bioRxiv

Journal: bioRxiv

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Cold Spring Harbor Laboratory

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Publications1 - 10 of 377

High-Throughput Antibody Neutralization Screening in Massively Parallel Droplet Arrays
Gutierrez-Gonzalez, Matias; Fahad, Ahmed S.; Delley, Cyrille L.; et al. (2025)
bioRxiv
Neutralizing antibodies provide rapid immune defense against infectious diseases, but are difficult to discover at scale because neutralization assays require live reporter cells and soluble monoclonal antibodies. Here we report Droplet Reporter Cell Testing for Neutralization (DrReCT-Neutralization) to screen antibody gene libraries for their ability to neutralize viral infections. We established the necessary engineered cell lines and validated the DrReCT screening platform using synthetic oligoclonal libraries, followed by an example discovery campaign that demonstrated scalable functional antibody data collection against viral diseases.
CXCL16 mediates nociception and inflammation in murine post-traumatic osteoarthritis
Lammlin, Lindsey; Redding, Stephen; Knights, Alexander J.; et al. (2025)
bioRxiv
Abstract This study investigates the role of the chemokine CXCL16 and its receptor, CXCR6, in post-traumatic osteoarthritis (PTOA) and joint nociception, highlighting the potential of targeting the CXCL16-CXCR6 axis for therapeutically managing joint inflammation and pain. Following joint injury in mice, the CXCL16-CXCR6 signaling axis is activated in synovium, driven by synovial fibroblasts and macrophages. Human OA synovium also exhibited increased CXCL16 and CXCR6 gene expression. CXCL16 stimulated a pro-inflammatory response in fibroblasts and macrophages, contrasting with an anti-inflammatory response observed in mesenchymal progenitor cells. In mice, repeated intra-articular CXCL16 injections induced histological synovitis and sex-dependent activation of inflammatory and fibrotic transcriptional programs in synovium. Repeated CXCL16 joint injections also induced knee hyperalgesia, which was mitigated by co-administration of the CXCR6 antagonist, ML339. A single intra-articular injection of CXCL16 induced acute knee hyperalgesia as early as 30 minutes post-injection, which was completely abrogated by ML339 co-treatment, suggesting direct CXCL16 binding to nociceptor-expressed CXCR6. In a murine PTOA model, systemic CXCR6 antagonism with ML339 alleviated knee hyperalgesia and altered circulating immune cell profiles. Direct stimulation of mouse dorsal root ganglion-derived nociceptive neurons with CXCL16 induced rapid calcium signaling, which was abolished by co-treatment with ML339. These findings establish CXCL16 as a regulator of joint inflammation and identifies the CXCL16-CXCR6 binding mechanism as key in mediating pain-related behaviors and nociceptor activation, offering a therapeutic target for PTOA-related inflammation and pain management.
The virulence gene ToxB is both amplified and disrupted by transposons in the wheat pathogen Pyrenophora tritici-repentis
Gourlie, Ryan; McDonald, Megan; Hafez, Mohamed; et al. (2025)
bioRxiv
Mechanisms that drive virulence gene duplication in plant pathogenic fungi remain poorly understood. In Pyrenophora tritici-repentis (Ptr), responsible for tan spot of wheat, ToxB is a multicopy virulence gene encoding a proteinaceous necrotrophic effector. ToxB exhibits a virulence dosage effect, where higher copy numbers are associated with increased disease severity. In this work, we sought to resolve a 25-year old question as to what drove the proliferation of ToxB within Ptr. To investigate this, 23 long-read assemblies were generated and analyzed from a collection of globally distributed isolates with various ToxB copy numbers, with a specific focus on regions containing ToxB. Extensive comparative alignments identified a Helitron-like element, ToxB-HLE, that appears to be driving the duplication of ToxB in an accessory region of chromosome 4. This region is entirely absent in isolates lacking ToxB or its nonfunctional homolog toxb. In addition to gene amplification by transposons, multiple independent transposon insertion events were identified in several isolates that disrupted the ToxB open reading frame creating inactive toxb haplotypes. This study provides strong evidence supporting the hypothesis that transposons play dual roles in the rapid evolution of fungal pathogenicity by both amplifying and disrupting a key virulence gene in a globally distributed plant pathogen
The Role of Vitamin D in Emiliania huxleyi: A Microalgal Perspective on UV-B Exposure
Eliason, Or; Malitsky, Sergey; Panizel, Irina; et al. (2023)
bioRxiv
An essential interaction between sunlight and eukaryotes involves the production of vitamin D through exposure to ultraviolet (UV) radiation. While extensively studied in vertebrates, the role of vitamin D in non-animal eukaryotes like microalgae remains unclear. To investigate the potential involvement of vitamin D in the response of microalgae to UV, we focus on Emiliania huxleyi, a microalga found in shallow ocean depths that are exposed to UV radiation. Our results show that E. huxleyi algae produce vitamin D2 and D3 in response to UV irradiation. We further demonstrate that E. huxleyi algae respond to external administration of vitamin D at the transcriptional level, regulating the expression of protective mechanisms that are also regulated in response to UV. Our data reveal that addition of vitamin D enhances the algal photosynthetic performance while reducing harmful reactive oxygen species buildup. This study contributes to understanding the function of vitamin D in E. huxleyi and sheds light on its role in non-animal eukaryotes, as well as its potential importance in marine ecosystems.
Travelling waves in somitogenesis: collective cellular properties emerge from time-delayed juxtacrine oscillation coupling
Tomka, Tomas; Iber, Dagmar; Boareto, Marcelo (2018)
bioRxiv
The sculpturing of the vertebrate body plan into segments begins with the sequential formation of somites in the presomitic mesoderm (PSM). The rhythmicity of this process is controlled by travelling waves of gene expression. These kinetic waves emerge from coupled cellular oscillators and sweep across the PSM. In zebrafish, the oscillations are driven by autorepression of her genes and are synchronized via Notch signalling. Mathematical modelling has played an important role in explaining how collective properties emerge from the molecular interactions. Increasingly more quantitative experimental data permits the validation of those mathematical models, yet leads to increasingly more complex model formulations that hamper an intuitive understanding of the underlying mechanisms. Here, we review previous efforts, and design a mechanistic model of the her1 oscillator, which represents the experimentally viable her7;hes6 double mutant. This genetically simplified system is ideally suited to conceptually recapitulate oscillatory entrainment and travelling wave formation, and to highlight open questions. It shows that three key parameters, the autorepression delay, the juxtacrine coupling delay, and the coupling strength, are sufficient to understand the emergence of the collective period, the collective amplitude, and the synchronization of neighbouring Her1 oscillators. Moreover, two spatiotemporal time delay gradients, in the autorepression and in the juxtacrine signalling, are required to explain the collective oscillatory dynamics and synchrony of PSM cells. The highlighted developmental principles likely apply more generally to other developmental processes, including neurogenesis and angiogenesis.
Versatile live-cell activity analysis platform for characterization of neuronal dynamics at single-cell and network level
Yuan, Xinyue; Schröter, Manuel; Obien, Marie Engelene J.; et al. (2020)
bioRxiv
Discrete regulation of β-catenin-mediated transcription governs identity of intestinal epithelial stem cells
Borrelli, Costanza; Valenta, Tomas; Handler, Kristina; et al. (2020)
bioRxiv
The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we demonstrate that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin’s transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by intrinsic and extrinsic stress signalling results in a process resembling aberrant “villisation” of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires discrete regulation of transcription by transcriptional co-factors.
Mutant SF3B1 promotes PDAC malignancy through TGF-β resistance
Simmler, Patrik T.; Mengis, Tamara; Lehmann, Kjong-Van; et al. (2022)
bioRxiv
The splicing factor SF3B1 is recurrently mutated in various tumors, including pancreatic ductal adenocarcinoma (PDAC). The impact of the hotspot mutation SF3B1K700E on the PDAC pathogenesis, however, remains elusive. Here, we demonstrate that Sf3b1K700E alone is insufficient to induce malignant transformation of the murine pancreas, but increases aggressiveness of PDAC if it co-occurs together with mutated KRAS and p53. We further demonstrate that SF3B1K700E reduces epithelial–mesenchymal transition (EMT) and confers resistance to TGF-β1-induced cell death, and provide evidence that this phenotype is in part mediated through aberrant splicing of Map3k7. Taken together, our work suggests that SF3B1K700E acts as an oncogenic driver in PDAC through enhancing resistance to the tumor suppressive effects of TGF-β.Competing Interest StatementThe authors have declared no competing interest.
A yeast mating-based platform enables the generation and screening of ultra large antibody libraries
Frei, Lester; Reddy, Sai T. (2025)
bioRxiv
Yeast display is a powerful platform for antibody engineering, offering eukaryotic protein folding and compatibility with fluorescence-activated sorting (FACS). However, conventional yeast display faces limitations in terms of library size and display heterogeneity due to plasmid-based expression. Here, we present a yeast display platform that combines genomic library integration and high-efficiency mating to generate ultra-diverse antibody libraries approaching the diversity of phage-libraries (>1011). We engineered two yeast strains, LFYa and LFYalpha, featuring genomically integrated landing pads for high-efficiency library insertion. To enable compatibility with deep sequencing, we integrated the recombinase BxB1, which links heavy (HC) and light chain (LC) information onto a single chromosome. Using our platform, we constructed synthetic antibody libraries with HC and LC diversities of 4.1×107 and 1.7×107 variants, respectively and with an improved mating protocol, we achieved a combinatorial library diversity exceeding 1011. We demonstrated that genomic integration yields uniform surface display. Screening this antibody library against multiple antigens resulted in the discovery of binders with affinities in the single-digit nanomolar to picomolar range, demonstrating the platform’s utility for the discovery of antibodies with therapeutically relevant affinities. This work establishes a robust and deep sequencing-compatible yeast display system that overcomes key limitations of previous mating-based platforms.
Proteome-wide analysis of phospho-regulated PDZ domain interactions through phosphomimetic proteomic peptide phage display
Sundell, Gustav N.; Arnold, Roland; Ali, Muhammad; et al. (2017)
bioRxiv

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