Biology subjects

Franklin, J. L.

Publications and source records attributed to Franklin, J. L..

9 recordsLinked to original sources

bioRxiv2026

The cost-benefit trade-off of peritrichous flagellation in bacteria

Many bacteria assemble multiple flagella, although building flagella imposes a substantial biosynthetic and energetic cost. We used the peritrichously flagellated model organism Salmonella enterica to quantify how flagellar abundance affects bacterial growth, proteome allocation, and motility. For this, we generated genetically modified strains with inducible or constitutive expression of the flagellar master regulator flhDC, resulting in a panel of strains ranging from nearly non-flagellated to hyperflagellated cells. We found that higher flagellar investment reduced growth rate and redirected proteome allocation, with an expansion of the flagellar sector occurring largely at the expense of the ribosomal sector. Growth analyses of flagellar assembly mutants, combined with cost modeling, suggested that flagellin biosynthesis dominated the energetic burden, whereas motor rotation contributed a smaller additional cost. Despite the associated cost, increased flagellation improved soft-agar spreading, single-cell swimming speed, effective diffusivity, and competitive fitness in spatially structured environments. A coarse-grained proteome-allocation model parametrized from these data reproduced the observed growth penalties, while simulations of navigation in dynamic chemical gradients predicted that motility benefits saturate near a flagellar investment of 3% of proteome mass. Beyond this point, rising biosynthetic costs outweigh diminishing motility gains. In summary, these results support a quantitative cost-benefit model in which heterogeneous, spatially structured environments favor an intermediate number of flagella by balancing motility benefits against the biosynthetic costs of building and operating multiple flagella.

microbiology↗

bioRxiv2025

Unbiased recording of clonal potency reveals species-specific regulation of mammalian intestine

The mammalian intestine regenerates rapidly after damage, yet the clonal dynamics and species-specific regulation of different populations remain poorly understood. Here we used synthetic or naturally occurring DNA alterations to reconstruct clonal histories of the mouse and human intestinal epithelium at single-cell resolution. In mice, we uncovered the clonal architecture of different cell types and their roles in regeneration, supporting a hierarchical regenerative response model. We identified a rare embryonic precursor population that persisted in the adult and was crucial for regeneration after irradiation. This population was marked by Tob2, which is required for nuclear transport of Ascl2. A parallel clonal analysis of 65 human colonic biopsies revealed secretory lineage bias and an age-associated decline in clonal diversity in the distal colon. Unlike highly proliferative murine Lgr5+ stem cells, human LGR5+ cells were found largely quiescent, revealing species-specific difference in clonal potency, and suggesting a distinct regulation of intestinal stemness.

genomics↗

bioRxiv2024

miR-100 and miR-125b Contribute to Enhanced Growth and Invasiveness

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b, we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b. Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

cancer biology↗

bioRxiv2024

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

molecular biology↗

bioRxiv2023

Detection of EGFR and its Activity State in Plasma CD63-EVs from Glioblastoma Patients: Rapid Profiling using an Anion Exchange Membrane Sensor

We present a novel quantitative immunoassay for CD63 EVs (extracellular vesicles) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane and a charged silica nanoparticle reporter. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins and fluorophore degradation, thus enabling direct plasma analysis. With a limit of detection of 30 EVs/L and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. Glioblastoma necessitates improved non-invasive diagnostic approaches for early detection and monitoring. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. This approach offers direct glioblastoma detection from untreated human patient samples. Analysis of glioblastoma clinical samples yielded an area-under-the-curve (AUC) value of 0.99 and low p-value of 0.000033, significantly surpassing the performance of existing assays and markers.

cancer biology↗

bioRxiv2023

NYUS.2: an Automated Machine Learning Prediction Model for the Large-scale Real-time Simulation of Grapevine Freezing Tolerance in North America

O_LIAccurate and real-time monitoring of grapevine freezing tolerance is crucial for the sustainability of the grape industry in cool climate viticultural regions. However, on-site data is limited. Current prediction models underperform under diverse climate conditions, which limits the large-scale deployment of these methods. C_LIO_LIWe combined grapevine freezing tolerance data from multiple regions in North America and generated a predictive model based on hourly temperature-derived features and cultivar features using AutoGluon, an automatic machine learning engine. Feature importance was quantified by AutoGluon and SHAP value. The final model was evaluated and compared with previous models for its performance under different climate conditions. C_LIO_LIThe final model achieved an overall 1.36 {degrees}C root-mean-square error during model testing and outperformed two previous models using three test cultivars at all testing regions. Two feature importance quantification methods identified five shared essential features. Detailed analysis of the features indicates that the model might have adequately extracted some biological mechanisms during training. C_LIO_LIThe final model, named NYUS.2, was deployed along with two previous models as an R shiny-based application in the 2022-2023 dormancy season, enabling large-scale and real-time simulation of grapevine freezing tolerance in North America for the first time. C_LI

plant biology↗

bioRxiv2021

Human colorectal pre-cancer atlas identifies distinct molecular programs underlying two major subclasses of pre-malignant tumors

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

cancer biology↗

bioRxiv2020

VIBRIO CHOLERAE ADAPTS TO SESSILE AND MOTILE LIFESTYLES BY CYCLIC DI-GMP REGULATION OF CELL SHAPE

The cell morphology of rod-shaped bacteria is determined by the rigid net of peptidoglycan forming the cell wall. While V. cholerae grows into a curved shape under most conditions, straight rods have been observed. However, the signals and regulatory pathways controlling cell shape transitions in V. cholerae and the benefits of switching between rod and curved shape have not been determined. We demonstrate that cell shape in V. cholerae is regulated by the bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) by repressing expression of crvA, a gene encoding an intermediate filament-like protein necessary for curvature formation in V. cholerae. This regulation is mediated by the transcriptional cascade that also induces production of biofilm matrix components, indicating that cell shape is coregulated with V. choleraes induction of sessility. Wild-type V. cholerae cells adhering to a surface lose their characteristic curved shape to become as straight as cells lacking crvA while genetically engineering cells to maintain high curvature reduced microcolony formation and biofilm density. Conversely, straight V. cholerae mutants have reduced speed when swimming using flagellar motility in liquid. Our results demonstrate regulation of cell shape in bacteria is a mechanism to increase fitness in planktonic or biofilm lifestyles.

microbiology↗

bioRxiv2019

Machine and deep learning single-cell segmentation and quantification of multi-dimensional tissue images

Increasingly, highly multiplexed in situ tissue imaging methods are used to profile protein expression at the single-cell level. However, a critical limitation is a lack of robust cell segmentation tools applicable for sections of tissues with a complex architecture and multiple cell types. Using human colorectal adenomas, we present a pipeline for cell segmentation and quantification that utilizes machine learning-based pixel classification to define cellular compartments, a novel method for extending incomplete cell membranes, quantification of antibody staining, and a deep learning-based cell shape descriptor. We envision that this method can be broadly applied to different imaging platforms and tissue types.

bioinformatics↗