Biology subjects

Mills, M.

Publications and source records attributed to Mills, M..

15 recordsLinked to original sources

bioRxiv2025

Molecular dynamics simulations reveal DNA gate opening mechanisms for M. smegmatis topoisomerase 1A

Type 1A topoisomerases relax torsional strain in DNA via a strand passage mechanism in which a protein mediated DNA gate must open during the enzymes catalytic cycle. This gate-open conformational state of the enzyme has been challenging to observe via experimental methods. In this study, we first used equilibrium Molecular Dynamics simulations to probe the structural properties of the gate closed state for the DNA-free apo system and a system with a ssDNA bound at the DNA binding site. For both systems, we followed the equilibrium simulations with Umbrella Sampling simulations. Umbrella sampling allowed us to bias the protein to adopt a gate-open state to study the properties of this conformation, as well as the pathways leading to it. We observed that several electrostatic interactions contribute to the closed-state stability of the protein which were broken during the gate opening. The gate opening comprised of three major domain motions that were determined from simulation trajectories and Principal Component Analysis. Finally, umbrella sampling results combined with the Weighted Histogram Analysis Method allowed us to reconstruct the free energy profiles of gate opening for all simulations. SIGNIFICANCEMulti-drug resistant bacterial strains necessitate newer ways of targeting bacteria. Type 1A topoisomerases are potential anti-bacterial drug targets. Current strategies are to either block the protein binding to DNA or prevent the cut DNA being religated during the catalytic cycle. A possible alternative to these methods would be to trap the protein at an open conformation. Structural information about the transient open state is crucial to this approach. Results of this study provide mechanistic details of gate opening. This information furthers the understanding of the topoisomerase catalytic cycle which will aid in anti-topoisomerase drug design efforts.

biophysics↗

bioRxiv2024

Mycobacterial Topoisomerase IA Energetically Suffers From C-terminal Deletions

Type IA topoisomerases relieve torsional stress in DNA by a strand-passage mechanism, using the strain in the DNA to drive relaxation. The topoisomerase IAs of the Mycobacterium genus have distinct C-terminal domains which are crucial for successful strand-passage. We used single-molecule magnetic tweezers to observe supercoil relaxation by wild type Mycobacterium smegmatis topoisomerase IA and two C-terminal truncation mutants. We recorded distinct behaviors from each truncation mutant. We calculated the free energy stored in the DNA as it is twisted under force to examine the differences between the proteins. Based on our results, we propose a modified model of the strand-passage cycle.

biophysics↗

bioRxiv2024

Forecasting the adoption and spread of a community-based marine management initiative using agent-based models

While many successful initiatives for conserving nature exist, efforts to take them to scale have been inadequate. Moreover, conservation science currently lacks a systematic methodology for determining if or when interventions will reach effective scales and how programmatic decisions will affect the scaling process. This paper presents a modelling framework that aims to address both issues by operationalizing Diffusion of Innovations theory and local knowledge using agent-based modelling and Bayesian inference. By applying our framework to existing data on the spatiotemporal adoption of a community-based marine management initiative in Fiji, we demonstrate that our approach can identify the mechanisms that govern the observed adoption patterns. In this case, the relative advantage of the intervention, village social networks, and perceived knowledge stand out as important drivers of adoption. Using the identified causal processes, our approach can forecast business-as-usual and counterfactual future scenarios and hence inform conservation policy. Finally, we highlight the importance of spatiotemporal data for making detailed scaling predictions. We structure the paper as a step-by-step guide, highlighting our modelling decisions and possible limitations. Thus, besides presenting a case study, this paper serves as a template for practitioners and researchers to better model the scaling process of other conservation interventions.

ecology↗

bioRxiv2023

Characterization of human XPD helicase activity with Single Molecule Magnetic Tweezers

XPD helicase is a DNA unwinding enzyme involved in multiple cellular processes. As part of TFIIH, XPD opens a repair bubble in DNA for access by proteins in the nucleotide excision repair pathway. XPD uses the energy from ATP hydrolysis to translocate in the 5 to 3 direction on one strand of duplex DNA, displacing the opposite strand in the process. We used magnetic tweezers assays to measure the double-stranded DNA (dsDNA) unwinding and single-stranded DNA (ssDNA) translocation activities of human XPD by itself. In our experimental setup, hXPD exhibits low unwinding processivity of ~14 bp and slow overall unwinding rate of ~0.3 bp/s. Individual unwinding and translocation events were composed of fast and slow runs and pauses. Analysis of these events gave similar mean run sizes and rates for unwinding and translocation, suggesting that unwinding is a reflection of translocation. The analysis also revealed that hXPD spent similar time stalling and unwinding. hXPD translocated on ssDNA at a similar overall rate as that of unwinding, pointing to an active helicase. However, we observed modest effects of DNA sequence on stalling and unwinding initiation position. Considering the slow unwinding rate, high probability of base pair separation at the ssDNA/dsDNA fork, and the observed DNA sequence dependences, we propose that hXPD is most likely a partially active helicase. Our results provide detailed information on the basal activity of hXPD which enhances our mechanistic understanding of hXPD activity. SIGNIFICANCEHuman XPD helicase is a major component of the general transcription factor TFIIH. TFIIH is essential in both transcription and nucleotide excision repair. Mutations in hXPD are associated with cancers and autosomal recessive disorders. Here we directly measured the dsDNA unwinding and ssDNA translocation of human XPD helicase by itself. Our measurements provide detailed information on the basal activity of human XPD, which enhance our mechanistic understanding of the activity of XPD in the cell, provide a basis for better understanding of the clinical phenotypes, and aid in drug design targeting hXPD related diseases.

biophysics↗

bioRxiv2022

Global loss of fine-scale chromatin architecture and rebalancing of gene expression during early colorectal cancer development

Although 3D genome architecture can be essential for gene regulation, the biological implications of long-range chromatin interactions in disease remain elusive. In this study, we traced the early evolution and malignant transformation of colorectal cancer by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth from polyps of Familial Adenomatous Polyposis (FAP) patients. Our analysis reveals a substantial progressive loss of genome-wide cis-regulatory connectivity at early stages of malignancy, which correlates with a non-linear effect on gene regulation. Genes with high promoter-enhancer (P-E) connectivity in unaffected mucosa are not correlated with elevated baseline expression, but instead tend to be up-regulated at advanced stages. Inhibition of highly connected promoters preferentially represses gene expression in colorectal cancer cells relative to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P-E connectivity from a redundant state to a rate-limiting one for transcriptional levels. Overall, our study illuminates the intricate interplay between 3D genome architecture and gene regulation during early colorectal cancer progression, and provides valuable insights for potential therapeutic interventions targeting the connectivity of cis-regulatory elements.

genomics↗

bioRxiv2022

MmCMS: Mouse models' Consensus Molecular Subtypes of colorectal cancer

BACKGROUNDColorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODSUsing transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n=577) and mouse (n=57 across n=8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTSWe developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONSWhen applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package with three options helps researchers select suitable mouse models of human CRC subtype for their experimental testing.

cancer biology↗

bioRxiv2021

Nanopore tweezers measurements of RecQ conformational changes reveal the energy landscape of helicase motion

Helicases are essential for nearly all nucleic acid processes across the tree of life. Using Nanopore Tweezers we observed the small, fast steps taken by single RecQ helicases as they step along and unwind DNA at ultrahigh spatiotemporal resolution. By directly measuring conformational substates of RecQ we determine the coupling between helicase domain motions and chemical reactions that together produce forward motion along the DNA. Application of assisting and opposing forces shows that RecQ has a highly asymmetric energy landscape that reduces its sensitivity to opposing mechanical forces that could be encountered in vivo by molecular roadblocks such as DNA bound proteins. This energy landscape enables RecQ to maintain speed against an opposing load.

biophysics↗

bioRxiv2021

Echoes of the late Pleistocene in a novel trophic cascade between cougars and feral donkeys

Introduced large herbivores have partly filled ecological gaps formed in the late Pleistocene, when many of the Earths megafauna were driven extinct. However, surviving predators are widely considered unable to influence introduced megafauna, leading them to exert unusually strong herbivory and disturbance-related effects. We report on a behaviorally-mediated trophic cascade between cougars (Puma concolor) and feral donkeys (Equus africanus asinus) at desert wetlands in North America. In response to predation of juveniles, donkeys shifted from nocturnal to almost exclusively diurnal, thereby avoiding peaks in cougar activity. Furthermore, donkeys reduced the time they spent at desert wetlands by 87%: from 5.5 hours a day to 0.7 hours at sites with predation. These shifts in activity were associated with increased activity and richness of other mammal species and reduced disturbance and herbivory-related effects on these ecologically-distinct wetland ecosystems, including 49% fewer trails, 35% less trampled bare ground, and 227% more canopy cover. Cougar predation on introduced donkeys rewires an ancient food web, with diverse implications for modern ecosystems.

ecology↗

bioRxiv2021

Single-cell analyses reveal a continuum of cell state and composition changes in the malignant transformation of polyps to colorectal cancer

To chart cell composition and cell state changes that occur during the transformation of healthy colon to precancerous adenomas to colorectal cancer (CRC), we generated 451,886 single-cell chromatin accessibility profiles and 208,557 single-cell transcriptomes from 48 polyps, 27 normal tissues, and 6 CRCs collected from patients with and without germline APC mutations. A large fraction of polyp and CRC cells exhibit a stem-like phenotype, and we define a continuum of epigenetic and transcriptional changes occurring in these stem-like cells as they progress from normal to CRC. Advanced polyps contain increasing numbers of stem-like cells, regulatory T-cells, and a subtype of FOX-regulated pre-cancer associated fibroblasts. In the cancerous state, we observe T-cell exhaustion, RUNX1-regulated cancer associated fibroblasts, and increasing accessibility associated with HNF4A motifs in epithelia. Methylation changes in sporadic CRC are strongly anti-correlated with accessibility changes along this continuum, further identifying regulatory markers for molecular staging of polyps.

genomics↗

bioRxiv2020

Genome-wide analysis identifies genetic effects on reproductive success and ongoing natural selection at the FADS locus.

Identifying genetic determinants of reproductive success may highlight mechanisms underlying fertility and also identify alleles under present-day selection. Using data in 785,604 individuals of European ancestry, we identify 43 genomic loci associated with either number of children ever born (NEB) or childlessness. These loci span diverse aspects of reproductive biology across the life course, including puberty timing, age at first birth, sex hormone regulation and age at menopause. Missense alleles in ARHGAP27 were associated with increased NEB but reduced reproductive lifespan, suggesting a trade-off between reproductive ageing and intensity. As NEB is one component of evolutionary fitness, our identified associations indicate loci under present-day natural selection. Accordingly, we find that NEB-increasing alleles have increased in frequency over the past two generations. Furthermore, integration with data from ancient selection scans identifies a unique example of an allele--FADS1/2 gene locus--that has been under selection for thousands of years and remains under selection today. Collectively, our findings demonstrate that diverse biological mechanisms contribute to reproductive success, implicating both neuro-endocrine and behavioural influences.

genetics↗

bioRxiv2020

Disconnects in global discourses - the unintended consequences of marine mammal protection on small-scale fishers

Globally, the populations of many marine mammals remain of critical concern after centuries of exploitation and hunting. However, some marine mammal populations (e.g. pinnipeds) have largely recovered from exploitation, and interactions between these species and fisheries--particularly small-scale fisheries--is once again of concern globally. The large scope and widespread scale of interactions highlights the local disconnect between two global policies: marine mammal conservation and small-scale fisheries protection. In this research, we explore these conflicting global policies by assessing the perceptions of coastal small-scale fishers in Peru and Chile regarding their interactions with pinnipeds, including the South American sea lion (Otaria flavescens) and South American fur seal (Arctocephalus australis). We surveyed 301 gill net fishers and assess perceptions using a best-worst scaling methodology. We find that fishers are chiefly concerned with the increase in pinniped populations, perceive that their interactions with pinnipeds have significantly increased over the past 80 years, and report pinniped-driven catch and income losses [≥] 26 per cent. Surprisingly, fishers do not believe that compensation schemes will resolve this issue--instead they overwhelmingly call for pinniped population culls. The reported number of pinnipeds illegally killed by fishers suggests the potential for large negative impacts on these protected species, and a loss of legitimacy in marine regulation. Collectively, our results portray a sense of marginalisation from fishers--that global policy treats them as less "important" than marine mammals. Our results highlight the increasing disconnect in global policy, which on one hand seeks to protect threatened marine mammal populations, and on the other seeks to promote the welfare of small-scale fishers.

ecology↗

bioRxiv2019

Using a residency index to estimate the economic value of saltmarsh provisioning services for commercially important fish species

Every year, 100 hectares of saltmarsh in the United Kingdom are lost due to sea level rise. The remaining areas are threatened by land conversion, agricultural activities, and climate change. There are important economic consequences to saltmarsh loss, as saltmarsh provides valuable ecosystem services including flood protection, carbon sequestration, and nursery habitat for commercially fished species. Quantifying the economic value of these ecosystem services can help target policies for saltmarsh restoration, or managed realignment, of new saltmarsh areas. In this study, we quantify the economic value of saltmarsh as a habitat for commercially fished species by developing a residency index. The residency index weights the relative importance of saltmarsh along a species lifecycle by explicitly incorporating the target species life histories and the estimated proportion of time it spends in saltmarsh at juvenile and adult life stages. Using this index, we estimate the value of saltmarsh to UK commercial fisheries landings. We find that UK saltmarsh contributes annually between 16.7% and 18.2% of total UK commercial landings for European seabass (Dicentrarchus labrax), European plaice (Pleuronectes platessa), and Common sole (Solea solea). Our findings highlight the importance of saltmarsh protection and restoration. Furthermore, our approach provides a general framework that integrates population ecology methods and economic analyses to assess the value of saltmarsh and other coastal habitats for fisheries worldwide.

ecology↗

bioRxiv2018

Wound-induced polyploidization is driven by Myc and supports tissue repair in the presence of DNA damage

Tissue repair requires either polyploid cell growth or cell division, but the molecular mechanism promoting polyploidy and limiting proliferation remains poorly understood. Here we find that injury to the adult Drosophila epithelium causes cells to enter the endocycle through the activation of Yorkie dependent genes (myc, e2f1, or cycE). Myc is even sufficient to induce the endocycle in the post-mitotic epithelium. As result, epithelial cells enter S phase but mitosis is blocked by inhibition of mitotic gene expression. The mitotic cell cycle program can be activated by simultaneously expressing the mitotic activator, Stg, while genetically depleting fzr. However, forcing cells to undergo mitosis is detrimental to wound repair as the adult fly epithelium accumulates DNA damage and mitotic errors ensue when cells are forced to proliferate. In conclusion, we find that wound-induced polyploidization enables tissue repair when cell division is not a viable option.

cell biology↗

bioRxiv2018

Direct Observation of Topoisomerase IA Gate Dynamics

Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

biophysics↗

bioRxiv2017

A suite of transgenic driver and reporter mouse lines with enhanced brain cell type targeting and functionality

Modern genetic approaches are powerful in providing access to diverse types of neurons within the mammalian brain and greatly facilitating the study of their function. We here report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

neuroscience↗