Biology subjects

Cen, L.

Publications and source records attributed to Cen, L..

8 recordsLinked to original sources

bioRxiv2025

Physiologically Relevant Coculture Model for Oral Microbial-Host Interactions

Understanding microbial-host interactions in the oral cavity is essential for elucidating oral disease pathogenesis and its systemic implications. In vitro bacteria-host cell coculture models have enabled fundamental studies to characterize bacterial infection and host responses in a reductionist yet reproducible manner. However, existing in vitro coculture models fail to replicate the physiological oxygen gradients critical for studying these interactions. Here, we present an asymmetric gas coculture system that simulates the oral microenvironment by maintaining distinct normoxic and anaerobic conditions for gingival epithelial cells and anaerobic bacteria, respectively. Using Fusobacterium nucleatum, a key oral pathobiont, we demonstrate that the system preserves bacterial viability and supports the integrity of telomerase-immortalized gingival keratinocytes. Compared to conventional models, this system enhanced bacterial invasion, elevated intracellular bacterial loads, and elicited more robust host pro-inflammatory responses, including increased secretion of CXCL10, IL-6, and IL-8. Additionally, the model enabled precise evaluation of antibiotic efficacy against intracellular pathogens. These results underscore the utility of this coculture platform for studying oral microbial pathogenesis and screening therapeutics, offering a physiologically relevant approach to advance oral and systemic health research. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/632380v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@3a5951org.highwire.dtl.DTLVardef@bdf8dcorg.highwire.dtl.DTLVardef@1d444forg.highwire.dtl.DTLVardef@16faae7_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

A pan-cancer gamma delta T cell repertoire

This report presents the largest collection of gamma-delta T cell receptor ({gamma}{delta} TCR) reads in human cancer to date, analyzing about 11,000 patient tumor samples across 33 cancer types using the TRUST4 algorithm. Despite {gamma}{delta} T cells being a small fraction of the T cell population, they play a key role in both innate and adaptive immunity. Our comprehensive analysis reveals their significant presence across all cancer types, specifically highlighting the diverse spectrum and clonality patterns of their {gamma}{delta} receptors. This research highlights the complex roles of {gamma}{delta} T cells in tumor tissues and their potential as prognostic biomarkers. We also demonstrate the utility of T cell receptor gamma (TRG) and delta (TRD) gene expression values from standard RNA-seq data. Ultimately, our work establishes a fundamental resource for future tumor-infiltrating {gamma}{delta} T cell research and may facilitate the development of novel {gamma}{delta}-T-cell-based therapeutic strategies. Together, we demonstrate the strong diversity and prognostic potential of {gamma}{delta} T cells in multiple cancer types. HighlightsComprehensive analysis of {gamma}{delta} TCRs from 11,473 tumor samples Significant variability and overall consistency in {gamma}{delta} gene expression and clonotype {gamma}{delta}TCR expression and diversity as prognostic biomarkers across multiple cancers Centralized {gamma}{delta} TCR repertoire database for future therapeutic discovery

genomics↗

bioRxiv2024

Identifying essential genes in Schaalia odontolytica using a highly-saturated transposon library

The unique epibiotic-parasitic relationship between Nanosynbacter lyticus type strain TM7x, a member of the newly identified Candidate Phyla Radiation, now referred to as Patescibacteria, and its basibiont, Schaalia odontolytica strain XH001 (formerly Actinomyces odontolyticus), require more powerful genetic tools for deeper understanding of the genetic underpinnings that mediate their obligate relationship. Previous studies have mainly characterized the genomic landscape of XH001 during or post TM7x infection through comparative genomic or transcriptomic analyses followed by phenotypic analysis. Comprehensive genetic dissection of the pair is currently cumbersome due to the lack of robust genetic tools in TM7x. However, basic genetic tools are available for XH001 and this study expands the current genetic toolset by developing high-throughput transposon insertion sequencing (Tn-seq). Tn-seq was employed to screen for essential genes in XH001 under laboratory conditions. A highly saturated Tn-seq library was generated with nearly 660,000 unique insertion mutations, averaging one insertion every 2-3 nucleotides. 203 genes, 10.5% of the XH001 genome, were identified as putatively essential.

microbiology↗

bioRxiv2024

Identification of P-type ATPase as a bacterial transporter for host-derived small RNA

Host-derived small RNAs represent a finely tuned host response to maintain the host-microbial homeostasis. Among these, an emerging class known as transfer RNA-derived small RNA (tsRNA) has been implicated in modulating microbial-host interaction. Our previous study showed that when challenged with an oral pathobiont, Fusobacterium nucleatum (Fn), an immortalized human oral keratinocyte cell line releases certain Fn-targeting tsRNAs that selectively inhibit the growth of Fn via their ribosome-targeting function. We also revealed the sequence- and energy-dependent uptake of tsRNAs by Fn. However, the mechanism underlying the tsRNA uptake at the molecular level remains elusive. In this study, using RNA affinity pull-down assay in combination with Mass Spectrometry, we identified a putative P-type ATPase transporter (PtaT) in Fn, which binds Fn-targeting tsRNAs in a sequence-specific manner. AlphaFold 3 simulation provides further evidence supporting the specific binding between PtaT and tsRNA compared to the scrambled control and the DNA counterpart. Through targeted mutagenesis and phenotypic characterization, we demonstrated the important role of PtaT in the uptake and antimicrobial capacity of tsRNAs against Fn in both ATCC 23726 type strain and a clinical tumor isolate (Fn CTI). Furthermore, global RNA sequencing and label-free Raman spectroscopy revealed the phenotypic differences between Fn wild type and PtaT-deficient mutant, highlighting the functional significance of PtaT in purine and pyrimidine metabolism. Collectively, our work identifies a bacterial transporter for tsRNAs and provides critical information for a fundamental understanding of how the host-generated tsRNAs specifically interact with its targeted bacteria at the molecular level.

microbiology↗

bioRxiv2023

Episymbiotic bacterium induces intracellular lipid droplet production in its host bacteria

Saccharibacteria (formerly TM7) Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Schaalia odontolytica, strain XH001 (formerly Actinomyces odontolyticus strain XH001). Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly living on the surface of its bacterial host to sustain this symbiotic lifestyle. To comprehend this intriguing, yet understudied interspecies interaction, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. In this study, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We found that the binding with TM7x led to a substantial alteration in the membrane fluidity of the host bacterium XH001. Unexpectedly, we revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature not commonly observed in oral bacteria cells. The TM7x-induced LD accumulation in XH001 was further confirmed by label-free non-invasive Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing host bacterium XH001 alone under various stress conditions showed that LD accumulation was a general response to stress. Intriguingly, a survival assay demonstrated that the presence of LDs likely plays a protective role in XH001, enhancing its overall survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and its host bacterium, highlighting the potential benefit conferred by TM7x to its host, and further emphasizing the context-dependent nature of symbiotic relationships.

microbiology↗

bioRxiv2022

Targeting Fusobacterium nucleatum through Chemical Modifications of Host-Derived Transfer RNA Fragments

Host mucosal barriers possess an arsenal of defense molecules to maintain mucosal health. In addition to well-established defense molecules such as antimicrobial peptides and immunoglobulins, a subset of extracellular host-derived small RNAs (sRNAs) also exhibits antimicrobial functions in a cross-kingdom fashion. We recently uncovered the sRNA-mediated crosstalk between human normal oral keratinocytes and Fusobacterium nucleatum (Fn), an opportunistic oral pathobiont with increasing implications in extra-oral diseases. Notably, when challenged with Fn, oral keratinocytes released Fn-targeting tRNA-derived sRNAs (tsRNAs), an emerging class of noncoding sRNAs with diverse functions in gene regulation. Additionally, synthetic mimics of the Fn-targeting tsRNAs exhibited highly selective antimicrobial activity against Fn. However, excess synthetic tsRNAs (in the micromolar range) were required to achieve growth inhibition, which limits their potential as antimicrobials. Here, we chemically modify nucleotides of the anti-Fn tsRNAs, termed MOD-tsRNAs, and demonstrate their species- and sequence-specific inhibition in the nanomolar range in various Fn type strains and clinical tumor isolates. In contrast, the same MOD-tsRNAs do not inhibit two representative oral bacteria, Porphoromonas gingivalis (Pg) and Streptococcus mitis (Sm). Additionally, MOD-tsRNAs are internalized by different Fn strains while exhibiting minimal uptake by Pg and Sm. Further RNA sequencing and affinity pull-down assays implicate MOD-tsRNAs as potential ribosome-targeting antimicrobials against Fn. Taken together, our work provides a framework to target opportunistic pathobionts through co-opting host-derived extracellular tsRNAs, whose potential applications may have been limited by their intrinsic instability as well as our limited understanding of the inhibition mechanism.

bioengineering↗

bioRxiv2022

Transcriptome of epibiont Saccharibacteria Nanosynbacter lyticus strain TM7x during establishment of symbiosis

Saccharibacteria Nanosynbacter lyticus strain TM7x is a member of the broadly distributed Candidate Phylum Radiation. These bacteria have ultrasmall cell size, reduced genomes and live as epibionts on the surface of other bacteria. The mechanisms by which they establish and maintain this relationship are not yet fully understood. The transcriptomes of the epibiont TM7x and its host bacteria Schaalia odontolyticus strain XH001 were captured across the establishment of symbiosis during both the initial interaction and stable symbiosis. The results showed a dynamic interaction with large shifts in gene expression for both species between the initial encounter and stable symbiosis, notably transporter genes. During stable symbiosis, the host XH001 showed higher gene expression for peptidoglycan biosynthesis, mannosylation, cell cycle and stress related genes, but lower expression of chromosomal partitioning genes. This was consistent with the elongated cell shape seen in XH001 infected with TM7x and our discovery that infection resulted in thickened cell walls. Within TM7x, increased pili, type IV effector gene, and arginine catabolism/biosynthesis gene expression during stable symbiosis implied a key role for these functions in the interaction. Consistent with its survival and persistence in the human microbiome as an obligate epibiont with reduced de novo biosynthetic capacities, TM7x also showed higher levels for energy production and peptidoglycan biosynthesis but lower expression of stress related genes during stable symbiosis. These results imply that TM7x and its host bacteria keep a delicate balance in order to sustain an episymbiotic lifestyle. IMPORTANCENanosynbacter lyticus type strain TM7x is the first cultivated member of the Saccharibacteria and the Candidate Phyla Radiation (CPR). It was discovered to have ultrasmall cell size with a highly reduced genome that establishes an obligate epibiotic relationship with its host bacterium. The CPR, now formally proposed as the Patescibacteria super-phylum, is a large monophyletic radiation of diverse bacteria with reduced genomes that includes Saccharibacteria. The vast majority of the CPR have yet to be cultivated in the laboratory and our insights into these unique organisms to date has been derived from only a few Saccharibacteria species. It is unknown however how these small obligate parasitic Saccharibacteria, that are missing many de novo biosynthetic pathways, are maintained at high prevalence within the human microbiome as well as in the environment. When TM7x infects its host bacterium there are distinct temporal phases, including an initial interaction, a killing phase, recovery phase, and finally stable symbiosis. Here we captured the gene expression of the host bacterium and epibiont during this dynamic interaction which represents the initial insights into the mechanisms of how these unique microbes may survive and persist.

microbiology↗

bioRxiv2021

The immunomodulatory properties of the HDAC6 inhibitor ACY241 supports robust anti-tumor response in NSCLC when coupled with the chemotherapy drug Oxaliplatin

BackgroundDurable treatments that benefit a wide pool of patients remain elusive for Non-small cell lung cancer (NSCLC). The success of immunotherapy in a subset of NSCLC patients highlights the potential contribution of immune response to anti-tumor immunity while underscoring a need for broadly applicable therapeutic strategies. HDAC inhibitors are a promising class of drugs whose immunomodulatory properties are now being appreciated. In the present study, we evaluated the effects of the HDAC6 inhibitor, ACY241 on lung tumor immune compartment with the goal of understanding the scope of its immunomodulatory properties and its therapeutic potential in combination with Oxaliplatin. MethodsLung adenocarcinoma-bearing mice were treated with ACY241 or vehicle after which the proportions and phenotype of tumor-associated T cells and macrophages were evaluated by comprehensive flow cytometric analysis. Bulk RNA-sequencing was also conducted on both cellular subsets to interrogate the transcriptomic changes associated with ACY241 treatment relative to vehicle controls. In vivo drug efficacy study was performed by administration of ACY241 and/or Oxaliplatin and assessing tumor growth and survival of tumor-bearing mice. Ex vivo functional studies was performed to assess tumor-associated T cell effector function as it correlates with measured outcomes. ResultsWe demonstrate that ACY241 promotes increased presence of T and NK cells in the lung tumors of treated mice. The tumor-associated T cells under ACY241 treatment displayed enhanced activation, proliferation, and effector profile. In addition, tumor-associated macrophages exhibited increased expression of MHC and co-stimulatory molecules while expression of inhibitory ligands were reduced. RNA-sequencing of both tumor-associated T cells and macrophages revealed significant genomic changes in both subsets that is consistent with ACY241-mediated enhancement of immune priming. These broad immunomodulatory properties of ACY241 were associated with significantly enhanced tumor-associated T cell effector functionality, robust anti-tumor response, and significantly prolonged survival of NSCLC-bearing mice when combined with the chemotherapy drug Oxaliplatin. ConclusionCollectively, our studies highlight the broad immunomodulatory effect of ACY241 as a promising HDAC6 inhibitor which coupled with Oxaliplatin promotes robust therapeutic outcomes in a pre-clinical model of NSCLC, providing compelling rationale for the clinical testing of this novel combinatorial regimen in NSCLC.

immunology↗