Biology subjects

Smith, V.

Publications and source records attributed to Smith, V..

15 recordsLinked to original sources

bioRxiv2026

A Humanized Mouse Model of DRPLA Recapitulates Epilepsy and Demonstrates Antisense Oligonucleotide-Mediated Disease Modification

Dentatorubral-pallidoluysian atrophy (DRPLA) is an ultra-rare progressive myoclonus epilepsy caused by a glutamine-coding CAG repeat expansion in atrophin-1 (ATN1). Mutant ATN1 (mATN1) expression results in symptoms that inversely correlate to CAG-expansion size: young-onset cases have the most CAG repeats and developmental delay, high-frequency treatment-resistant seizures, and early mortality. We sought to characterize the seizure susceptibility of a fully humanized ATN1 mouse expressing human mATN1 with 112 CAG repeats to then assess therapeutic antisense oligonucleotide (ASO) intervention on seizure-related outcomes. Atn1Q112/+ or wild-type (WT) mice were treated with investigational ASO targeting human, CAG-expanded, ATN1 or saline (P2 and then again at 4 weeks-old) before acute seizure testing, ataxia profiling, and chronic in vivo video-EEG recording for spontaneous seizures up to 8 weeks-old. Saline-treated Atn1Q112/+ mice were more seizure prone with early death. However, ASO administration to Atn1Q112/+ mice normalized seizure threshold, decreased ataxia, and protected against early death. Atn1Q112/+ mice exhibited spontaneous behavioral seizures recorded over 2 weeks, which were absent in ASO-treated Atn1Q112/+ and WT mice, with a specific mATN1 expression-driven decrease in delta and theta EEG frequencies. Atn1Q112/+ mice are highly seizure prone, validating this model for DRPLA epilepsy. ATN1 represents a novel epilepsy-related risk factor for therapeutic investigation.

neuroscience↗

bioRxiv2026

Variable zinc concentrations among commercially available Mueller-Hinton Broth brands affects SIR interpretations during broth microdilution in vitro antimicrobial susceptibility testing with the novel metallo-beta-lactamase inhibitor APC148

The well-known discrepancy between the in vitro and in vivo efficacy of {beta}-lactam antibiotics in metallo-{beta}-lactamase (MBL) containing Gram-negative bacteria has during recent years been found to be at least partially explained by zinc levels at infection sites being much lower than those found in conventional cation-adjusted Mueller-Hinton Broth (caMHB) media used for in vitro susceptibility testing. Previous studies have also demonstrated that a high variability exists with respect to zinc content in caMHB from different manufacturers, potentially leading to differences in SIR interpretations for {beta}-lactam antibiotics with MBL-carrying isolates, depending on the brand of caMHB used for testing. APC148 is a zinc-chelating compound acting as an inhibitor of MBL enzymes and is currently undergoing phase one in clinical trials. In this study, ten clinical isolates of Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii harbouring MBLs (NDM, n = 6; VIM, n = 4; IMP, n = 1) were tested in a broth microdilution assay with meropenem and APC148, employing caMHB of various brands. One K. pneumoniae strain carrying only a serine-{beta}-lactamase (KPC-2) was included as control. Antimicrobial susceptibility testing (AST) by broth microdilution was performed according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). MICs of meropenem alone and in combination with inhibitors were tested in four cation-adjusted Mueller Hinton II (caMHB).The four caMHBs used were analysed by ICP-MS/MS and found to have highly vaiable zinc content (in the range 0.4 to 1.9 {micro}g/mL, corresponding to 5.6 to 29.4 {micro}M). At 16{micro}g/ml APC148 lowered the MIC in nearly all strains and in caMHBs from all manufacturers. At lower concentrations of APC148 (4 or 8 {micro}g/mL), this MIC reduction could however only be retained when the zinc concentration in the broth was low, indicating that higher concentration of inhibitor is needed during in vitro Mic testing when using caMHB from certain manufacturers. The present work clearly shows that not taking the zinc concentration of the caMHB used into consideration when estimating MIC performance of compounds functioning through interactions with zinc may be a considerable source of error, and specifically when investigating potential inhibitors of metallo-{beta}-lactamase (MBL) enzymes. The present work supports the call for standardising zinc content in caMHB to be used for this purpose, to ensure that MIC results for drug combinations involving the use of zinc-chelating compounds are consistent and reproducible across laboratories.

microbiology↗

bioRxiv2026

Refined USP25/28 inhibitors with improved selectivity towards c-Myc driven squamous lung cancer cells

The ubiquitin specific protease 28 (USP28) is implicated in tumorigenesis by controlling the turnover of substrates including the oncogene c-MYC and the ubiquitin ligase FBW7. Here, we describe small molecule inhibitors of USP25 and USP28, leading to cancer cell cycle arrest and death. However, genetic deletion of USP25/28 does not replicate this effect. An integrated -omics approach revealed off-target effects for thienopyridine and thienopyrazine carboxamide compounds in protein translation. Chemoproteomics and biochemical analyses suggested binding of such compounds to a region near the ribosome complex polypeptide exit tunnel. Structural analysis of a USP28-inhibitor complex enabled the design of modified USP25/28 inhibitor molecules which minimized translation-related off-target effects. In distinction to earlier compounds, the optimized inhibitors were non-toxic to breast cancer cells yet retained potent anti-proliferative activity in squamous lung carcinoma cells, where USP28 is associated with disease progression. Together, our results demonstrate that refined USP25/28 inhibitors can selectively suppress tumor growth by targeting c-MYC driven pathways, offering a more precise therapeutic strategy for treating squamous lung cancers whilst minimizing undesired cytotoxicity.

cancer biology↗

bioRxiv2026

In vitro activity of combination formulations of the novel metallo-β-lactamase (MBL) inhibitor APC148 with comparator treatments against 176 MBL-containing Enterobacterales isolates from the SENTRY Antimicrobial Surveillance Program (2019-2022)

The global dissemination of Enterobacterales producing both metallo-{beta}-lactamases (MBLs) and serine {beta}-lactamases (SBLs) represents a critical threat to modern medicine, as no currently marketed antibiotics effectively target MBL-mediated resistance. APC148 is a novel, selective zinc-chelating MBL inhibitor designed to restore {beta}-lactam activity in MBL positive isolates, when used in combination with a broad-spectrum carbapenem. In this study, we evaluated the in vitro efficacy of APC148 in triple combinations with either meropenem-avibactam (APC301) or cefepime-avibactam (APC302) against a diverse global collection (JMI collection) of 176 MBL- and SBL-producing Enterobacterales isolates (including NDM, VIM, and IMP variants). Using broth microdilution, the triple combinations were compared against several newly approved and late-stage pipeline antibiotic products. Both APC301 and APC302 demonstrated superior potency, achieving a MIC90 of 0.12 {micro}g/mL. When applying CLSI breakpoint interpretive criteria for the parent {beta}-lactams, 99.4% of the MBL and SBL-containing isolates were susceptible to APC301, while 97.2% were susceptible to APC302. These results indicate that the addition of a selective MBL inhibitor to an SBL-inhibitor/{beta}-lactam antibiotic effectively bypasses complex co-existing {beta}-lactam resistance mechanisms in multidrug-resistant (MDR) pathogens. Given that MDR Enterobacterales frequently harbor multiple {beta}-lactamase classes simultaneously, these triple combinations constitute a highly promising clinical strategy to address the therapeutic void in MBL-mediated resistance

microbiology↗

bioRxiv2025

Hypoxia-induced extracellular matrix changes are conserved in cancer and directly impact radiotherapy benefit

Our overarching aim was to determine how hypoxia affects the extracellular matrix (ECM). Transcriptomic analysis (21,941 patients; 10 cancer types) identified ECM remodelling as the predominant pathway affected by hypoxia. Multi-omics confirmed that hypoxia impacts ECM organisation and collagen degradation; 53 ECM genes were affected, of which 74% were HIF1/HIF2-regulated. Spatial transcriptomics highlighted different hypoxia remodelling processes in tumour and stroma. Five ECM genes commonly affected in tumour and in vitro constituted a signature. This signature was independently prognostic and independently predictive of radiotherapy benefit in multiple malignancies. Patients within either high or low hypoxic-ECM score tertiles benefited from radiotherapy versus surgery. Hypoxic ECMs generated in vitro increased adhesion and decreased migration of cancer cells, an effect enhanced by irradiation. Immunofluorescence demonstrated that hypoxia decreased collagen fibre number, and irradiation decreased cell-ECM interactions. Taken together, these findings demonstrate hypoxia induces pan-cancer ECM changes, directly impacting radiotherapy benefit.

cancer biology↗

bioRxiv2024

Noise Reduction and Localization Accuracy in a Mobile Magnetoencephalography System

Magnetoencephalography (MEG) non-invasively provides important information about human brain electrophysiology. The growing use of optically pumped magnetometers (OPM) for MEG, as opposed to fixed arrays of cryogenic sensors, has opened the door for innovation in system design and use cases. For example, cryogenic MEG systems are housed in large shielded rooms to provide sufficient space for the system dewar. Here, we investigate the performance of OPM recordings inside of a cylindrical shield with a 1x2 m2 footprint. The efficacy of shielding was measured in terms of field attenuation and isotropy, and the value of post hoc noise reduction algorithms was also investigated. Localization accuracy was quantified for 104 OPM sensors mounted on a fixed helmet array based on simulations and recordings from a bespoke current dipole phantom. Passive shielding attenuated direct current (DC) and power line (60 Hz) fields by approximately 60 dB, and all other frequencies by 80-90 dB. Post hoc noise reduction provided an additional 5-15 dB attenuation. Substantial field anisotropy remained in the volume encompassing the sensor array. The consistency of the anisotropy over months suggests that a field nulling solution could be readily applied. A current dipole phantom generating source activity at an appropriate magnitude for the human brain generated field fluctuations on the order of 0.5-1 pT. Phantom signals were localized with 3 mm localization accuracy and no significant bias in localization was observed, which is in line with performance for cryogenic and OPM MEG systems. This validation of the performance of a small footprint MEG system opens the door for lower cost MEG installations in terms of raw materials and facility space, as well as mobile imaging systems (e.g., truck-based). Such implementations are relevant for global adoption of MEG outside of highly resourced research and clinical institutions.

biophysics↗

bioRxiv2023

The staphylococcal type VII secretion system impacts daptomycin sensitivity through controlling bacterial cell envelope integrity

The human pathogen Staphylococcus aureus encodes a specialised type VII secretion system (T7SS), which plays an important role in bacterial virulence during infection. However, the functions the T7SS during infection and in bacterial physiology remain unclear. Here we demonstrate that S. aureus strains lacking the the T7SS effector EsxC ({Delta}esxC) was highly sensitive to the important last resort drug, daptomycin, as well as other membrane-targeting antibiotics, including gramicidin and bithionol. To understand how EsxC mediates increased antibiotic sensitivity, we investigated its functions in the staphylococcal cell envelope. Scanning electron microscopy analysis of an esxC mutant revealed a distinct cell surface morphology. Interestingly, {Delta}esxC displayed a decrease in membrane fluidity, altered membrane protein profiles and altered cell wall synthesis. The esxC mutant demonstrated enhanced daptomycin binding which correlated with the increased negative charge of mutant membranes. Calcium ions, which can bind membranes affecting charge, impacted growth of {Delta}esxC and sensitivity to daptomycin, suggesting that EsxC may modulate calcium binding to membranes. Furthermore, the esxC mutant displayed a heightened susceptibility to daptomycin during intracellular infection, and in a murine skin infection model. Thus, our data show that the T7SS effector EsxC impacts sensitivity of S. aureus to membrane-acting drugs such as daptomycin through modulation of cell membrane integrity, indicating its potential as a drug target. Author SummaryT7SS has a range of functions in bacteria including specific roles in bacterial physiology including DNA uptake, membrane integrity and bacterial development. In S. aureus T7SS has been shown to be critical for bacterial virulence, intra-species competition and in host cell interactions, although their functions in bacterial physiology are not clear. Here we report a role of the staphylococcal T7SS effector EsxC in the modulation of the cell membrane and surface integrity, which impacts the activity of membrane targeting drugs like daptomycin. Our data indicate that targeting this system could potentially enhance activity of existing therapeutic agents.

microbiology↗

bioRxiv2023

Genomic differences and species delimitation: a case for two species in the zoonotic cestode Dipylidium caninum

Dipylidium caninum (Linnaeus, 1758) is a common zoonotic cestode of dogs and cats worldwide. Previous studies have demonstrated the existence of largely host associated canine and feline genotypes based on infection studies, genetic differences at the nuclear 28S rDNA gene and complete mitochondrial genomes. There have been no comparative studies at a genome-wide scale. Here, we sequenced the genomes of a dog and cat isolate of Dipylidium caninum from the United States using the Illumina platform and conducted comparative analyses with the reference draft genome. Complete mitochondrial genomes were used to confirm the genotypes of the isolates. D. caninum canine and feline genomes generated in this study had mean coverage depths of 45x and 26x and an average identity of 98% and 89% respectively when compared to the reference genome. SNPs were 20 times higher in the feline isolate. Comparison and species delimitation using universally conserved orthologs and protein coding mitochondrial genes revealed that the canine and feline isolates are different species. Data from this study builds a base for future integrative taxonomy. Further genomic studies from geographically diverse populations are necessary to understand implications for taxonomy, epidemiology, veterinary clinical medicine, and anthelmintic resistance.

microbiology↗

bioRxiv2022

Genetic characterization of the zoonotic parasite Ancylostoma caninum in the central and eastern USA

Ancylostoma caninum is the most common nematode parasite of dogs in the United States. This study aimed to describe the molecular epidemiology of A. caninum isolates from the central and eastern states of the U.S. using the partial mitochondrial cox1 gene and to compare them with those reported globally. We isolated eggs from fecal samples of dogs and characterized each isolate based on cox1 sequences. A total of 60 samples originating from Kansas, Iowa, New York, Florida, and Massachusetts were included. 25 haplotypes were identified in the U.S. dataset with high haplotype diversity (0.904). Sequence data were compared to sequences from other world regions available in GenBank. Global haplotype analysis demonstrated 35 haplotypes with a haplotype diversity of 0.931. Phylogenetic and network analysis provide evidence for the existence of moderate geographical structuring of A. caninum haplotypes. Our results provide an updated summary of A. caninum haplotypes and data for neutral genetic markers with utility for tracking hookworm populations. Sequences have been deposited in GenBank (ON980650 - ON980674). Further studies of isolates from other regions are essential to understand the genetic diversity of this parasite.

microbiology↗

bioRxiv2021

LungMAP Portal Ecosystem: Systems-Level Exploration of the Lung

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular, imaging and pathological datasets. To centralize and standardize information across broad lung research efforts we expanded the LungMAP.net website into a gateway portal. This portal connects a broad-spectrum of research networks, bulk and single-cell multi-omics data and a diverse collection of image data that span mammalian lung development and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, mouse), to enable consistent queries across technologies, cohorts, age, disease and drug treatment. These atlases are provided as independent and integrated queriable datasets, with an emphasis on dynamic visualization, figure generation and reference-based classification of user-provided datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, data portals and datasets from LungMAP and external research efforts.

systems biology↗

bioRxiv2021

Fluid intelligence and naturalistic task impairments after focal brain lesions

Classical executive tasks, such as Wisconsin card-sorting and verbal fluency, are widely used as tests of frontal lobe control functions. Since the pioneering work of Shallice and Burgess (1991), it has been known that complex, naturalistic tasks can capture deficits that are missed in these classical tests. Matching this finding, deficits in several classical tasks are predicted by loss of fluid intelligence, linked to damage in a specific cortical "multiple-demand" (MD) network, while deficits in a more naturalistic task are not. To expand on these previous results, we examined the effect of focal brain lesions on three new tests - a modification of the previously-used Hotel task, a new test of task switching after extended delays, and a test of decision-making in imagined real-life scenarios. As potential predictors of impairment we measured volume of damage to a priori MD and default mode (DMN) networks, as well as cortical damage outside these networks. Deficits in the three new tasks were substantial, but were not explained by loss of fluid intelligence, or by volume of damage to either MD or DMN networks. Instead, deficits were associated with diverse lesions, and not strongly correlated with one another. The results confirm that naturalistic tasks capture cognitive deficits beyond those measured by fluid intelligence. We suggest, however, that these deficits may not arise from specific control operations required by complex behaviour. Instead, like everyday activities, complex tasks combine a rich variety of interacting cognitive components, bringing many opportunities for processing to be disturbed.

neuroscience↗

bioRxiv2020

MogR is a ubiquitous transcriptional repressor affecting motility, biofilm formation and virulence in the Bacillus cereus group

Flagellar motility is considered an important virulence factor in different pathogenic bacteria. In Listeria monocytogenes the transcriptional repressor MogR regulates motility in a temperature-dependent manner, directly repressing flagellar- and chemotaxis genes. The only other bacteria known to carry a mogR homolog are members of the Bacillus cereus group, which includes motile species such as B. cereus and Bacillus thuringiensis as well as the non-motile species Bacillus anthracis, Bacillus mycoides and Bacillus pseudomycoides. Furthermore, the main motility locus in B. cereus group bacteria, carrying the genes for flagellar synthesis, appears to be more closely related to L. monocytogenes than to Bacillus subtilis, which belongs to a separate phylogenetic group of Bacilli and does not carry a mogR ortholog. Here, we show that in B. thuringiensis, MogR overexpression results in non-motile cells devoid of flagella. Global gene expression profiling showed that 110 genes were differentially regulated by MogR overexpression, including flagellar motility genes, but also genes associated with virulence, stress response and biofilm lifestyle. Accordingly, phenotypic assays showed that MogR also affects cytotoxicity and biofilm formation in B. thuringiensis. Overexpression of a MogR variant mutated in two amino acids within the putative DNA binding domain restored phenotypes to those of an empty vector control. In accordance, introduction of these mutations resulted in complete loss in MogR binding to its candidate flagellar locus target site in vitro. In contrast to L. monocytogenes, MogR appears to be regulated in a growth-phase dependent and temperature-independent manner in B. thuringiensis 407. Interestingly, mogR was found to be conserved also in non-motile B. cereus group species such as B. mycoides and B. pseudomycoides, which both carry major gene deletions in the flagellar motility locus and where in B. pseudomycoides mogR is the only gene retained. Furthermore, mogR is expressed in non-motile B. anthracis. Altogether this provides indications of an expanded set of functions for MogR in B. cereus group species, beyond motility regulation. In conclusion, MogR constitutes a novel B. thuringiensis pleiotropic transcriptional regulator, acting as a repressor of motility genes, and affecting the expression of a variety of additional genes involved in biofilm formation and virulence.

microbiology↗

bioRxiv2020

The deubiquitylase USP9X controls ribosomal stalling

When a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

cell biology↗

bioRxiv2019

Allosteric regulation of glycogen phosphorylase solution phase structural dynamics at high spatial resolution

Glycogen phosphorylase (GlyP) was the first allosteric enzyme to be described. Yet, the precise dynamic changes in solution phase structure and stability that underpin functional regulation have remained elusive. We have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. This enabled measurements of the solution phase local structural dynamics involved in allosteric regulation of GlyP. Here, we quantify GlyP structural dynamics in solution, describing correlated changes in structure in the activated (pSer14) and inhibited (glucose-6-phosphate bound) forms of the enzyme. The sensitivity of these measurements discerned that the 250s loop is natively disordered in the apo T-state, adopting a more ordered conformation in the active state. The quantitative change in stability of the 280s loop is identified, providing the first direct evidence of the entropic switch that sterically regulates substrate access to the active site.\n\nSignificance StatementWe have developed a new fully-automated and highly flexible implementation of hydrogen/deuterium-exchange mass spectrometry, operating in the millisecond regime. Measurements of glycogen phosphorylase quantify the solution phase stability of local structure at near-amino acid structural resolution and with no appreciable lower limit of stability. This uncovered the highly-resolved local alterations in stability which provides direct evidence of the entropic mechanism by which access to the active site is gated by the 280s loop.\n\nFootnotesAuthor contributions: M.K., V.S., S.S., N.L., F.V., N.B., L.C. and J.J.P. designed research; M.K., V.S., S.S., L.C. and J.J.P. performed research; M.K., V.S., S.S., L.C. and J.J.P. analyzed data; and M.K. and J.J.P. wrote the manuscript.

biophysics↗

bioRxiv2018

Role of the default mode network in cognitive transitions

A frequently repeated finding is that the default mode network (DMN) shows activation decreases during externally-focused tasks. This finding has led to an emphasis in DMN research on internally-focused self-relevant thought processes. A recent study, in contrast, implicates the DMN in substantial externally-focused task switches. Using functional magnetic resonance imaging, we scanned 24 participants performing a task switch experiment. Whilst replicating previous DMN task switch effects, we also found large DMN increases for brief rests as well as task restarts after rest. Our findings are difficult to explain using theories strictly linked to internal or self-directed cognition. In line with principal results from the literature, we suggest that the DMN encodes scene, episode or context, by integrating spatial, self-referential and temporal information. Context representations are strong at rest, but re-reference to context also occurs at major cognitive transitions.

neuroscience↗