Biology subjects

Campbell, A. E.

Publications and source records attributed to Campbell, A. E..

12 recordsLinked to original sources

bioRxiv2026

Multi-lineage emergence of USA300 MRSA in in Chile and a potential role for environmental copper resistance

Methicillin-resistant S. aureus (MRSA) is prevalent worldwide, but individual strains of MRSA tend to spread and expand in relatively confined geographic areas. The USA300 lineage of MRSA is most well-known for its rapid epidemic spread in North America during the first decade of the 2000's leading to an overall increase in serious skin infections and the emergence of methicillin resistance in the majority of community acquired S. aureus infections. A distinct but closely related South American epidemic clade of MRSA USA300 (USA300-SAE), first reported in 2006, proliferated rapidly in Northern South America but was rarely found in other Latin American countries. In a new survey of 274 invasive MRSA isolates from 2022 collected in Chile, we saw a marked increase in USA300-SAE in Southern South America. We found that Chilean USA300-SAE is phylogenetically diverse and was likely introduced multiple times from multiple different South American countries over the course of more than a decade, but only recently became a highly prevalent clone. Using novel molecular diagnostic methods for isolates collected in 2023, we estimate that USA300-SAE made up more than 22% of invasive MRSA strains in Chile in that year and is likely still increasing. We show that copper-associated genes in the signature copper and mercury resistance mobile element (COMER) of USA300-SAE are strongly conserved, and that isogenic mutants of the copper resistance genes in COMER are highly susceptible to copper killing at concentrations seen in tap water. These findings suggest that environmental exposure to copper may provide a fitness advantage that enhances spread of this lineage.

microbiology↗

bioRxiv2025

Splice-switching antisense oligonucleotides correct cryptic exon inclusion and restore SDCCAG8 protein in Bardet-Biedl Syndrome

Bardet-Biedl Syndrome (BBS) is a ciliopathy often associated with progressive blindness and obesity. A patient presenting with BBS was discovered to have two mutations within 55bp of each other in intron 7 of SDCCAG8 (BBS16). One of the biallelic mutations, c.740+356C>T, causes inclusion of cryptic exon(s) containing premature termination codons, while c.740+301G>A has not been characterized. We hypothesized that antisense oligonucleotides (ASOs) complementary to the patients mutations or to the cryptic exon splice sites would correct the splicing of SDCCAG8 between exons 7 and 8 to prevent cryptic exon inclusion and restore SDCCAG8 expression. We systematically screened 20nt-long ASOs tiled across each mutation and ASOs targeting the 3' splice sites of the cryptic exons in patient-derived fibroblasts, using RT-PCR assays to assess exon 7 and 8 splicing. We identified one ASO for each mutation and a cryptic exon-targeting ASO that restored the splicing pattern to that observed in an unaffected cell line. Lead ASOs were further investigated through RT-PCR, RNA sequencing, and western blotting to confirm ASO-mediated restoration of wild-type transcript and protein. Notably, ASO 20, which targets the cryptic exon 7a/7a' splice site rather than patient-specific mutations, achieved the greatest rescue effect, increasing exon 7-8 splicing from 0% to an average of 26% and restoring SDCCAG8 protein from undetectable levels to approximately 40% of wild-type expression. This mutation-agnostic approach could benefit multiple patients with cryptic exon inclusion in this region of SDCCAG8, expanding therapeutic impact beyond traditional N-of-1 ASO strategies. These findings establish a molecular foundation for clinical development of ASO therapy for BBS caused by SDCCAG8 splicing defects.

genetics↗

bioRxiv2024

Activation loop phosphorylation of Cdk11 is restrained by PNUTS-PP1 and regulates Cdk11 activity and function

Organisation of the transcription cycle is facilitated by the reversible phosphorylation of the C-terminal domain of RNA Polymerase II (RNAPII-CTD) and its accessory factors. The PNUTS-PP1 protein phosphatase is crucial for mRNA synthesis and processing, yet the complete spectrum of its physiological targets in these processes remain elusive. Here, using quantitative phosphoproteomics, we discover that Cdk11, in addition to various spliceosomal and RNA processing factors, associates with PNUTS, and that disruption of PP1-binding results in hyperphosphorylation of Cdk11 at an evolutionarily conserved Serine residue, seven amino acid residues C-terminal to DFG residues in the activation loop. In vitro experiments reveal a role for Ser DFG+7 in modulating Cdk11 kinase activity towards RNAPII-CTD Ser5. Making use of a novel technique to conditionally disrupt PP1 binding, we show that PNUTS-PP1 normally serves to restrain Cdk11 phosphorylation in vivo. Mutational analysis shows that cdk11 is not only essential for survival but also plays a widespread role in regulating normal mRNA expression and splicing. Notably, we find that a phosphomimetic mutation in cdk11 exhibits distinct biological effects compared to loss of cdk11 function, including defective processing of intronic small nucleolar RNAs, diminished intronic RNA Pol II velocity, and a decrease in intergenic transcription. These findings underscore physiologically significant roles of Cdk11 dephosphorylation by PNUTS-PP1 in the regulation of mRNA transcription and processing.

molecular biology↗

bioRxiv2024

Peptidyl-tRNA hydrolysis rate influences the efficiency of nonsense-mediated mRNA decay

Nonsense variants underlie many genetic diseases. The phenotypic impact of nonsense variants is determined by nonsense-mediated mRNA decay (NMD), which degrades transcripts with premature termination codons (PTCs). Despite its clinical importance, the factors controlling transcript-specific and context-dependent variation in NMD activity remain poorly understood. Through analysis of human genetic datasets, we discovered that the amino acid preceding the PTC strongly influences NMD activity. Notably, glycine codons promote robust NMD efficiency and show striking enrichment before PTCs but depletion before normal termination codons (NTCs). This glycine-PTC enrichment is particularly pronounced in genes tolerant to loss-of-function variants, suggesting evolutionary selection or neutrality conferred by efficient elimination of truncated proteins from non-essential genes. Using biochemical assays and massively parallel reporter analysis, we demonstrated that the peptide release rate during translation termination varies substantially with the identity of the preceding amino acid and serves as the primary determinant of NMD activity. We propose a "window of opportunity" model where translation termination kinetics modulate NMD efficiency. By revealing how sequence context shapes NMD activity through translation termination dynamics, our findings provide a mechanistic framework for improved clinical interpretation of nonsense variants.

molecular biology↗

bioRxiv2023

A system of reporters for comparative investigation of EJC-independent and EJC-enhanced nonsense-mediated mRNA decay

Nonsense-mediated mRNA decay (NMD) is a network of pathways that degrades transcripts that undergo premature translation termination. In mammals, NMD can be divided into the exon junction complex (EJC)-enhanced and EJC-independent branches. Fluorescence- and luminescence-based reporters have long been effective tools to investigate NMD, yet existing reporters largely focus on the EJC-enhanced pathway. Here, we present a system of reporters for comparative studies of EJC-independent and EJC-enhanced NMD. This system also enables the study of NMD-associated outcomes such as premature termination codon (PTC) readthrough and truncated protein degradation. These reporters are compatible with fluorescence or luminescence-based readouts via transient transfection or stable integration. Using this reporter system, we show that EJC-enhanced NMD RNA levels are reduced by 2- or 9-fold and protein levels are reduced by 7- or 12-fold compared to EJC-independent NMD, depending on the reporter gene used. Additionally, the extent of readthrough induced by G418 and SMG1i, alone and in combination, varies across NMD substrates. When combined, G418 and SMG1i increase readthrough product levels in an additive manner for EJC-independent reporters, while EJC-enhanced reporters show a synergistic effect. We present these reporters as a valuable toolkit to deepen our understanding of NMD and its associated mechanisms.

molecular biology↗

bioRxiv2023

Wound microbiota-mediated correction of matrix metalloproteinase expression promotes re-epithelialization of diabetic wounds

Chronic wounds are a common and costly complication of diabetes, where multifactorial defects contribute to dysregulated skin repair, inflammation, tissue damage, and infection. We previously showed that aspects of the diabetic foot ulcer microbiota were correlated with poor healing outcomes, but many microbial species recovered remain uninvestigated with respect to wound healing. Here we focused on Alcaligenes faecalis, a Gram-negative bacterium that is frequently recovered from chronic wounds but rarely causes infection. Treatment of diabetic wounds with A. faecalis accelerated healing during early stages. We investigated the underlying mechanisms and found that A. faecalis treatment promotes re-epithelialization of diabetic keratinocytes, a process which is necessary for healing but deficient in chronic wounds. Overexpression of matrix metalloproteinases in diabetes contributes to failed epithelialization, and we found that A. faecalis treatment balances this overexpression to allow proper healing. This work uncovers a mechanism of bacterial-driven wound repair and provides a foundation for the development of microbiota-based wound interventions.

microbiology↗

bioRxiv2022

Staphyloxanthin production by Staphylococcus aureus promotes resistance to oxidative stress to delay diabetic wound healing

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus that burden patients and health care systems. Staphylococcus aureus is prevalent and abundant in the DFU microbiome, and strain-level differences in S. aureus may drive clinical outcomes. To identify mechanisms underlying strain-specific outcomes in DFU with S. aureus, we performed high-throughput phenotyping screens on a collection of 221 S. aureus cultured isolates from clinically uninfected DFU. Of the 4 phenotypes examined (in vitro biofilm formation and production of staphylokinase, staphyloxanthin, and siderophores), we discovered that isolates from non-healing wounds produced more staphyloxanthin, a carotenoid cell membrane pigment. In a murine diabetic wound healing model, staphyloxanthin-producing isolates delayed wound closure significantly compared to staphyloxanthin-deficient isolates. Staphyloxanthin promoted resistance to oxidative stress in vitro and enhanced bacterial survival in human neutrophils. Comparative genomic and transcriptomic analysis of genetically similar clinical isolates with disparate staphyloxanthin phenotypes revealed a mutation in the Sigma B regulatory pathway that resulted in marked differences in stress response gene expression. Our findings suggest that staphyloxanthin production delays wound healing by protecting S. aureus from neutrophil-mediated oxidative stress, and may provide a target for therapeutic intervention in S. aureus-positive wounds.

microbiology↗

bioRxiv2022

Human DUX4 and mouse Dux interact with STAT1 and broadly inhibit interferon-stimulated gene induction

DUX4 activates the first wave of zygotic gene expression in the early embryo. Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral dystrophy (FSHD), whereas expression in cancers suppresses IFN{gamma}-induction of MHC Class I and contributes to immune evasion. We show that the DUX4 protein interacts with STAT1 and broadly suppresses expression of IFN{gamma} stimulated genes by decreasing bound STAT1 and Pol-II recruitment. Transcriptional suppression of ISGs requires conserved (L)LxxL(L) motifs in the carboxyterminal region of DUX4 and phosphorylation of STAT1 Y701 enhances interaction with DUX4. Consistent with these findings, expression of endogenous DUX4 in FSHD muscle cells and the CIC-DUX4 fusion containing the DUX4 CTD in a sarcoma cell line inhibit IFN{gamma}-induction of ISGs. Mouse Dux similarly interacted with STAT1 and suppressed IFN{gamma} induction of ISGs. These findings identify an evolved role of the DUXC family in modulating immune signaling pathways with implications for development, cancers, and FSHD.

developmental biology↗

bioRxiv2021

The myopathic transcription factor DUX4 induces the production of truncated RNA-bindingproteins in human muscle cells

Nonsense-mediated RNA decay (NMD) is a surveillance mechanism that degrades both canonical and aberrant transcripts carrying premature translation termination codons. NMD is thought to have evolved to prevent the synthesis of toxic truncated proteins. However, whether global inhibition of NMD results in widespread production of truncated proteins is unknown. A human genetic disease, facioscapulohumeral muscular dystrophy (FSHD) features acute inhibition of NMD upon expression of the disease-causing transcription factor, DUX4. Here, using a cell-based model of FSHD, we show the production of hundreds of truncated proteins from physiological NMD targets. Using ribosome profiling, we map the precise C-terminal end of these aberrant truncated proteins and find that RNA-binding proteins are especially enriched for aberrant truncations. The stabilized NMD isoform of one RNA-binding protein, SRSF3, is robustly translated to produce a stable truncated protein, which can also be detected in FSHD patient-derived myotubes. Notably, ectopic expression of truncated SRSF3 alone confers toxicity and its downregulation is cytoprotective. Our results demonstrate the genome-scale impact of NMD inhibition. This widespread production of potentially deleterious truncated proteins has implications for FSHD biology as well as other genetic diseases where NMD is therapeutically modulated.

genomics↗

bioRxiv2021

Regulation of CHK1 inhibitor resistance by a c-Rel and USP1 dependent pathway

We previously discovered that deletion of c-Rel in the E-Myc mouse model of lymphoma results in earlier onset of disease, a finding that contrasted with the expected function of this NF-{kappa}B subunit in B-cell malignancies. Here we report that c-rel -/- E{micro}-Myc cells have an unexpected and major defect in the CHK1 pathway, with almost undetectable levels of CHK1 and CLSPN protein leading to therapeutic resistance to the highly specific CHK1 inhibitor (CHK1i) CCT244747. Similar downregulation of CHK1 levels was also seen in CCT244747 resistant U20S osteosarcoma cells. Further investigation revealed that downregulation of the deubiquitinase USP1 is responsible, at least in part, for these effects. Importantly, we demonstrate that c-rel -/- E{micro}-Myc lymphoma cells survive though upregulation of compensatory PI3K/AKT pathway activity. Moreover, targeting this pathway with Pictilisib (GDC-0941) effectively killed c-rel -/- E{micro}-Myc in vivo, while having no effect on wild type E-Myc cells. This data reveals an NF-{kappa}B regulated pathway controlling CHK1 activity in cancer cells and identifies a potential mechanism for both acquiring and overcoming CHK1i resistance in cancer patients.

cancer biology↗

bioRxiv2019

DUX4-induced bidirectional HSATII satellite repeat transcripts form intranuclear double stranded RNA foci in human cell models of FSHD

The DUX4 transcription factor is normally expressed in the cleavage stage embryo and regulates genes involved in embryonic genome activation. Mis-expression of DUX4 in skeletal muscle, however, is toxic and causes facioscapulohumeral muscular dystrophy (FSHD). We recently showed DUX4-induced toxicity is due, in part, to the activation of the double-stranded RNA (dsRNA) response pathway and the accumulation of intranuclear dsRNA foci. Here, we determined the composition of DUX4-induced dsRNAs. We found that a subset of DUX4-induced dsRNAs originate from inverted Alu repeats embedded within the introns of DUX4-induced transcripts and from DUX4-induced dsRNA-forming intergenic transcripts enriched for endogenous retroviruses, Alu and LINE-1 elements. However, these repeat classes were also represented in dsRNAs from cells not expressing DUX4. In contrast, pericentric human satellite II (HSATII) repeats formed a class of dsRNA specific to the DUX4 expressing cells. Further investigation revealed that DUX4 can initiate the bidirectional transcription of normally heterochromatin-silenced HSATII repeats. DUX4 induced HSATII RNAs co-localized with DUX4-induced nuclear dsRNA foci and with intranuclear aggregation of EIF4A3 and ADAR1. Finally, gapmer-mediated knockdown of HSATII transcripts depleted DUX4-induced intranuclear ribonucleoprotein aggregates and decreased DUX4-induced cell death, suggesting that HSATII formed dsRNAs contribute to DUX4 toxicity.

molecular biology↗

bioRxiv2019

Saccade countermanding reflects automatic inhibition as well as top-down cognitive control

Countermanding behavior has long been seen as a cornerstone of executive control - the human ability to selectively inhibit undesirable responses and change plans. In recent years, however, scattered evidence has emerged that stopping behavior is entangled with simpler automatic stimulus-response mechanisms. Here we give flesh to this idea by merging the latest conceptualization of saccadic countermanding with a versatile neural network model of visuo-oculomotor behavior that integrates bottom-up and top-down drives. This model accounts for all fundamental qualitative and quantitative features of saccadic countermanding, including neuronal activity. Importantly, it does so by using the same architecture and parameters as basic visually guided behavior and automatic stimulus-driven interference. Using simulations and new data, we compare the temporal dynamics of saccade countermanding with that of saccadic inhibition (SI), a hallmark effect thought to reflect automatic competition within saccade planning areas. We demonstrate how SI accounts for a large proportion of the saccade countermanding process when using visual signals. We conclude that top-down inhibition acts later, piggy-backing on the quicker automatic inhibition. This conceptualization fully accounts for the known effects of signal features and response modalities traditionally used across the countermanding literature. Moreover, it casts different light on the concept of top-down inhibition, its timing and neural underpinning, as well as the interpretation of stop-signal reaction time, the main behavioral measure in the countermanding literature.

animal behavior and cognition↗