Biology subjects

Francis, D.

Publications and source records attributed to Francis, D..

9 recordsLinked to original sources

bioRxiv2026

Improved Protein Semi-Synthesis Enables Biophysical Studies of Thioamide Destabilization of β-Sheet Interactions

Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the {beta}-sheet region of the B1 domain of protein G (GB1) as well as {beta}-strand interactions in amyloid fibrils of the Parkinsons disease protein -synuclein. In both contexts, we observe dramatic destabilization of the {beta}-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of -synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen bonding networks and prompt future study of both systems.

biochemistry↗

bioRxiv2026

Crowdsourced riboregulators reveal design principles for programmable RNA switching

RNA-based sensors offer powerful and programmable control of gene expression, yet our understanding of the structural principles that govern their potential design space remains incomplete. Here, we challenged a community of designers to generate novel riboregulators capable of activating translation in response to specific RNA targets. Participants submitted diverse sequence architectures, which were synthesized and evaluated in a cell-free transcription-translation system. Across 100 designs, community-generated riboregulators displayed wide variability in activation dynamics, fold change, and structural features, outperforming some canonical toehold-switch designs and achieving up to 80-fold activation. Structural ensemble analyses identified accessibility patterns near the ribosome binding site that distinguish high- from low-performing regulators, highlighting the central role of RBS sequestration and release in modulating expression. Together, we demonstrate community-driven design can expand the accessible structural space of riboregulators and uncover mechanistic features governing translational activation. Our findings establish quantitative links between RNA folding energetics and gene expression output, providing design principles for next-generation programmable RNA sensors.

synthetic biology↗

bioRxiv2025

Pooled CRISPRi screening reveals fungal-specific vulnerabilities across environments and genetic backgrounds

The rising rate of drug-resistant fungal infections and the emergence of fungal pathogens with intrinsic resistance phenotypes are a growing concern. The close evolutionary distance between mammals and fungi complicates the design of new antifungals and increases the chances of toxic off-target effects. As such, antifungal drug development usually focuses on fungal-specific proteins when considering potential new targets. Ideal drug targets should mediate essential cell processes and be highly sensitive to inhibition. Targeted gene repression can serve as a model for drug-mediated inhibition and for determining the dosage-sensitivity profile of genes of interest. In the fungal pathogen Candida albicans, classical approaches for gene repression can be labour-intensive and limited to one genetic background due to low throughput. Here, we adapt pooled CRISPRi screening in C. albicans for the first time and exploit this technique for large-scale functional genomic analysis. Through pooled CRISPRi screening, we test the repression sensitivity of over a hundred essential genes conserved in fungi but absent in humans, and successfully identify highly dosage-sensitive genes across multiple cell components and pathways. By extending our analysis to ten diverse environmental conditions, we show how the environment influences dosage-sensitivity profiles. Finally, we extend our experiments to two clinical drug-resistant C. albicans strain backgrounds and demonstrate that many of the fitness defects we observed are conserved in resistant clinical isolates. Together, our results highlight a set of genes that are highly dosage-sensitive across different genetic and environmental contexts, making them attractive targets for further investigation. By facilitating rapid, efficient large-scale functional genomics assays across diverse genetic backgrounds, CRISPRi pooled screening will open new frontiers in C. albicans biology.

microbiology↗

bioRxiv2025

Robust Colonization of Nasal Microbiota Across the Respiratory Tract and the Gut Modulates Innate Immune Response in a Germ-Free Pig Model

The use of antibiotics in swine production has contributed to improved health and growth rates but also led to significant challenges, including the emergence of antibiotic-resistant bacteria and disruptions in the microbiome. This study employs a germ-free pig model to investigate the transplantation of the nasal microbiome into the respiratory tract, exploring its potential to colonize multiple body sites and its impact on host immunity. Using germ-free piglets inoculated with nasal microbiota from healthy donors, we examined the dynamics of microbial community formation across the respiratory and gastrointestinal tracts and assessed the effects of antibiotic intervention. Our findings reveal that specific taxa, predominantly from the phyla Proteobacteria and Firmicutes, cross colonized both respiratory as well as gastrointestinal tract. This cross- colonization highlights the adaptability of nasal microbiota and suggests a significant role in modulating host immunity through the gut-lung axis. The administration of the antibiotic tulathromycin on day 11 post-inoculation altered both nasal and gut microbiota compositions, reducing microbial diversity and impacting immune gene expression and cytokine profiles. These results underscore the complex interactions within the gut-lung axis and emphasize the implications of antibiotic use on microbial health and host immunity. Understanding these interactions provides crucial insights into alternative strategies for managing health in swine production without relying on antibiotics. Our study advocates for a reevaluation of antibiotic practices and supports the development of microbiome-centered interventions in swine health management.

microbiology↗

bioRxiv2024

Modulation of SpyCatcher Ligation Kinetics by SpyTag Thioamide Substitution

Thioamide substitutions have been shown to impart valuable properties on peptides for biophysical experiments as well as cell or in vivo studies, but a rational understanding of thioamide effects on protein structure and protein-protein interactions is lacking. To elucidate their effects in {beta}-sheet structures, we have used SpyCatcher003-SpyTag003 as a host-guest system to study individual thioamide incorporation at eight different positions in the SpyTag peptide. We have demonstrated that incorporating thioamides into SpyTag at specific positions can result in a [~]2-fold faster ligating complex, as well as >2000-fold slower ligating complex. Biophysical analysis and structural modeling provide a reasonable explanation for most of the thioamide effects, altering hydrogen bond networks as well as modulating an n[->]{pi}* interaction within the SpyTag peptide. Our findings have important implications for potential applications of thioamide SpyTag variants, where the thioamide could impart protease stability in cells while also controlling the rate of ligation to SpyCatcher. These SpyCatcher-SpyTag host-guest experiments will also help to build a database for predicting thioamide effects on protein structure and function.

biochemistry↗

bioRxiv2024

A role for the putative error-prone polymerase REV1 in DNA damage and antifungal drug resistance in Candida albicans

Antimicrobial-induced DNA damage, and subsequent repair via upregulation of DNA repair factors, including error-prone translesion polymerases, can lead to the increased accumulation of mutations in the microbial genome, and ultimately increased risk of acquired mutations associated with antimicrobial resistance. While this phenotype is well described in bacterial species, it is less thoroughly investigated amongst microbial fungi. Here, we monitor DNA damage induced by antifungal agents in the fungal pathogen Candida albicans, and find that commonly used antifungal drugs are able to induce DNA damage, leading to the upregulation of transcripts encoding predicted error-prone polymerases and related factors. We focus on REV1, encoding a putative error-prone polymerase, and find that while deleting this gene in C. albicans leads to increased sensitivity to DNA damage, it also unexpectedly renders cells more likely to incur mutations and evolve resistance to antifungal agents. We further find that deletion of REV1 leads to a significant depletion in the uncharacterized protein Shm1, which itself plays a role in fungal mutagenesis. Together, this work lends new insight into previously uncharacterized factors with important roles in the DNA damage response, mutagenesis, and the evolution of antifungal drug resistance.

microbiology↗

bioRxiv2024

Essential conserved neuronal motors kinesin-1 and kinesin-3 regulate Abeta42 toxicity in vivo

Alzheimers Disease is the leading cause of dementia and the most common neurodegenerative disorder. Understanding the molecular pathology of Alzheimers Disease may help identify new ways to reduce neuronal damage. In the past decades Drosophila has become a powerful tool in modelling mechanisms underlying human diseases. Here we investigate how the expression of the human 42-residue {beta}-amyloid (A{beta}) carrying the E22G pathogenic "Arctic" mutation (A{beta}42Arc) affects axonal health and behaviour of Drosophila. We find that A{beta}42Arc flies present aberrant neurons, with altered axonal transport of mitochondrial and an increased number of terminal boutons at neuromuscular junctions. We demonstrate that the major axonal motor proteins kinesin-1 and kinesin-3 are essential for the correct development of neurons in Drosophila larvae and similar findings are replicated in human iPSC-derived cortical neurons. We then show that the over-expression of kinesin-1 or kinesin-3 restores the correct number of terminal boutons in A{beta}42Arc expressing neurons and that this is associated with a rescue of the overall neuronal function, measured by negative geotaxis locomotor behavioural assay. We therefore provide new evidence in understanding the mechanisms of axonal transport defects in Alzheimers Disease, and our results indicate that kinesins should be considered as potential drug targets to help reduce dementia-associated disorders.

cell biology↗

bioRxiv2020

Integrated glycoproteomics identifies a role of N-glycosylation and galectin-1 on myogenesis and muscle development

ABSTRACTMany cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development but our molecular understanding of the precise glycans, catalytic enzymes and lectins involved remain only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins most notably the up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the up-regulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.Competing Interest StatementThe authors have declared no competing interest.AbbreviationsAAV adeno-associated virusANOVAanalysis of variationCBDscarbohydrate binding domainsCDGscongenital disorders of glycosylationCRISPRclustered regularly interspaced short palindromic repeatsCIDcollisional induced dissociationDTTdithiothreitolECMextracellular matrixEThcDelectron transfer dissociation with higher collisional dissociation supplemental activationFBSfetal bovine serumFDRfalse discovery rateFAformic acidGBPsglycan-binding proteinsHCDhigher collisional dissociationHILIChydrophilic interaction liquid chromatographyMCSmultiple cloning siteMeCNacetonitrileNMJneuromuscular junctionsPCAprincipal component analysisPGCporous graphitized carbonPSMpeptide spectral matchTEABtriethylammonium bicarbonateTFAtrifluoroacetic acidTMTtandem mass tagsView Full Text

biochemistry↗

bioRxiv2019

Genome-wide analysis of diet and gene interactions in Drosophila uncovers the glucose transporter, CG4607, as a diet-responsive gene.

Genetic and environmental factors play a major role in metabolic health. However, they do not act in isolation, as a change in an environmental factor such as diet may exert different effects based on an individuals genotype. Here, we sought to understand how such gene-diet interactions influenced nutrient storage and utilisation, a major determinant of metabolic disease. We subjected the Drosophila Genetic Reference Panel (DGRP), comprising 200 genetically divergent inbred fly strains, to diets varying in sugar, fat and protein. We assessed starvation resistance, a holistic phenotype of nutrient storage and utilisation that can be robustly measured. Diet influenced the starvation resistance of each strain, but this effect varied markedly between strains. This demonstrates that genetics plays a major role in the response to diet. Furthermore, heritability analysis revealed that the greatest variability arose from diets either high in sugar or high in protein. To uncover the genetic underpinnings of this variation, we mapped 1,239 diet-responsive SNPs in 534 genes, 325 of which have human orthologues. Using whole-body knockdown, we confirmed that 30 candidate genes were required for glucose tolerance, storage and utilization. In particular, we characterised CG4607, a GLUT6/GLUT8 homolog, as a key protein involved in sugar tolerance. Overall, this provides strong evidence that genetics is a major contributor to how individuals respond to diets of varying nutrient composition. It is likely that a similar principle may be applied to metabolic disease in higher organisms thus supporting the case for nutrigenomics as an important health strategy.

genetics↗