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Sanders, C.

Publications and source records attributed to Sanders, C..

At least 19 recordsLinked to original sources
bioRxiv2025

Pharmacological Tools to Modulate Ordered Membrane Domains and Order-Dependent Protein Function

Ordered membrane nanodomains colloquially known as "lipid rafts" have many proposed cellular functions. However, pharmacological tools to modulate protein affinity for rafts and to manipulate raft formation are currently lacking. We screened 24,000 small molecules for compounds that impact the raft affinity of a known raft-preferring protein, peripheral myelin protein 22 (PMP22), in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and also tested for their impact on raft stability. We identified three chemically distinct tools for manipulating lipid rafts. Two compounds were seen to both decrease PMP22 raft partitioning and to destabilize ordered domains (VU0607402 and VU0519975) while a third (primaquine diphosphate) increased PMP22 partitioning and stabilized ordered domains. While discovered in a PMP22-focused screen, all three were seen to modulate raft formation in a protein-independent manner by altering lipid-lipid interactions and membrane fluidity. Acute treatment of live cells with the raft destabilizing compound, VU0607402 was seen to modulate TRPM8 channel function, highlighting the utility of this compound in live-cell experiments for dissecting the role that membrane order and fluidity play in cell signaling. These compounds provide novel pharmacological tools for probing lipid raft properties and function in biophysical experiments and in living cells.

biophysics↗
bioRxiv2025

Comprehensive characterization of human color discrimination thresholds

Color discrimination thresholds--the smallest detectable color differences--provide a benchmark for models of color vision, enable quantitative evaluation of eye diseases, and inform the design of display technologies. Despite their importance, a comprehensive characterization of these thresholds has long been considered intractable due to the psychophysical curse of dimensionality. Here, we address this challenge using a novel semi-parametric Wishart Process Psychophysical Model (WPPM), which leverages the feature that the internal noise limiting color discrimination varies smoothly across stimulus space. The model was fit to data collected with a non-parametric adaptive trial-placement procedure, enabling efficient stimulus selection. Together, through the combination of adaptive trial placement and post hoc WPPM fitting, we achieved a comprehensive characterization of color discrimination in the isoluminant plane with only [~]6,000 trials per participant (N = 8). Once fit, the WPPM allows readouts of discrimination performance for any stimulus pair. We validated these readouts against 25 probe psychometric functions, measured with an additional 6,000 trials per participant held out from model fitting. In conclusion, our study provides a foundational dataset for color vision, and our approach generalizes beyond color to any domain in which the internal noise limiting performance varies smoothly across stimulus space, offering a powerful and efficient method for comprehensively characterizing various perceptual discrimination thresholds.

neuroscience↗
bioRxiv2025

Lack of Vector Competence in UK Culex pipiens molestus for Oropouche Virus

Oropouche virus (OROV) is an orthobunyavirus (Peribunyaviridae) that has caused recurrent outbreaks in South America and has recently expanded into the Caribbean, with various biting midge and mosquito species considered vectors. Recent imported cases to Europe and North America have raised concerns about the potential for local transmission in non-endemic areas. To assess this risk in the United Kingdom, we investigated the vector competence of Culex pipiens molestus (Cx. molestus), a human-biting mosquito common in urban environments. Laboratory-reared adult females were fed a bloodmeal containing a Cuban 2024 OROV outbreak strain (240023) and maintained at 27{degrees}C. Of 64 individuals tested at 12-s14 days post-infection via plaque assay, none were positive for OROV in bodies, indicating no evidence of infection and subsequently limited or no vector competence potential. These results provide evidence that UK populations of Cx. molestus are unlikely to support OROV transmission, thereby refining assessments of OROV emergence risk in temperate settings. Further studies are needed to test other putative UK mosquito vectors, as well as Culicoides biting midge species to fully assess the potential for OROV transmission in this region.

microbiology↗
bioRxiv2025

Functional profiling of KCNE1 variants informs population carrier frequency of Jervell and Lange-Nielsen syndrome type 2

Congenital long-QT syndrome (LQTS) is most often associated with pathogenic variants in KCNQ1 encoding the pore-forming voltage-gated potassium channel subunit of the slow delayed rectifier current (IKs). Generation of IKs requires assembly of KCNQ1 with an auxiliary subunit encoded by KCNE1, which is also associated with LQTS but causality of autosomal dominant disease is disputed. By contrast, KCNE1 is an accepted cause of recessive type 2 Jervell and Lange-Nielson syndrome (JLN2). The functional consequences of most KCNE1 variants have not been determined and the population prevalence of JLN2 is unknown. Methods: We determined the functional properties of 95 KCNE1 variants co-expressed with KCNQ1 in heterologous cells using high-throughput voltage-clamp recording. Experiments were conducted with each KCNE1 variant expressed in the homozygous state and then a subset was studied in the heterozygous state. The carrier frequency of JLN2 was estimated by considering the population prevalence of dysfunctional variants. Results: There is substantial overlap between disease-associated and population KCNE1 variants. When examined in the homozygous state, 68 KCNE1 variants exhibited significant differences in at least one functional property compared to WT KCNE1, whereas 27 variants did not significantly affect function. Most dysfunctional variants exhibited loss-of-function properties. We observed no evidence of dominant-negative effects. Most variants were scored as variants of uncertain significance (VUS) and inclusion of functional data resulted in revised classifications for only 14 variants. The population carrier frequency of JLN2 was calculated as 1 in 1034. Peak current density and activation voltage-dependence but no other biophysical properties were correlated with findings from a mutational scan of KCNE1. Conclusions: Among 95 disease-associated or population KCNE1 variants, many exhibit abnormal functional properties but there was no evidence of dominant-negative behaviors. Using functional data, we inferred a population carrier frequency for recessive JLN2. This work helps clarify the pathogenicity of KCNE1 variants.

genetics↗
bioRxiv2024

Small-molecule modulators of lipid raft stability and protein-raft partitioning

Development of an understanding of membrane nanodomains colloquially known as "lipid rafts" has been hindered by a lack of pharmacological tools to manipulate rafts and protein affinity for rafts. We screened 24,000 small molecules for modulators of the affinity of peripheral myelin protein 22 (PMP22) for rafts in giant plasma membrane vesicles (GPMVs). Hits were counter-screened against another raft protein, MAL, and tested for impact on raft, leading to two classes of compounds. Class I molecules altered the raft affinity of PMP22 and MAL and also reduced raft formation in a protein-dependent manner. Class II molecules modulated raft formation in a protein-independent manner. This suggests independent forces work collectively to stabilize lipid rafts. Both classes of compounds altered membrane fluidity in cells and modulated TRPM8 channel function. These compounds provide new tools for probing lipid raft function in cells and for furthering our understanding of raft biophysics. TeaserCompounds have been discovered that modulate the affinity of membrane proteins for lipid rafts as well as raft formation.

biophysics↗
bioRxiv2024

Transfer RNA acetylation regulates in vivo mammalian stress signaling

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2 phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.

developmental biology↗
bioRxiv2023

A transmission chain linking Mycobacterium ulcerans with Aedes notoscriptus mosquitoes, possums and human Buruli ulcer cases in southeastern Australia

In temperate southeastern Australia over the past two decades there has been a marked progressive increase in human cases of Buruli ulcer, an infection of subcutaneous tissue caused by Mycobacterium ulcerans. Native possums are the major local environmental reservoir of M. ulcerans as they not only develop Buruli lesions but they also shed M. ulcerans in their excreta. However the way humans acquire M. ulcerans from possums has not been determined. Previous case-control studies, insect field surveys and vector competence studies have suggested a role for mosquitoes in M. ulcerans transmission between possums and humans. To explore these links we conducted an extensive, 4-month structured mosquito field survey and four ad hoc field surveys across an area of 350km2 on the Mornington Peninsula, an area endemic for Buruli ulcer to the south of the major metropolitan city of Melbourne. We then compared spatial and temporal patterns of M. ulcerans-positive mosquito occurrence with M. ulcerans-positive possums (established by previous possum excreta surveys) and human Buruli ulcer cases across the region. We used metabarcoding to assess mosquito blood-feeding host preference and to reconstruct M. ulcerans genomes from positive mosquitoes to test epidemiological inferences. We collected 66,325 mosquitoes spanning 26 different species from 180 repeatedly sampled traps over a 4-month period. Culex molestus and Aedes notoscriptus were the dominant species (42% and 35% of trapped mosquitoes, respectively). PCR screening 25% of trapped mosquitoes revealed a significant association between M. ulcerans and Ae. notoscriptus (p<0.0001) with a maximum likelihood estimate (MLE) of 5.88 M. ulcerans positive mosquitoes per 1,000 tested. Using spatial scanning statistics, we also observed significant overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and human Buruli ulcer cases. Metabarcoding analyses of blood-fed Ae. notoscriptus showed individual mosquitoes had fed both on humans and native possums. Enrichment genome sequencing from PCR-positive mosquitoes confirmed shared M. ulcerans genome single-nucleotide polymorphism (SNP) profiles between mosquitoes, possum excreta and clinical human isolates within the same regions. These findings indicate that Ae. notoscriptus likely transmit M. ulcerans in southeastern Australia and highlight mosquito control as a plausible means to control the Buruli ulcer epidemic in our region.

microbiology↗
bioRxiv2023

Evaluation of non-modified wireframe DNA origami for acute toxicity and biodistribution in mice

Wireframe DNA origami can be used to fabricate virus-like particles for a range of biomedical applications, including the delivery of nucleic acid therapeutics. However, the acute toxicity and biodistribution of these wireframe nucleic acid nanoparticles (NANPs) have not previously been characterized in animal models. In the present study, we observed no indications of toxicity in BALB/c mice following therapeutically relevant dosage of unmodified DNA-based NANPs via intravenous administration, based on liver and kidney histology, liver biochemistry, and body weight. Further, the immunotoxicity of these NANPs was minimal, as indicated by blood cell counts and type-I interferon and pro-inflammatory cytokines. In an SJL/J model of autoimmunity, we observed no indications of NANP-mediated DNA-specific antibody response or immune-mediated kidney pathology following the intraperitoneal administration of NANPs. Finally, biodistribution studies revealed that these NANPs accumulate in the liver within one hour, concomitant with substantial renal clearance. Our observations support the continued development of wireframe DNA-based NANPs as next-generation nucleic acid therapeutic delivery platforms.

pharmacology and toxicology↗
bioRxiv2023

Nucleus Accumbens Local Circuit for Cue-Dependent Aversive Learning

Response to threatening environmental stimuli requires detection and encoding of important environmental features that dictate threat. Aversive events are highly salient which promotes associative learning about stimuli that signal this threat. The nucleus accumbens is uniquely positioned to process this salient, aversive information and promote motivated output, through plasticity on the major projection neurons in the brain area. We uncovered a nucleus accumbens core local circuit whereby excitatory plasticity facilitates learning and recall of discrete aversive cues. We demonstrate that putative nucleus accumbens substance P release and long-term excitatory plasticity on dopamine 2 receptor expressing projection neurons is required for cue-dependent fear learning. Additionally, we found fear learning and recall were dependent on distinct projection-neuron subtypes. Our work demonstrates a critical role for Nucleus Accumbens substance P in cue-dependent aversive learning.

neuroscience↗
bioRxiv2020

Glycosylation Limits Forward Trafficking of the Tetraspan Membrane Protein PMP22

Peripheral myelin protein 22 (PMP22) folds and traffics inefficiently, a phenomenon closely related to the mechanisms by which this tetraspan membrane protein causes Charcot-Marie-Tooth disease (CMTD). We report that elimination of N-glycosylation results in a 3-fold increase in the cell surface trafficking of wild type (WT) PMP22 and a 10-fold increase in trafficking of the unstable L16P disease mutant form. Studies of the interactions of PMP22 with oligosaccharyltransferases A and B as well as quantitative proteomic experiments established that critical endoplasmic reticulum (ER) quality control decisions occur earlier in the biogenesis to cell surface trafficking pathway for the L16P mutant than for WT. CRISPR knock-out cell lines for ER proteins calnexin, RER1, and UGGT1 illuminated the role of each protein in glycosylation dependent and independent surface trafficking of WT PMP22, as well as for a series of disease mutants of varying folding stabilities. One Sentence SummaryN-linked glycosylation was seen to dramatically limit the cell surface trafficking of PMP22, with some key quality control factors in PMP22 biogenesis being identified.

cell biology↗
bioRxiv2020

Disease-Linked Super-Trafficking of a Mutant Potassium Channel

Gain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface--"super-trafficking". We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

biophysics↗
bioRxiv2020

Quantifying the acquisition and retention of lumpy skin disease virus by haematophagus insects and the implications for transmission and control.

Lumpy skin disease virus (LSDV), a poxvirus that causes severe disease in cattle, has in the last few years rapidly extended its distribution from Africa and the Middle East into Europe, Russia, and across Asia. LSDV is believed to be primarily spread mechanically by blood-feeding arthropods, however the exact mode of arthropod transmission, the relative ability of different arthropod species to acquire and retain the virus, as well as their comparative importance for LSDV transmission, remain poorly characterised. Since the vector-borne nature of LSDV transmission is believed to have enabled the rapid geographic expansion of this virus, the lack of quantitative evidence on LSDV transmission has impeded effective control of the disease during the current epidemic. Obtaining high quality data on virus transmission by arthropods is challenging, and practical limitations often result in inadequate arthropod numbers or model hosts, limiting the transferability of experimental findings to the natural transmission scenario. We have addressed these limitations in this study. Using a highly representative bovine experimental model of lumpy skin disease we allowed four representative vector species (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans and Culicoides nubeculosus) to blood-feed on LSDV-inoculated cattle in order to examine the acquisition and retention of LSDV by these species in unprecedented detail. We found the probability of LSDV transmission from clinical cattle to vector correlated with disease severity. Subclinical disease was more common than clinical disease in the inoculated cattle, however the probability of vectors acquiring LSDV from subclinical animals was very low. All four potential vector species studied had a similar rate of acquisition of LSDV after feeding on the host, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in the in-vivo transmission experiments subsequently enabled enhanced modelling approaches to determine the basic reproduction number of LSDV in cattle mediated by each of the insect species. This was highest for Stomoxys calcitrans (19.1), C. nubeculosus (7.4), and Ae. aegypti (2.4), surprisingly indicating these three species are all potentially efficient transmitters of LSDV. These results reveal that currently applied LSDV control measures such as stamping out of all cattle on affected premises or insect control measures targeting single species need to be urgently reconsidered. Overall our studies have highlighted that the combination of highly relevant in-vivo experiments and mathematical modelling can be directly applied to devise evidence-based proportionate and targeted control programmes.

microbiology↗
bioRxiv2020

Structural Determinants of Cholesterol Recognition in Helical Integral Membrane Proteins

Cholesterol (CLR) is an integral component of mammalian membranes. It has been shown to modulate membrane dynamics and alter integral membrane protein (IMP) function. However, understanding the molecular mechanisms of these processes is complicated by limited and conflicting structural data: Specifically, in co-crystal structures of CLR-IMP complexes it is difficult to distinguish specific and biologically relevant CLR-IMP interactions from a nonspecific association captured by the crystallization process. The only widely recognized search algorithm for CLR-IMP interaction sites is sequence-based, i.e. searching for the so-called CRAC or CARC motifs. While these motifs are present in numerous IMPs, there is inconclusive evidence to support their necessity or sufficiency for CLR binding. Here we leverage the increasing number of experimental CLR-IMP structures to systematically analyze putative interaction sites based on their spatial arrangement and evolutionary conservation. From this analysis we create three-dimensional representations of general CLR interaction sites that form clusters across multiple IMP classes and classify them as being either specific or nonspecific. Information gleaned from our characterization will eventually enable a structure-based approach for prediction and design of CLR-IMP interaction sites. SIGNIFICANCECLR plays an important role in composition and function of membranes and often surrounds and interacts with IMPs. It is a daunting challenge to disentangle CLRs dual roles as a direct modulator of IMP function through binding or indirect actor as a modulator of membrane plasticity. Only recently studies have delved into characterizing specific CLR-IMP interactions. We build on this previous work by using a combination of structural and evolutionary characteristics to distinguish specific from nonspecific CLR interaction sites. Understanding how CLR interacts with IMPs will underpin future development towards detecting and engineering CLR-IMP interaction sites.

biophysics↗
bioRxiv2019

A novel multidimensional reinforcement task in mice elucidates sex-specific behavioral strategies

BackgroundSex is a critical biological variable in the neuropathology of psychiatric disease, and in many cases, women represent a vulnerable population. It has been hypothesized that sex differences in neuropsychiatric disorders are manifestations of differences in basic reward processing. However, preclinical models often present rewards in isolation, ignoring that ethologically, reward seeking requires the consideration of potential aversive outcomes.\n\nMethodsWe developed a Multidimensional Cue Outcome Action Task (MCOAT) to dissociate motivated action from cue learning and valence. Mice are trained in a series of operant tasks. In phase 1, mice acquire positive and negative reinforcement in the presence of discrete discriminative stimuli. In phase 2, both discriminative stimuli are presented concurrently allowing us to parse innate behavioral strategies based on reward seeking and shock avoidance. Phase 3 is punished responding where a discriminative stimulus predicts that nose-poking for sucrose occurs concurrently with footshock, allowing for the assessment of how positive and negative outcomes are relatively valued.\n\nResultsFemales prioritize avoidance of negative outcomes over seeking positive, while males have the opposite strategy. In cases where rules are uncertain, males and females employ different strategies, with females demonstrating bias for shock avoidance.\n\nConclusionsThe MCOAT has broad utility for neuroscience research where pairing this task with recording and manipulation techniques will allow for the definition of the discrete information encoded within cellular populations. Ultimately, we show that making conclusions from unidimensional data leads to inaccurate generalizations about sex-specific behaviors that do not accurately represent ground truth.

animal behavior and cognition↗
bioRxiv2018

Using Flow Cytometry and Multistage Machine Learning to Discover Label-Free Signatures of Algal Lipid Accumulation

--Most applications of flow cytometry or cell sorting rely on the conjugation of fluorescent dyes to specific biomarkers. However, labeled biomarkers are not always available, they can be costly, and they may disrupt natural cell behavior. Label-free quantification based upon machine learning approaches could help correct these issues, but label replacement strategies can be very difficult to discover when applied labels or other modifications in measurements inadvertently modify intrinsic cell properties. Here we demonstrate a new, but simple approach based upon feature selection and linear regression analyses to integrate statistical information collected from both labeled and unlabeled cell populations and to identify models for accurate label-free single-cell quantification. We verify the methods accuracy to predict lipid content in algal cells (Picochlorum soloecismus) during a nitrogen starvation and lipid accumulation time course. Our general approach is expected to improve label-free single-cell analysis for other organisms or pathways, where biomarkers are inconvenient, expensive, or disruptive to downstream cellular processes.

systems biology↗
bioRxiv2018

Evidence of reduced viremia, pathogenicity and vector competence in a re-emerging European strain of bluetongue virus serotype 8 in sheep

The outbreak of bluetongue virus (BTV) serotype 8 (BTV-8) during 2006-2009 in Europe was the most costly epidemic of the virus in recorded history. In 2015, a BTV-8 strain re-emerged in France which has continued to circulate since then. To examine anecdotal reports of reduced pathogenicity and transmission efficiency, we investigated the infection kinetics of a 2007 UK BTV-8 strain alongside the re-emerging BTV-8 strain isolated from France in 2017. Two groups of eight BTV-naive British mule sheep were inoculated with 5.75 log10TCID50 ml-1 of either BTV-8 strain. BTV RNA was detected by 2 dpi in both groups with peak viremia occurring between 5-9 dpi. A significantly greater amount of BTV RNA was detected in sheep infected with the 2007 strain (6.0-8.8 log10 genome copies mL-1) than the re-emerging BTV-8 strain (2.9-7.9 log10 genome copies mL-1). All infected sheep developed BTV-specific antibodies by 9 dpi. BTV was isolated from 2 dpi to 12 dpi for 2007 BTV-8-inoculated sheep and from 5 to 10 dpi for sheep inoculated with the remerging BTV-8. In Culicoides sonorensis feeding on the sheep over the period 7-12 dpi, vector competence was significantly higher for the 2007 strain than the re-emerging strain. Both the proportion of animals showing moderate (as opposed to mild or no) clinical disease (6/8 vs 1/8) and the overall clinical scores (median 5.25 vs 3) were significantly higher in sheep infected with the 2007 strain, compared to those infected with the re-emerging strain. However, one sheep infected with the re-emerging strain was euthanized at 16 dpi having developed severe lameness. This highlights the potential of the re-emerging BTV-8 to still cause illness in naive ruminants with concurrent costs to the livestock industry.\n\nSummaryThe re-emerging Bluetongue virus serotype 8 still presents a threat to naive ruminants in Europe despite reduced virulence

epidemiology↗
bioRxiv2018

Visualizing APP trafficking and processing reveals its association with neuronal membrane cholesterol

The amyloid precursor protein (APP) has been extensively studied because of its association with Alzheimers disease (AD). However, APP distribution across different subcellular membrane compartments and its function in neurons remains unclear. We generated an APP fusion protein with a pH-sensitive green fluorescent protein at its ectodomain and a pH-insensitive blue fluorescent protein at its cytosolic domain and used it to measure APPs distribution, subcellular trafficking and cleavage in live neurons. This reporter, closely resembling endogenous APP, revealed only a limited correlation between synaptic activities and APP trafficking. However, the synaptic surface distribution of APP was inversely correlated to membrane cholesterol levels, a phenomenon that involves APPs cholesterol-binding site. Mutations within this site not only altered surface APP and cholesterol levels in a dominant negative manner, but also increased synaptic vulnerability to moderate membrane cholesterol reduction. Our results reveal reciprocal modulation of APP and membrane cholesterol levels at synaptic boutons.

neuroscience↗