst neurons. In vivo Neuropixels recordings in dehydrated and starved neonatal mice revealed that maternal presentation leads to a relative increase in activity which is suppressed by feeding on short timescales, particularly in hypothalamic and thalamic neurons. Changes in activity become more heterogeneous on longer timescales. Lastly, within neonatal regions that respond to both hunger and thirst, subpopulations of neurons respond distinctly to one or the other need. Combining food and water into a liquid diet throughout the animals life does not alter the distinct representations of hunger and thirst in the adult brain. Thus, neural representations of hunger and thirst in mice become distinct before food- and water-seeking behaviors mature and are robust to environmental changes in food and water sources.

neuroscience↗

bioRxiv2024

BST2 induces vascular smooth muscle cell plasticity and phenotype switching during cancer progression

BackgroundSmooth muscle cell (SMC) plasticity and phenotypic switching play prominent roles in the pathogenesis of multiple diseases, but their role in tumorigenesis is unknown. We investigated whether and how SMC diversity and plasticity plays a role in tumor angiogenesis and the tumor microenvironment. Methods and ResultsWe use SMC-specific lineage-tracing mouse models and single cell RNA sequencing to observe the phenotypic diversity of SMCs participating in tumor vascularization. We find that a significant proportion of SMCs adopt a phenotype traditionally associated with macrophage-like cells. These cells are transcriptionally similar to resolution phase M2b macrophages, which have been described to have a role in inflammation resolution. Computationally predicted by the ligand-receptor algorithm CellChat, signaling from BST2 on the surface of tumor cells to PIRA2 on SMCs promote this phenotypic transition; in vitro SMC assays demonstrate upregulation of macrophage transcriptional programs, and increased proliferation, migration, and phagocytic ability when exposed to BST2. Knockdown of BST2 in the tumor significantly decreases the transition towards a macrophage-like phenotype, and cells that do transition have a comparatively higher inflammatory signal typically associated with anti-tumor effect. ConclusionAs BST2 is known to be a poor prognostic marker in multiple cancers where it is associated with an M2 macrophage-skewed TME, these studies suggest that phenotypically switched SMCs may have a previously unidentified role in this immunosuppressive milieu. Further translational work is needed to understand how this phenotypic switch could influence the response to anti-cancer agents and if targeted inhibition of SMC plasticity would be therapeutically beneficial.

cancer biology↗

bioRxiv2024

Spatiotemporal Single-Cell Roadmap of Human Skin Wound Healing

Wound healing is vital for human health, yet the details of cellular dynamics and coordination in human wound repair remain largely unexplored. To address this, we conducted single-cell and spatial transcriptomics analyses on human skin and acute wound tissues through inflammation, proliferation, and remodeling phases of wound repair from the same individuals, monitoring the cellular and molecular dynamics of human skin wound healing at an unprecedented spatiotemporal resolution. This singular roadmap reveals the cellular architecture of the wound margin and identifies FOSL1 as a critical driver of re-epithelialization. It shows that pro-inflammatory macrophages and fibroblasts sequentially support keratinocyte migration like a relay race across different healing stages. Comparison with single-cell data from venous and diabetic foot ulcers uncovers a link between failed keratinocyte migration and impaired inflammatory response in chronic wounds. Additionally, comparing human and mouse acute wound transcriptomes underscores the indispensable value of this roadmap in bridging basic research with clinical innovations. HighlightsO_LIA spatiotemporal atlas to explore human in vivo gene expression across wound healing C_LIO_LIWound margin architecture unveils a model for human re-epithelialization C_LIO_LIDistinct healing challenges in venous ulcers and diabetic foot ulcers C_LIO_LIUnique human healing traits emerge in cellular heterogeneity and gene expression C_LI

cell biology↗

bioRxiv2024

Dimensionality reduction simplifies synaptic partner matching in an olfactory circuit

The distribution of postsynaptic partners in three-dimensional (3D) space presents complex choices for a navigating axon. Here, we discovered a dimensionality reduction principle in establishing the 3D glomerular map in the fly antennal lobe. Olfactory receptor neuron (ORN) axons first contact partner projection neuron (PN) dendrites at the 2D spherical surface of the antennal lobe during development, regardless of whether the adult glomeruli are at the surface or interior of the antennal lobe. Along the antennal lobe surface, axons of each ORN type take a specific 1D arc-shaped trajectory that precisely intersects with their partner PN dendrites. Altering axon trajectories compromises synaptic partner matching. Thus, a 3D search problem is reduced to 1D, which simplifies synaptic partner matching and may generalize to the wiring process of more complex brains.

developmental biology↗

bioRxiv2024

Early Inflammation and Interferon Signaling Direct Enhanced Intestinal Crypt Regeneration after Proton FLASH Radiotherapy

Ultra-high dose rate ("FLASH") radiotherapy ([≥]40-60 Gy/s) is a promising new radiation modality currently in human clinical trials. Previous studies showed that FLASH proton radiotherapy (FR) improves toxicity of normal tissues compared to standard proton radiotherapy (SR) without compromising anti-tumor effects. Understanding this normal tissue sparing effect may offer insight into how toxicities from cancer therapy can be improved. Here, we show that compared to SR, FR resulted in improved acute weight recovery and survival in mice after whole-abdomen irradiation. Improved morbidity and mortality after FR were associated with greater proliferation of damage-induced epithelial progenitor cells followed by improved tissue regeneration. FR led to the accelerated differentiation of revival stem cells (revSCs), a rare damage-induced stem cell required for intestinal regeneration, and to qualitative and quantitative changes in activity of signaling pathways important for revSC differentiation and epithelial regeneration. Specifically, FR resulted in greater infiltration of macrophages producing TGF-{beta}, a cytokine important for revSC induction, that was coupled to augmented TGF-{beta} signaling in revSCs. In pericryptal fibroblasts, FR resulted in greater type I IFN (IFN-I) signaling, which directly stimulates production of FGF growth factors supporting revSC proliferation. Accordingly, the ability of FR to improve epithelial regeneration and morbidity was dependent on IFN-I signaling. In the context of SR, however, IFN-I had a detrimental effect and promoted toxicity. Thus, a tissue-level signaling network coordinated by differences in IFN-I signaling and involving stromal cells, immune cells, and revSCs underlies the ability of FLASH to improve normal tissue toxicity without compromising anti-tumor efficacy. One Sentence SummaryFLASH radiation improves normal tissue toxicity without compromising anti-tumor efficacy through a stroma, immune, and revSC signaling network coordinated by IFN-I.

cell biology↗

bioRxiv2024

Comprehensive assembly of monoclonal and mixed antibody sequences

The elucidation of antibody sequence information is crucial for understanding antigen binding and advancing therapeutic and research applications. However, complete de novo assembly of monoclonal antibody sequences remains challenging due to accuracy and robustness limitations. To address this issue, we introduce Fusion, an innovative de novo assembler that integrates overlapping peptides and template information into complete sequences using a beam search strategy. We demonstrate Fusions performance by reconstructing multiple human and murine antibodies with highest accuracy (100% and over 99%, respectively). Biological validation of the recombinantly expressed AFS98 antibody with unknown sequences further supports its effectiveness. Furthermore, current methods are applicable only to traditional monoclonal antibody sequencing assembly, presenting a significant bottleneck in achieving higher throughput. In contrast, Fusion can assemble peptide sequences from mixtures of two or three monoclonal antibodies into complete individual sequences with the same accuracy as traditional sequencing, significantly enhancing throughput. To our knowledge, this is the first study enabling high-throughput sequencing of multiple antibodies using only bottom-up mass spectrometry. The duration, expense, and reagent consumption of mass spectrometry detection are comparable to those required for sequencing a single monoclonal antibody. In summary, Fusions superior performance in handling the complex antibody sequencing represents a significant advancement in antibody research.

bioinformatics↗

bioRxiv2024

Concerted modulation of spontaneous behavior and time-integrated whole-brain neuronal activity by serotonin receptors

Serotonin neurons from the raphe nuclei project across the entire brain and modulate diverse physiology and behavior by acting on over a dozen receptors. Here, we took a step towards dissecting this complex process by examining the effects of agonists and antagonists of four widely expressed serotonin receptors (2A, 2C, 1A, and 1B) on spontaneous mouse behavior, which we related to time-integrated whole-brain neuronal activity as assessed by the expression of Fos, a canonical immediate-early gene product. Low-dimensional representations of behavioral and Fos map data revealed the dominant factors of variation in each domain, captured predictable differences across drug groups, and enabled predictions of behavioral changes following perturbations in Fos maps and vice versa. Our study provides a rich resource describing the effects of manipulating serotonin receptors on animal behavior and whole-brain integrated neuronal activity. It also establishes an experimental and analysis paradigm for interrogating the relationship between behavior and neuronal activity across different time scales.

neuroscience↗

bioRxiv2024

Colicin Receptor CirA Enhances Salmonella Typhimuriums Resistance to Colicin Ib in the Absence of the Cognate Immunity Protein

Intestinal microbiota play a central role in colonisation resistance providing a fundamental barrier to infection to enteric pathogens. An important mechanism of colonisation resistance involves the production of antimicrobial peptides, such as colicins. Pore-forming colicins, synthesised by Escherichia coli (E. coli) strains, target competing bacteria in their environmental niche, whilst the producing cells are safeguarded by specific immunity proteins. Notably, non-typhoidal Salmonella Typhimurium strains can produce a narrow-spectrum protein toxin colicin IB (ColIb) providing a competitive edge against susceptible Enterobacteriaceae strains. However, the multi-drug resistant and systemically invasive iNTS (invasive non-Typhoidal Salmonella) S. Typhimurium D23580 strain poses an interesting case. The strain lacks colicin Ib production and the corresponding immunity protein, but its potential vulnerability in a colicin-rich gastrointestinal milieu remains uninvestigated. In this study, S. Typhimurium D23580 exhibited resistance to colicin Ib under tested conditions, despite the absence of the immunity gene imm. Intriguingly, in colicin Ib-producing S. Typhimurium strains, the imm gene appeared functionally redundant in contrast to our current understanding. ColIb binds to the outer membrane protein CirA and is translocated to the inner membrane where it forms a pore in sensitive bacteria dissipating the electrochemical potential. Through a series of experimental approaches, including the use of Escherichia coli and S. Typhimurium cirA deletion mutants, promoter-swap techniques, and gene complementation, we identified that the colicin resistance phenotype in S. Typhimurium was partly attributable to the CirA receptor. This finding suggests a complex interplay in the microbial resistance to colicins, highlighting the intricacies of microbial interactions within the gastrointestinal environment.

microbiology↗

bioRxiv2024

Molecular Signatures for Microbe-Associated Colorectal Cancers

BackgroundGenetic factors and microbial imbalances play crucial roles in colorectal cancers (CRCs), yet the impact of infections on cancer initiation remains poorly understood. While bioinformatic approaches offer valuable insights, the rising incidence of CRCs creates a pressing need to precisely identify early CRC events. We constructed a network model to identify continuum states during CRC initiation spanning normal colonic tissue to pre-cancer lesions (adenomatous polyps) and examined the influence of microbes and host genetics. MethodsA Boolean network was built using a publicly available transcriptomic dataset from healthy and adenoma affected patients to identify an invariant Microbe-Associated Colorectal Cancer Signature (MACS). We focused on Fusobacterium nucleatum (Fn), a CRC-associated microbe, as a model bacterium. MACS-associated genes and proteins were validated by RT-qPCR, RNA seq, ELISA, IF and IHCs in tissues and colon-derived organoids from genetically predisposed mice (CPC-APCMin+/-) and patients (FAP, Lynch Syndrome, PJS, and JPS). ResultsThe MACS that is upregulated in adenomas consists of four core genes/proteins: CLDN2/Claudin-2 (leakiness), LGR5/leucine-rich repeat-containing receptor (stemness), CEMIP/cell migration-inducing and hyaluronan-binding protein (epithelial-mesenchymal transition) and IL8/Interleukin-8 (inflammation). MACS was induced upon Fn infection, but not in response to infection with other enteric bacteria or probiotics. MACS induction upon Fn infection was higher in CPC-APCMin+/- organoids compared to WT controls. The degree of MACS expression in the patient-derived organoids (PDOs) generally corresponded with the known lifetime risk of CRCs. ConclusionsComputational prediction followed by validation in the organoid-based disease model identified the early events in CRC initiation. MACS reveals that the CRC-associated microbes induce a greater risk in the genetically predisposed hosts, suggesting its potential use for risk prediction and targeted cancer prevention.

microbiology↗

bioRxiv2024

Embryonically Active Piriform Cortex Neurons Promote Intracortical Recurrent Connectivity during Development

Neuronal activity plays a critical role in the maturation of circuits that propagate sensory information into the brain. How widely does early activity regulate circuit maturation across the developing brain? Here, we used Targeted Recombination in Active Populations (TRAP) to perform a brain-wide survey for prenatally active neurons in mice and identified the piriform cortex as an abundantly TRAPed region. Whole-cell recordings in neonatal slices revealed preferential interconnectivity within embryonically TRAPed piriform neurons and their enhanced synaptic connectivity with other piriform neurons. In vivo Neuropixels recordings in neonates demonstrated that embryonically TRAPed piriform neurons exhibit broad functional connectivity within piriform and lead spontaneous synchronized population activity during a transient neonatal period, when recurrent connectivity is strengthening. Selectively activating or silencing of these neurons in neonates enhanced or suppressed recurrent synaptic strength, respectively. Thus, embryonically TRAPed piriform neurons represent an interconnected hub-like population whose activity promotes recurrent connectivity in early development.

neuroscience↗

bioRxiv2024

Comprehensive evaluation and prediction of editing outcomes for near-PAMless adenine and cytosine base editors

Base editors enable the direct conversion of target bases without inducing double-strand breaks, showing great potential for disease modeling and gene therapy. Yet, their applicability has been constrained by the necessity for specific protospacer adjacent motif (PAM). We generated four versions of near-PAMless base editors and systematically evaluated their editing patterns and efficiencies using an sgRNA-target library of 45,747 sequences. Near-PAMless base editors significantly expanded the targeting scope, with both PAM and target flanking sequences as determinants for editing outcomes. We developed BEguider, a deep learning model to accurately predict editing results for near-PAMless base editors. We also provided experimentally measured editing outcomes of 20,541 ClinVar sites, demonstrating that variants previously inaccessible by NGG PAM base editors can now be precisely generated or corrected. We have made our predictive tool and data available online to facilitate development and application of near-PAMless base editors in both research and clinical settings.

bioinformatics↗

bioRxiv2024

Interaction between ciliary component proteins from Chlamydomonas revealed by CRISPR/CAS9, cryo-electron tomography and mass spectrometry

To understand molecular mechanism of ciliary beating motion, knowledge of location, interaction and dynamics of >400 component proteins are indispensable. While recent progress of structural biology revealed conformation and localization of >100 proteins, we still need to investigate their networking, art of their interaction and assembly mechanism. We applied CRISPR/CAS9 genome editing technique to the green algae Chlamydomonas to engineer a deletion mutant of a ciliary component, FAP263, located at the distal protrusion, and examined it structurally by cryo-electron tomography (cryo-ET) and mass spectrometry (MS). Cryo-ET and atomic model fitting demonstrated that the FAP263 deletion mutant lacks additional components, FAP78, and FAP184. Unassigned density near FAP263 in the cryo-ET map of WT cilia is likely FAP151, as suggested by cross-linking mass spectrometry. Based on the structure, we modeled how these four proteins might form a complex. Furthermore, it was shown that dynein f phosphorylation is inhibited in the FAP263 mutant, indicating an important role of this protein complex for dynein f phosphorylation. Our study demonstrates a novel approach to investigate protein networking inside cilia.

cell biology↗

bioRxiv2024

MALAT1 mediates miR-206/CDC42/PAK1/Paxillin signaling axis to alleviate erectile dysfunction in rats

Erectile dysfunction (ED) is a male sexual dysfunction with a gradually increasing prevalence, and current treatments are ineffective. This study aims to find a safer and more effective treatment for erectile dysfunction. Bone marrow-derived mesenchymal stem cells (BM-MSCs) treated with VEGFA. The expression of endothelial markers vWF, VE-cadherin and eNOS were determined by overexpressing MALAT1 and CDC42, respectively. The results showed that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs and the expression of endothelial marker-related proteins. Moreover, MALAT1 induced differentiation of BM-MCs to ECs through the CDC42/PAK1/Paxillin pathway was explored by transfecting si-MALAT1 and overexpressing CDC42 as well as PAK1 in the BM-MSCs. Next, miR-206 was overexpressed within BM-MCs to determine CDC42 expression. The binding sites of MALAT1, miR-206 and CDC42 were reported using luciferase. it was found that the MALAT1 competes with CDC42 3-UTR for binding miR-206, which in turn is involved in differentiating BM-MSCs to ECs. Finally, DMED rat modeling success was demonstrated by APO experiments. MALAT1 overexpression-modified BM-MSCs had significant therapeutic effects in DMED rats.

molecular biology↗

bioRxiv2024

Intercontinental dissemination and diversification of Xanthomonas perforans in tomato production

Emerging and re-emerging plant diseases continue to present multifarious threats to global food security. Considerable recent efforts are therefore being channeled towards understanding the nature of pathogen emergence, their spread and evolution. Xanthomonas euvesicatoria pv. perforans (Xep), one of the causal agents of bacterial spot of tomato, rapidly emerged and displaced other bacterial spot xanthomonads in tomato production regions around the world. In less than three decades, it has become a dominant xanthomonad pathogen in tomato production systems across the world and presents a model for understanding diversification of recently emerged bacterial plant pathogens. Although Xep has been continuously monitored in Florida since its discovery, the global population structure and evolution at the genome-scale is yet to be fully explored. The objectives of this work were to determine genetic diversity globally to ascertain if different tomato production regions contain genetically distinct Xep populations, to examine genetic relatedness of strains collected in tomato seed production areas in East Asia and other production regions, and to evaluate variation in type III effectors, which are critical pathogenicity and virulence factors, in relationship to population structure. We used genome data from 270 strains from 13 countries for phylogenetic analysis and characterization of Xop effector gene diversity among strains. Our results showed notable genetic diversity in the pathogen. We found genetically similar strains in distant tomato production regions, including seed production regions, and diversification over the past 100 years, which is consistent with intercontinental dissemination of the pathogen in hybrid tomato production chains. Evolution of the Xep pangenome, including the acquisition and loss of type III secreted effectors, is apparent within and among phylogenetic lineages. The apparent long-distance movement of the pathogen, together with variants that may not yet be widely distributed, poses risks of emergence of new variants in tomato production.

microbiology↗