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Nichols, A.

Publications and source records attributed to Nichols, A..

12 recordsLinked to original sources
bioRxiv2026

Using Latent Chemical Recognition in an Evolved Periplasmic Binding Protein Family to Diversify Biosensors

Genetically encoded fluorescent biosensors have gained traction in neuroscience as continuous, reagentless reporters of cellular activity in situ. These sensors, often soluble, can also provide time-resolution in multiple biosensing form factors: benchtop and wearable devices, lyophilized powder tests, and smartphone-based diagnostic tests. These biosensors often take advantage of naturally occurring conformation-switching but require extensive screening to optimize the linkers to a fluorescent reporter. Each new target ligand often requires its own engineering campaign. We asked whether sensors evolved towards a particular target retain useful recognition scope for others. We screened a family of 18 OpuBC-cpGFP sensors evolved toward nicotinic agonists, SSRIs, opioids, and other neural drugs, against 63 structurally diverse compounds. We found that 24 ligands activated at least one biosensor with {Delta}F/F0 > 0.3, sufficient to begin directed evolution, with 8 of those ligands activating at least one biosensor with {Delta}F/F0 > 1.0, the regime of dynamic range usable in end applications. With 124 ligand-biosensor pairs in total, we found multiple leads suitable for directed evolution. Most notably, ligands participating in hits spanned well beyond neural drugs and included DEHP, ergothioneine, ciprofloxacin, thiamine, betahistine, L-carnitine, and L-thyroxine. Across the biosensor family, mutation distance weakly predicted substrate scope. In particular, we observed sequence-function cliffs that could be exploited for future protein engineering campaigns. Thus, broad screening of performant scaffolds offers rapid bootstrapping in biosensor engineering particularly for exogenous molecules.

bioengineering↗
bioRxiv2026

Squamous-state excursions activate APOBEC3A in cancer

The cytidine deaminase APOBEC3A is a major endogenous mutagen in human cancer, yet is rarely captured in bulk tumor RNA or protein profiles despite the prominent mutational scars it leaves in cancer genomes. The origin of these episodic mutational bursts has remained unclear, with prevailing models emphasizing sustained inflammatory signaling. Here, we show that APOBEC3A is induced in a rare subpopulation of cancer cells engaging a transient squamous differentiation program, linking APOBEC3A mutagenesis to lineage-state plasticity rather than persistent inflammatory signaling. Across breast and lung cancer cell lines and patient tumors, keratinocyte differentiation markers, including the stress keratins KRT6A and KRT16, are the strongest correlates of endogenous APOBEC3A expression. These days-long squamous-state excursions explain how APOBEC3A can leave durable mutational scars while remaining largely invisible to bulk tumor RNA and protein profiling. APOBEC3A catalytic activity reinforces selected components of this program through uracil excision and JNK-AP-1 signaling. The squamous differentiation transcription factor ZNF750 promotes APOBEC3A induction during squamous-state engagement in breast and lung cancer models. In established human squamous tumors, however, ZNF750 loss-of-function is associated with elevated APOBEC3A expression and APOBEC mutagenesis, revealing lineage-context-dependent regulation. These findings identify a transient differentiation state as a mutagenic intermediate, coupling cell-state plasticity to cancer genome evolution.

cancer biology↗
bioRxiv2026

Radiant DIA: A Fast, Sensitive, and Accurate Search Engine for Quantitative Proteomics

In mass spectrometry-based proteomics, robust and efficient search engines are essential for accurate peptide and protein identification and quantification. Advances in sample preparation and instrumentation have increased the demand for highly scalable processing tools, with datasets comprising hundreds or thousands of samples in single-cell and population studies. Here we present Radiant DIA, a novel Data-Independent Acquisition search engine which achieves 4x faster processing and 10x lower cloud compute costs for large experiments while ensuring rigorous control of false discovery rate (FDR) and maintaining similar sensitivity, precision, and quantitative accuracy to widely-used tools. The Radiant DIA search engine is paired with a modular pipeline deployable on cloud and desktop environments comprising individual modules for distributed re-scoring, FDR estimation, protein inference and quantification. Unlike traditional monolithic applications, this architecture enables high-performance, cloud-scale analysis without sacrificing local usability. Together, the Radiant DIA and Fulcrum Pipeline tools enhance computational efficiency to facilitate biological discovery in large-scale proteomics, as demonstrated by analyses of real-world experiments up to thousands of MS acquisitions.

bioinformatics↗
bioRxiv2025

Mitotic transcription ensures ecDNA inheritance through chromosomal tethering

Extrachromosomal DNA (ecDNA) are circular DNA bodies that play critical roles in tumor progression and treatment resistance by amplifying oncogenes across a wide range of cancer types. ecDNA lack centromeres and are thus not constrained by typical Mendelian segregation, enabling their unequal accumulation within daughter cells and associated increases in copy number. Despite intrinsic links to their oncogenic potential, the fidelity and mechanisms of ecDNA inheritance are poorly understood. Here, we show that ecDNA are protected against cytosolic mis-segregation through mitotic clustering and by tethering to the telomeric and subtelomeric regions of mitotic chromosomes. ecDNA-chromosome tethering depends on BRD4 transcriptional co-activation and mitotic transcription of the long non-coding RNA PVT1, which is co-amplified with MYC in colorectal and prostate cancer cell lines. Disruption of ecDNA-chromosome tethering through BRD4 inhibition, PVT1 depletion, or inhibiting mitotic transcription results in cytosolic mis-segregation, ecDNA reintegration, and the formation of homogeneously staining regions (HSRs). We propose that nuclear inheritance of ecDNA is facilitated by an RNA-mediated physical tether that links ecDNA to mitotic chromosomes and thus protects against cytosolic mis-segregation and chromosomal integration.

molecular biology↗
bioRxiv2024

Cloud-enabled Scalable Analysis of Large Proteomics Cohorts

Rapid advances in depth and throughput of untargeted mass-spectrometry-based proteomic technologies are enabling large-scale cohort proteomic and proteogenomic analyses. As such studies scale, the data infrastructure and search engines required to process data must also scale. This challenge is amplified in search engines that rely on library-free match between runs (MBR) search, which enable enhanced depth-per-sample and data completeness. However, to-date, no MBR-based search could scale to process cohorts of thousands or more individuals. Here, we present a strategy to deploy search engines in a distributed cloud environment without source code modification, thereby enhancing resource scalability and throughput. Additionally, we present an algorithm, Scalable MBR, that replicates the MBR procedure of the popular DIA-NN software for scalability to thousands of samples. We demonstrate that Scalable MBR can search thousands of MS raw files in a few hours compared to days required for the original DIA-NN MBR procedure and demonstrate that the results are almost indistinguishable to those of DIA-NN native MBR. The method has been tested to scale to over 15,000 injections and is available for use in the Proteograph Analysis Suite.

bioinformatics↗
bioRxiv2023

The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and [~]7000 new gene cluster families. Selected examples demonstrate how the NPDC Portals strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

microbiology↗
bioRxiv2023

Lanthanide Cathodophores for Multicolor Electron Microscopy

Electron microscopy (EM) and fluorescence imaging are indispensable techniques that provide complementary information on cellular organization. Combining these two modalities is a long-standing challenge in bioimaging. In principle, it should be possible to use the electron beam both for ultrastructural imaging and for molecular localization. The latter could be accomplished by directly exciting suitable biomolecular labels and detecting their luminescence - a process termed cathodoluminescence (CL). Here, we achieve multicolor, single-particle CL imaging of sub-20-nm lanthanide nanocrystals (cathodophores) in the same field of view on the surface of a mammalian cell while simultaneously imaging cellular ultrastructure. In pursuit of this goal, we have developed a comprehensive framework for single-particle CL imaging of lanthanide nanocrystals. By mitigating nonlocal excitation due to secondary electrons, we achieved single-particle detection of multiple spectrally distinct types of sub-20-nm cathodophores. The smallest detectable cathodophores were sub-12 nm in diameter. We found that the CL emission rate scaled linearly with nanocrystal diameter. Furthermore, even in the absence of inert shells, cathodophores were not quenched in the context of mammalian cells processed for EM imaging using heavy-metal staining and sputter-coating. These findings establish cathodophores as promising biomolecular tags for multicolor EM. Moreover, our results inform general design rules for precise control and rational engineering of future generations of single-particle cathodoluminescent nanoprobes.

biophysics↗
bioRxiv2023

Interactive computational and experimental approaches improve the sensitivity of periplasmic binding protein-based nicotine biosensors for measurements in biofluids

We developed fluorescent protein sensors for nicotine with improved sensitivity. For iNicSnFR12 at pH 7.4, the proportionality constant for {Delta}F/F0 vs [nicotine] ({delta}-slope, 2.7 M-1) is 6.1-fold higher than the previously reported iNicSnFR3a. The activated state of iNicSnFR12 has a fluorescence quantum yield of at least 0.6. We measured similar dose-response relations for the nicotine-induced absorbance increase and fluorescence increase, suggesting that the absorbance increase leads to the fluorescence increase via the previously described nicotine-induced conformational change, the "candle snuffer" mechanism. Molecular dynamics (MD) simulations identified a binding pose for nicotine, previously indeterminate from experimental data. MD simulations also showed that Helix 4 of the periplasmic binding protein (PBP) domain appears tilted in iNicSnFR12 relative to iNicSnFR3a, likely altering allosteric network(s) that link the ligand binding site to the fluorophore. In thermal melt experiments, nicotine stabilized the PBP of the tested iNicSnFR variants. iNicSnFR12 resolved nicotine in diluted mouse and human serum at 100 nM, the peak [nicotine] that occurs during smoking or vaping, and possibly at the decreasing levels during intervals between sessions. NicSnFR12 was also partially activated by unidentified endogenous ligand(s) in biofluids. Improved iNicSnFR12 variants could become the molecular sensors in continuous nicotine monitors for animal and human biofluids.

bioengineering↗
bioRxiv2021

PGfinder, a novel analysis pipeline for the consistent, reproducible and high- resolution structural analysis of bacterial peptidoglycans

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan, the essential and major component of bacterial cell envelopes. It is comprised of glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGfinder for the analysis of peptidoglycan structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial peptidoglycans. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

microbiology↗
bioRxiv2019

Fibrotic human lung extracellular matrix as a disease-specific substrate for 3D in-vitro models of pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is an irreversible and uniformly fatal lung disease marked by destruction and scarring of the lung parenchyma and progressive loss of respiratory function. IPF affects nearly 3 million people worldwide, and annual mortality in the US alone exceeds 40,000. Nintedanib and pirfenidone, the only drugs approved for the treatment of IPF, slow progression but do not cure the disease. Consequently, there is a pressing need for effective treatments beside lung transplantation. Unfortunately, predictive models of IPF are not available, underscoring the critical need for physiologically relevant in-vitro substrates that enable quantitative and mechanistic studies of human IPF. Here we report the development and characterization of a human pulmonary fibrosis-specific cell culture substrate comprised of intact fibrotic lung extracellular matrix that recapitulates the human IPF disease environment in vitro. We document the activation and disease-specific phenotype of human lung fibroblasts cultured in the IPF disease-specific substrate, and establish feasibility of testing antifibrotic agents using this substrate. Altogether, our results demonstrate the applicability of this fibrosis-specific substrate for 3D in-vitro models of IPF and cell-based assays in early-stage drug discovery.

cell biology↗
bioRxiv2019

Investigating Transfusion-Related Sepsis using Culture-Independent Metagenomic Sequencing

BackgroundTransfusion-related sepsis remains an important hospital infection control challenge. Investigating septic transfusion events is often restricted by the limitations of bacterial culture in terms of time requirements and low yield in the setting of prior antibiotic administration.\n\nMethodsIn three Gram-negative septic transfusion cases, we performed mNGS of direct clinical blood specimens in addition to standard culture-based approaches utilized for infection control investigations. Pathogen detection leveraged IDSeq, a new open-access microbial bioinformatics portal. Phylogenetic analysis was performed to assess microbial genetic relatedness and understand transmission events.\n\nResultsmNGS of direct clinical blood specimens afforded precision detection of pathogens responsible for each case of transfusion-related sepsis, and enabled discovery of a novel Acinetobacter species in a platelet product that had become contaminated despite photochemical pathogen reduction. In each case, longitudinal assessment of pathogen burden elucidated the temporal sequence of events associated with each transfusion-transmitted infection. We found that informative data could be obtained from culture-independent mNGS of residual platelet products and leftover blood specimens that were either unsuitable or unavailable for culture, or that failed to grow due to prior antibiotic administration. We additionally developed methods to enhance accuracy for detecting transfusion-associated pathogens sharing taxonomic similarity to contaminants commonly found in mNGS library preparations.\n\nConclusionsCulture-independent mNGS of blood products afforded rapid and precise assessment of pathogen identity, abundance and genetic relatedness. Together, these challenging cases demonstrated the potential for metagenomics to advance existing methods for investigating transfusion-transmitted infections.

microbiology↗