Biology subjects

Maxwell, A.

Publications and source records attributed to Maxwell, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Development and characterization of a miniaturized focused ultrasound extraction (FUSE) system to enable rapid, point-of-care genetic testing

Point-of-care (POC) molecular detection has enabled rapid and accessible testing at or near the site of need, facilitating timely decision-making in clinical, field, and industrial settings. Despite these advancements, molecular detection platforms remain limited in their ability to prepare DNA from complex tissue matrices, restricting their applicability to a narrow range of sample types. To address sample preparation inefficiencies, focused ultrasound extraction (FUSE) has been developed for the rapid release of DNA from robust tissue matrices. FUSE disintegrates target tissue and releases DNA by delivering high pressure ultrasound pulses that generate a cavitation bubble cloud. In prior work, FUSE was performed using large, laboratory-based equipment. Here, a miniaturized FUSE device was designed and tested to enable POC DNA sample preparation. Design constraints were defined based on prior FUSE experiments, and based on these metrics, a 750 kHz cylindrical transducer was fabricated. The pressure output was measured, and high-speed optical imaging demonstrated that the transducer generated sustained cavitation within the defined focal region. The feasibility of DNA extraction from piscine and timber tissues was evaluated, and results show that the device can release DNA with quality suitable for PCR-based detection from both tissue types. These results demonstrate the potential of this device to rapidly extract DNA from complex samples and expand the applicability of molecular detection platforms.

bioengineering↗

bioRxiv2026

DNA gyrase in live bacteria forms liquid condensates through weak multivalent bonding of excess GyrB

Type IIA bacterial topoisomerase DNA gyrase, a GyrA/GyrB heterotetramer, has crucial roles maintaining transcription and DNA replication by relaxing positive DNA supercoils through introducing negative supercoils. However, rates of gyrase-catalysed supercoiling in vitro cannot explain much higher rates required in vivo. To address this puzzle, we used high-speed single-molecule fluorescence imaging of GyrA/GyrB reporters in live Escherichia coli, indicating that cells contain [~]40% more GyrB than GyrA expressed in a diffuse pool or in clusters whose mobility depends on whether they are bound to DNA. Unexpectedly, we discovered that clusters are non-stoichiometric containing [~]150% more GyrB than GyrA, significantly greater than the cellular average, with fluorescence recovery after photobleaching revealing that clustered GyrA and GyrB behave as a liquid whose abundance can be increased by applying gyrase-targeting antibiotics. Structural docking indicates that the liquid state is stabilised through excess GyrB progressively binding to existing clusters via weak, multivalent interactions. By operating in liquid condensates, A2B2 that dissociates from DNA can rebind rapidly instead of diffusing away, increasing enzyme processivity to enable multiple rounds of catalysis that can keep pace with transcription and DNA replication in vivo. This demonstrates a new role for condensates of overcoming kinetic limitations imposed by diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/744598v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1514d4aorg.highwire.dtl.DTLVardef@a95ceeorg.highwire.dtl.DTLVardef@11417daorg.highwire.dtl.DTLVardef@2a2856_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Single-molecule insights into DNA gyrase in live bacteria

Molecular motors travelling along DNA introduce positive supercoils that present as barriers to replication leading to genome instability. To counter these, bacterial cells express DNA gyrase, a topoisomerase that introduces negative supercoils. While much is known about DNA gyrase from genetic and in vitro biochemical studies, the spatiotemporal dynamics of this enzyme remain a mystery. Only recently have we been able to observe the in vivo spatiotemporal dynamics down to single molecule level using advanced super-resolution microscopy techniques. We used Slimfield microscopy, a cutting-edge molecule microscopy technique to address the gap in our knowledge. We analysed a dual fluorescently labelled Escherichia coli strain expressing the replisome marker DnaN-mCherry along with mYPet-GyrB as the enzyme marker. We performed sequential Slimfield microscopy of the labelled proteins from the same strain and analysed in vivo GyrB dynamics in live E. coli cells in relation to the replisome. We find that the majority of replisomes are associated with GyrB. Inhibition of gyrase activity reduces the proportion of replisomes associated with GyrB. Interestingly, GyrB behaviour is distinct from that observed for GyrA in a previous study. Our results reveal the previously unknown dynamics of GyrB inside living bacterial cells highlighting the advantages of in vivo single molecule investigations. Our findings also demonstrate the importance of analysing all subunits of a functional enzyme complex to gain comprehensive understanding of its in vivo mechanisms. This study demonstrates the utility of single-molecule super-resolved microscopy as a valuable underpinning technology to understand in vivo behaviour of biomedically important molecules. Our insights will help impact discovery and development of novel antibiotics that interfere with gyrase function, thus contributing to tackling the growing problem of antimicrobial resistance.

biophysics↗

bioRxiv2024

Terrestrial 3D Laser Scanning for Ecosystem and Fire Effects Monitoring

Long-term terrestrial ecosystem monitoring is a critical component of documenting outcomes of land management actions, assessing progress towards management objectives, and guiding realistic long-term ecological goals, all through repeated observation and measurement. Traditional monitoring methods have evolved for specific applications in forestry, ecology, and fire and fuels management. While successful monitoring programs have clear goals, trained expertise, and rigorous sampling protocols, new advances in technology and data management can help overcome the most common pitfalls in data quality and repeatability. This paper presents Terrestrial Laser Scanning (TLS), a specific form of LiDAR (Light Detection and Ranging), as an emerging sampling method that can complement and enhance existing monitoring methods. TLS captures in high resolution the 3D structure of a terrestrial ecosystem (forest, grassland, etc.), and is increasingly efficient and affordable (<$30,000). Integrating TLS into ecosystem monitoring can standardize data collection, improve efficiency, and reduce bias and error. Streamlined data processing pipelines can rigorously analyze TLS data and incorporate constant improvements to inform management decisions and planning. The approach described in this paper utilizes portable, push-button TLS equipment that, when calibrated with initial transect sampling, captures detailed forestry, fuels, and ecological features in less than 5 minutes per plot. We also introduce an interagency automated processing pipeline and dashboard viewer for instant, user-friendly analysis, and data retrieval of hundreds of metrics. Forest metrics and inventories produced with these methods offer effective decision-support data for managers to quantify landscape-scale conditions and respond with efficient action. This protocol further supports interagency compatibility for efficient natural resource monitoring across jurisdictional boundaries with uniform data, language, methods, and data analysis. With continued improvement of scanner capabilities and affordability, these data will shape the future of terrestrial ecosystem monitoring as an important means to address the increasingly fast pace of ecological change facing natural resource managers.

ecology↗

bioRxiv2023

An ex vivo model of interactions between extracellular vesicles and peripheral mononuclear blood cells in whole blood

Extracellular vesicles (EVs) can be loaded with therapeutic cargo and engineered for retention by specific body sites; therefore, they have great potential for targeted delivery of biomolecules to treat diseases. However, the pharmacokinetics and biodistribution of EVs in large animals remain relatively unknown, especially in primates. We recently reported that when cell culture-derived EVs are administered intravenously to Macaca nemestrina (pig-tailed macaques), they differentially associate with specific subsets of peripheral blood mononuclear cells (PBMCs). More than 60% of CD20+ B cells were observed to associate with EVs for up to 1 hr post-intravenous administration. To investigate these associations further, we developed an ex vivo model of whole blood collected from healthy pig-tailed macaques. Using this ex vivo system, we found that labeled EVs preferentially associate with B cells in whole blood at levels similar to those detected in vivo. This study demonstrates that ex vivo blood can be used to study EV-blood cell interactions.

immunology↗

bioRxiv2023

An inverse agonist of orphan receptor GPR61 reveals a novel allosteric mechanism

GPR61 is a biogenic amine receptor-related orphan GPCR associated with phenotypes relating to appetite and thus, is of interest as a druggable target to treat disorders of metabolism and body weight, such as obesity and cachexia. To date, lack of structural information or a known biological ligand or tool compound has hindered comprehensive efforts to study its structure and function. Here, we report the first ever structural characterization of GPR61, in both its active-like complex with heterotrimeric G protein and in its inactive state. Moreover, we report the discovery of a potent and selective small-molecule inverse agonist against GPR61 and structural elucidation of its unprecedented allosteric site and mode of action. These findings offer key mechanistic insights into an orphan GPCR, while providing both a new structural framework and tool compound to support further studies of GPR61 function and modulation.

biophysics↗

bioRxiv2023

Terrestrial laser scan metrics predict surface vegetation biomass and consumption in a frequently burned southeastern U.S. ecosystem

Fire-prone landscapes found throughout the world are increasingly managed with prescribed fire for a variety of objectives. These frequent low-intensity fires directly impact lower forest strata, and thus estimating surface fuels or understory vegetation is essential for planning, evaluating, and monitoring management strategies and studying fire behavior and effects. Traditional fuel estimation methods have applications for stand-level and canopy fuel loading; however, local-scale understory biomass remains challenging because of complex within-stand heterogeneity and fast recovery post-fire. Previous studies have demonstrated how single location terrestrial laser scanning (TLS) can be used to estimate plot-level vegetation characteristics and impacts from prescribed fire. To build upon this methodology, co-located single TLS scans and physical biomass measurements were used to generate linear models for predicting understory vegetation and fuel biomass as well as consumption by fire in a southeastern U.S. pineland. A variable selection method was used to select the six most important TLS-derived structural metrics for each linear model, where model fit ranged in R2 from 0.61 to 0.74. This study highlights a prospect for efficiently estimating vegetation and fuel characteristics relevant to prescribed burning via the integration of a single-scan TLS method adaptable by managers and relevant for coupled fire-atmosphere models.

ecology↗

bioRxiv2022

Rapid, DNA-induced subunit exchange by DNA gyrase

DNA gyrase, a ubiquitous bacterial enzyme, is a type IIA topoisomerase formed by heterotetramerisation of 2 GyrA subunits and 2 GyrB subunits, to form the active complex. GyrA is usually found as a dimer in solution, whereas GyrB can exist as a monomer. DNA gyrase is able to loop DNA around the C-terminal domains (CTDs) of GyrA and pass one DNA duplex through a transient double-strand break (DSB) established in another duplex. This results in the conversion of a positive loop into a negative one, thereby introducing negative supercoiling into the bacterial genome, an activity essential for DNA replication and transcription. The strong protein interface in the GyrA dimer must be broken to allow passage of the transported DNA segment and it is generally assumed that the interface is usually stable and only opens when DNA is transported, to prevent the introduction of deleterious DSBs in the genome. In this paper we show that DNA gyrase can exchange its DNA-cleaving interfaces between two active heterotetramers. This so-called interface "swapping" or "exchange" (IS) can occur within a few minutes in solution. We also show that bending of DNA by gyrase is essential for cleavage but not for DNA binding per se and favors IS. interface swapping is also favored by DNA wrapping and an excess of GyrB. We suggest that proximity, promoted by GyrB oligomerization and binding and wrapping along a length of DNA, between two heterotetramers favors rapid interface exchange. This exchange does not require ATP, can occur in the presence of fluoroquinolones, and raises the possibility of non-homologous recombination solely through gyrase activity. The ability of gyrase to undergo interface swapping also explains how gyrase heterodimers, containing a single active-site tyrosine, can carry out double-strand passage reactions and therefore suggests an alternative explanation to the recently proposed "swivelling" mechanism for DNA gyrase (Gubaev, Weidlich, and Klostermeier 2016).

biochemistry↗

bioRxiv2022

DNA Release from Complex Plant Tissue Using Focused Ultrasound Extraction (FUSE)

Sample preparation in genomics is a critical step that is often overlooked in molecular workflows and impacts the success of downstream genetic applications. This study explores the use of a recently developed focused ultrasound extraction (FUSE) technique to enable the rapid release of DNA from plant tissues for genetic analysis. FUSE generates a dense acoustic cavitation bubble cloud that pulverizes targeted tissue into acellular debris. This technique was applied to leaf samples of American chestnut (Castanea dentata), tulip poplar (Liriodendron tulipifera), red maple (Acer rubrum), and chestnut oak (Quercus montana). We observed that FUSE can extract high quantities of DNA in 9-15 minutes, compared to the 30 minutes required for conventional DNA extraction. FUSE extracted DNA quantities of 24.33 {+/-} 6.51 ng/mg and 35.32 {+/-} 9.21 ng/mg from American chestnut and red maple, respectively, while conventional methods yielded 6.22 {+/-} 0.87 ng/mg and 11.51 {+/-} 1.95 ng/mg, respectively. The quality of the DNA released by FUSE allowed for successful amplification and next-generation sequencing. These results indicate that FUSE can improve DNA extraction efficiency for leaf tissues. Continued development of this technology aims to adapt to field-deployable systems to increase the cataloging of genetic biodiversity, particularly in low-resource biodiversity hotspots.

genetics↗

bioRxiv2021

FAN1 nuclease activity affects CAG expansion and age at onset of Huntington's disease

The age at onset of motor symptoms in Huntingtons disease (HD) is driven by HTT CAG repeat length but modified by other genes. We used exome sequencing of 683 HD patients with extremes of onset or phenotype relative to CAG length to identify rare variants associated with clinical effect. We identified damaging coding variants in candidate modifier genes from prior genome-wide association studies associated with altered HD onset or severity. Variants in FAN1 clustered in its DNA-binding and nuclease domains and were associated predominantly with earlier onset HD. Nuclease activities of these variants correlated with residual age at motor onset of HD. Mutating endogenous FAN1 to a nuclease-inactive form in an induced pluripotent stem cell model of HD led to rates of CAG expansion comparable to those observed with complete FAN1 knock out. Together, these data implicate FAN1 nuclease activity in slowing somatic repeat expansion and hence onset of HD.

neuroscience↗

bioRxiv2021

Topoisomerase VI is a chirally-selective, preferential DNA decatenase

DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates, DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a dramatic increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90{degrees}.

biochemistry↗

bioRxiv2021

Essential Role of Placenta Derived Interferon Stimulated Gene 20 Against ZIKA Virus Infection

Zika virus is a positive-sense single-stranded RNA virus, which can be transmitted across the placenta and leads to adverse effects on fetal development during pregnancy. The severity of these complications highlights the importance of prevention and treatment. However, no vaccines or drugs are currently available. In this study, we characterized the IFN{beta}-mediated antiviral response in trophoblast cells in order to identify critical components that are necessary for the successful control of viral replication; and determined whether we could use the components of the IFN-induced response as a replacement therapy for ZIKA viral infection during pregnancy. We identified and characterized interferon stimulated gene 20 (ISG20), playing a central role in controlling Zika infection in trophoblast cells, and successfully established a recombinant ISG20-Fc protein that effectively decrease viral titers in vitro and in vivo by maintaining its exonuclease activity and displaying immune modulatory functions. Therefore, rISG20-Fc is a promising anti-viral/immune modulatory approach for the control and prevention of RNA viral infections such as ZIKA virus.

immunology↗

bioRxiv2020

CM1-driven assembly and activation of Yeast γ-Tubulin Small Complex underlies microtubule nucleation

Microtubule (MT) nucleation is regulated by the {gamma}-tubulin ring complex ({gamma}TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, {gamma}TuRC is composed of seven identical {gamma}-tubulin small complex ({gamma}TuSC) sub-assemblies which associate helically to template microtubule growth. {gamma}TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here we combine cross-linking mass spectrometry (XL-MS), X-ray crystallography and cryo-EM structures of both monomeric and dimeric {gamma}TuSCs, and open and closed helical {gamma}TuRC assemblies in complex with Spc110p to elucidate the mechanisms of {gamma}TuRC assembly. {gamma}TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent {gamma}TuSCs. By providing the highest resolution and most complete views of any {gamma}TuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human {gamma}TuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan {gamma}TuRC.

biochemistry↗

bioRxiv2020

Treating Porcine Abscesses With Histotripsy: A Pilot Study

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections, and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical wash-out when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity, for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

bioengineering↗

bioRxiv2020

Syringeal vocal folds do not have a voice in zebra finch vocal development

Vocal behaviour can be dramatically changed by both neural circuit development and postnatal maturation of the body. During song learning in songbirds, both the song system and syringeal muscles are functionally changing, but it is unknown if maturation of sound generators within the syrinx contributes to vocal development. Here we densely sample the respiratory pressure control space of the zebra finch syrinx in vitro. We show that the syrinx produces sound very efficiently and that key acoustic parameters, minimal fundamental frequency, entropy and source level, do not change over development in both sexes. Thus, our data suggests that the observed acoustic changes in vocal development must be attributed to changes in the motor control pathway, from song system circuitry to muscle force, and not by material property changes in the avian analog of the vocal folds. We propose that in songbirds, muscle use and training driven by the sexually dimorphic song system are the crucial drivers that lead to sexual dimorphism of the syringeal skeleton and musculature. The size and properties of the instrument are thus not changing, while its player is.

neuroscience↗

bioRxiv2020

Does participation in acoustic experiments provide enrichment to animals under human care? A case study of three grey seals (Halichoerus grypus)

Both mental and physiological conditions determine the well-being state in an animal. Enrichment is a way to increase an animals well-being and may require problem solving through thinking, tolerance of ambiguity, openness, and intrinsic motivation. It is unclear if it is enriching when an animal participates in different types of research. Therefore, it is important to answer the question of whether research can be used as an enrichment tool in zoological facilities. Here, we examine if participation in psychophysical research affected the mental stimulation of three grey seals under human care. The effects varied amongst the three individuals that took part in the research, and indicated that their participation in the research task was dependent on their individual personalities and life history. Two seals indicated that their involvement in the research was positive and motivating, and therefore can be considered enriching. In comparison, the third seal displayed a tendency for frustration and low motivation. Our results indicate that research can be a powerful enrichment tool with animals that find research motivating.

animal behavior and cognition↗

bioRxiv2020

One-to-one innervation of vocal muscles allows precise control of birdsong

The motor control resolution of any animal behavior is limited to the minimal force step available when activating muscles, which is set by the number and size distribution of motor units (MUs) and muscle specific force [1, 2]. Birdsong is an excellent model system for understanding sequence learning of complex fine motor skills [3], but we know surprisingly little how the motor pool controlling the syrinx is organized [4] and how MU recruitment drives changes in vocal output [5]. Here we combine measurements of syringeal muscle innervation ratios with muscle stress and an in vitro syrinx preparation to estimate MU size distribution and the control resolution of fundamental frequency (fo), a key vocal parameter, in zebra finches. We show that syringeal muscles have extremely small MUs, with 50% innervating [≤] 3, and 13 - 17% innervating a single muscle fiber. Combined with the lowest specific stress (5 mN/mm2) known to skeletal vertebrate muscle, small force steps by the major fo controlling muscle provide control of 50 mHz to 4.2 Hz steps per MU. We show that the song system has the highest motor control resolution possible in the vertebrate nervous system and suggest this evolved due to strong selection on fine gradation of vocal output. Furthermore, we propose that high-resolution motor control was a key feature contributing to the radiation of songbirds that allowed diversification of song and speciation by vocal space expansion.

neuroscience↗

bioRxiv2019

Combining high-resolution AFM with MD simulations shows that DNA supercoiling induces kinks and defects that enhance flexibility and recognition.

In the cell, DNA is arranged into highly-organised and topologically-constrained (supercoiled) structures. It remains unclear how this supercoiling affects the detailed double-helical structure of DNA, largely because of limitations in spatial resolution of the available biophysical tools. Here, we overcome these limitations, by a combination of atomic force microscopy (AFM) and atomistic molecular dynamics (MD) simulations, to resolve structures of negatively-supercoiled DNA minicircles at base-pair resolution. We observe that negative superhelical stress induces local variation in the canonical B-form DNA structure by introducing kinks and defects that affect global minicircle structure and flexibility. We probe how these local and global conformational changes affect DNA interactions through the binding of triplex-forming oligonucleotides to DNA minicircles. We show that the energetics of triplex formation is governed by a delicate balance between electrostatics and bonding interactions. Our results provide mechanistic insight into how DNA supercoiling can affect molecular recognition, that may have broader implications for DNA interactions with other molecular species.

biophysics↗