Biology subjects

Lewis, O.

Publications and source records attributed to Lewis, O..

14 recordsLinked to original sources

bioRxiv2026

QuadCleave: Enzymatic cleavage and analysis of DNA G-quadruplexes.

Prediction of G-quadruplexes (G4), noncanonical DNA secondary structures containing guanine tetrads, can have profound relevance across cancer, neurodegenerative and other genetic diseases, but require experimental validation by instrument intensive methods including CD spectroscopy, fluorescence, NMR, or crystallography. We report a novel, inexpensive, efficient and scalable 2-enzyme system for confirmation of G4 formation and copy number repeat count. First, QuadCleave, in which Mismatch Endonuclease I was used to repeatably, site-specifically and selectively cleave oligodeoxynucleotides containing exclusively G4-forming sequences. Second, HaeIII enzyme identified G4-forming sequences that can also generate GG/CC base pairs, and a sequences cleavage by both established it in C9ORF72, the most mutated gene in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These findings establish ubiquitously accessible experimental validation of G4 forming sequences and their copy number repeat assessment, with specificity to C9ORF72 oligodeoxynucleotides whose repeat expansion has critical clinical implications in a subpopulation of ALS and FTD patients.

molecular biology↗

bioRxiv2026

A novel qPCR assay to detect the presence of Anopheles gambiae complex mosquitoes

BackgroundAnopheles mosquitoes vector pathogens responsible for more than 600,000 human deaths annually. Ecological studies of these insects are important to guide effective vector-control campaigns and to understand their broader ecological consequences. Molecular ecology methods, particularly qPCR, provide a valuable tool in such studies. By detecting trace DNA of a taxon of interest within mixed or environmental samples, qPCR can facilitate identification of prey taxa of interest in the diets of consumers. However, no protocol for the detection of An. gambiae complex mosquitoes in dietary samples has been available. MethodsWe introduce a new set of qPCR primers (Agam_CO1_F1 and Agam_CO1_R1) and a probe-based assay for detection of Anopheles gambiae-complex mosquitoes, even with short reads common in dietary and environmental samples. The primers were tested in vitro for their specificity and sensitivity, and in silico using Primer-BLAST to assess potential off-target amplification. ResultsThe qPCR primers amplified An. gambiae DNA even at low starting concentrations (5 copies {micro}l-1). The primers did not amplify any non-target DNA in either the in vitro or in silico tests, but consistently amplified An. gambiae complex DNA. The primers can therefore provide reliable tests for the presence or absence of An. gambiae complex in dietary or eDNA samples. ConclusionsThe new qPCR primers should allow advances in research into mosquito ecology by allowing detection of even trace amounts of An. gambiae DNA in dietary and environmental samples. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/707393v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1fcce3corg.highwire.dtl.DTLVardef@47e5f5org.highwire.dtl.DTLVardef@4a7063org.highwire.dtl.DTLVardef@1188d60_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. To gain further insight into how the ubiquitin system regulates the DDR, we performed a CRISPR-Cas9 knockout screen focused on E3 ligases and related proteins with the DNA topoisomerase I inhibitor, camptothecin. This uncovered the CTLH ubiquitin E3 ligase complex -- and particularly one of its core subunits, MAEA -- as a critical regulator of the cellular response to single-ended DNA double-strand breaks (seDSBs) and replication stress. In tandem, we identified patients with variants in MAEA who present with neurodevelopmental deficits including global developmental delay, dysmorphic facial features, brain abnormalities, intellectual disability, and abnormal movement. Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart as a likely cause of disease. We propose that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which compromises genome integrity and cell division during development.

genetics↗

bioRxiv2025

Thermally activated irreversible homogenization of G-quadruplexes in an ALS/FTD-associated nucleotide expansion

A significant proportion of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases exhibit a substantial copy number expansion of the hexanucleotide GGGGCC/GGCCCC sequence in the C9ORF72 gene. The GGGGCC sequence forms a non-canonical DNA structure called a G-quadruplex (G4) which has been associated with the disease states and with nucleic acid condensate formation. G4s can fold into various topologies, which can differentially impact fidelity of DNA synthesis. However, how G4 conformational heterogeneity and its regulation impact hexanucleotide repeat expansion is unclear, and important clues may lie in the thermodynamic properties of different G4 topologies. Here, we use temperature-swept CD spectroscopy to observe configurational homogenization of an initially heterogeneous population of G4s over a small range of temperatures, demonstrating thermally activated behavior. We further show that this reaction is irreversible, since subsequent temperature sweeps do not show CD shifts from non-parallel to parallel G4 topologies. Finally, we provide an analytical theory based on a two-state thermodynamic model which is compatible with experimental evidence, and we discuss alternate mechanisms for the homogenization transition. These findings suggest that kinetic regulation of non-canonical DNA structures may play a role in cellular homeostasis or disease pathogenesis. SIGNIFICANCEThe GGGGCC repeats in the C9ORF72 gene expand in copy number in certain neurodegenerative diseases, forming non-canonical DNA structures called G-quadruplexes (G4) which are associated with the pathological state. However, why the repeat expansion occurs is not known, and a key may lie in the thermodynamic stability of certain G4 conformations. Here, we use CD spectroscopy to experimentally report thermally activated heat-induced G4 conformation homogenization, from a heterogeneous population to the parallel configuration. We derive an analytical biophysical theory which is compatible with this experimental observation, which is shown to be irreversible. Our in vitro tuning of the free energy landscape that modulates G4 conformational fidelity motivates a search for possible in vivo enzymatic regulators.

biophysics↗

bioRxiv2024

Multiple factors regulate i-motif and G-quadruplex structures in vitro: analysis of repeated and non-repeated polyG/polyC clusters by circular dichroism

The B-form of DNA in the genome contains thousands of sequences that can form various noncanonical structures. Of particular interest are two structures namely G-quadruplex (G4), formed by two or more stacks of four guanine residues in a plane, and intercalating-motif (i-motif, iM) formed by alternately arranged C-C+ pairs. Circular dichroism (CD) spectroscopy is a fast biophysical technique to analyze G4s and iMs. We conducted a CD analysis of two types of DNA sequences, one containing tandem repeats and one without, for the generation of G4s and iMs under various environmental conditions, which include pH, buffer composition, boiling, with flanking sequences, complimentary DNA strands, and single-stranded DNA binding protein (SSB). Changes in pH and boiling caused drastic variations in the CD spectra of DNA containing tandem repeats of GGGGCC and GGCCCC from the C9ORF72 gene, although some changes in G4/iM-forming DNA from promoter-proximal regions of several oncogenes also occur. An increase in the number of hexanucleotide repeats generated complex CD patterns at specific pH due to the presence of both G and C bases. The presence of flanking sequences affects CD pattern of a mixture of G4- and iM-forming sequences of the c-MYC promoter-proximal region. SSB disassembled G4 and iMs of all sequences suggesting an in vivo role for SSBs in disassembly of G4s and iMs during various DNA transactions.

biophysics↗

bioRxiv2024

Temporal variability in host availability alters the outcome of competition between two parasitoid species

O_LIVariability in the availability of resources through time is a common attribute in trophic interactions, but its effects on the fitness of different consumer species and on interspecific competition between them are not clearly understood. C_LIO_LITo investigate this, we allowed two parasitoid species, Trichopria drosophilae and Pachycrepoideus vindemiae, to exploit Drosophila host pupae under three temporal variability treatments, either on their own or simultaneously. C_LIO_LIWhen tested individually (in the absence of interspecific competition), both parasitoid species had lower fitness when hosts were exposed for a short duration at high density than when exposed for a long duration at low density. When both parasitoid species exploited hosts simultaneously, interspecific competition significantly decreased the number of offspring for both parasitoid species. The outcome of this interspecific competition depended on host temporal variability, with T. drosophilae or P. vindemiae dominating in short and long host exposure treatments, respectively. C_LIO_LIThese results can be explained by the combination of host availability and egg load of female adult parasitoids. When abundant hosts are provided for a short period, the ample mature eggs of the proovigenic T. drosophilae enable them to exploit hosts more efficiently than P. vindemiae, which is synovigenic. However, P. vindemiae is an intrinsically superior competitor and dominates when multiparasitism occurs. Multiparasitism is more frequent when hosts are at low levels relative to the egg load of the parasitoids. C_LIO_LIOur results clearly demonstrate that resource temporal availability can alter the outcome of competition between consumers with different reproductive traits. C_LI

ecology↗

bioRxiv2023

ExoChew: An exonuclease technique to generate single-stranded DNA libraries

Although DNA in the genome is double-stranded, single-stranded DNA is generated during various processes including DNA replication and repair. Some single-stranded DNAs can form noncanonical structures. Various proteins bind to the single-stranded DNAs site-specifically and/or structure-specifically to regulate various DNA transactions. Because of the transient nature of single-stranded DNAs in the cell, current in vivo techniques may not reveal all such sequences, structures, and protein-DNA complexes. To explore such sequences and structures genome-wide, it is necessary to generate single-stranded DNA libraries. Current in vitro methods involve heat denaturation of libraries of double-stranded DNA fragments followed by cooling to prevent reannealing; however, a significant amount of DNA can reanneal to regenerate double-stranded DNAs. In ExoChew method, double-stranded DNA fragment libraries are enzymatically converted to single-stranded DNA libraries. Genomic DNA is sonicated to generate pools of double-stranded DNA fragments of required size. Each pool of double-stranded DNA fragments is then treated with either T7 exonuclease or E. coli exonuclease III. which recognize and cleave double-stranded DNA from 5 ends or 3 ends, generating single-stranded DNA pools, respectively. The enzymatically generated single-stranded DNA pools can be used for genome-wide studies of protein-DNA interactions and structural studies of DNA.

molecular biology↗

bioRxiv2023

Experimental heatwaves and warming cause distinctive community responses through their interactions with a novel species

As mean temperatures increase and heatwaves become more frequent, species are expanding their distributions to colonise new habitats. The resulting novel species interactions will simultaneously shape the temperature-driven reorganization of resident communities. The interactive effects of climate change and climate change-facilitated invasion have rarely been studied in multi-trophic communities, and are likely to differ depending on the nature of the climatic driver (i.e. climate extremes or constant warming). We recreated under laboratory conditions a host-parasitoid community typical of high-elevation rainforest sites in Queensland, Australia, comprising four Drosophila species and two associated parasitoid species. We subjected these communities to climate change in the form of either heatwaves or constant warming, in combination with an invasion treatment involving a novel host species from lower-elevation habitats. The two parasitoid species were sensitive to both warming and heatwaves, while the demographic responses of Drosophila species were highly idiosyncratic, reflecting the combined effects of thermal tolerance, parasitism, competition, and facilitation. After multiple generations, heatwaves (but not constant warming) promoted the establishment of low-elevation species in upland communities. The introduction of this invading species correlated negatively with the abundance of one of the parasitoid species, leading to cascading effects on its hosts and their competitors. Our study, therefore, reveals differing, sometimes contrasting, impacts of extreme temperatures and constant warming on community composition. It also highlights how the scale and direction of climate impacts could be further modified by range-expanding species within a bi-trophic community network.

ecology↗

bioRxiv2022

Factors determining distributions of rainforest Drosophila shift from interspecific competition to high temperature with decreasing elevation

Species turnover with elevation is a widespread phenomenon and provides valuable information on why and how ecological communities might reorganize as the climate warms. Tropical mountains typically have pronounced thermal gradients and intense species interactions, providing a testing ground for investigating the relationship between thermal tolerances and biotic interactions as the proximate factors influencing species distributions. We investigated temperature and interspecific competition as causes of species turnover and abundance changes of the nine most abundant species of Drosophila along elevational gradients in the Australian Wet Tropics. Thermal performance curves revealed that species distributions were better explained by their performance at extreme temperatures, rather than their thermal optima. Upper thermal limits varied less among species than lower thermal limits. Nonetheless, these small differences were associated with differences in centred elevation of distribution, consistent with environmental sorting as a driver of community composition at low-elevation sites. In contrast, community composition at cool, high elevations was driven by temperature-dependent interspecific competition rather than tolerance to low temperatures. These results run counter to common assumptions about the role of abiotic and biotic factors in structuring communities along thermal gradients, and indicate that tropical insects may be highly vulnerable to future warming. Our study illustrates the importance of experimental, quantitative tests across biological levels (i.e., individuals to populations) and temporal scales (i.e., within-generation to multi-generation) for characterizing effects of climate on a guild of closely-interacting species.

ecology↗

bioRxiv2021

DROP: Molecular voucher database for identification of Drosophila parasitoids

Molecular identification is increasingly used to speed up biodiversity surveys and laboratory experiments. However, many groups of organisms cannot be reliably identified using standard databases such as GenBank or BOLD due to lack of sequenced voucher specimens identified by experts. Sometimes a large number of sequences are available, but with too many errors to allow identification. Here we address this problem for parasitoids of Drosophila by introducing a curated open-access molecular reference database, DROP (Drosophila parasitoids). Identifying Drosophila parasitoids is challenging and poses a major impediment to realize the full potential of this model system in studies ranging from molecular mechanisms to food webs, and in biological control of Drosophila suzukii. In DROP (http://doi.org/10.5281/zenodo.4519656), genetic data are linked to voucher specimens and, where possible, the voucher specimens are identified by taxonomists and vetted through direct comparison with primary type material. To initiate DROP, we curated 154 laboratory strains, 856 vouchers, 554 DNA sequences, 16 genomes, 14 transcriptomes, and 6 proteomes drawn from a total of 183 operational taxonomic units (OTUs): 114 described Drosophila parasitoid species and 69 provisional species. We found species richness of Drosophila parasitoids to be heavily underestimated and provide an updated taxonomic catalogue for the community. DROP offers accurate molecular identification and improves cross-referencing between individual studies that we hope will catalyze research on this diverse and fascinating model system. Our effort should also serve as an example for researchers facing similar molecular identification problems in other groups of organisms.

genomics↗

bioRxiv2020

Altered parasitism of a butterfly assemblage associated with a range-expanding species

AimBiotic interactions are an important factor structuring ecological communities but data scarcity limits our understanding of the impact of their response to climate and land use changes on communities. We studied the impact of a change in species assemblage on biotic interactions in a community of closely-related butterflies. Specifically, we examined the impact of the recent range expansion of Araschnia levana on the resident species, with a particular focus on natural enemies, parasitoids, shared with other butterfly species in the assemblage. LocationSweden. Time periodTwo years (2017-2018). Major taxa studiedNettle-feeding butterflies (Aglais urticae, Aglais io, Araschnia levana, and Vanessa atalanta) and their parasitoids. MethodsWe assessed parasitism in 6777 butterfly larvae sampled in the field from 19 sites distributed along a 500 km latitudinal gradient, and every two weeks throughout species reproductive seasons. We identified the parasitoid complex of each butterfly species and their overlap, and analysed how parasitism rates were affected by species assemblage, variations in abundance, time, and the arrival of A. levana. ResultsParasitoids caused high mortality, with substantial overlap across the four host species. The composition of the host community influenced parasitism rates and this effect was specific to each species. In particular, the rate of parasitism in resident species was comparatively higher at sites where A. levana has been established for longer. Main conclusionsParasitoid pressure is a significant source of mortality in the nettle-feeding butterfly community studied. Variations in butterfly species assemblages are associated with substantial variations in rates of parasitism. This is likely to affect the population dynamics of their butterfly host species, and, potentially, the larger number of species with which they interact.

ecology↗

bioRxiv2019

Finding missing links in interaction networks

Documenting which species interact within ecological communities is challenging and labour-intensive. As a result, many interactions remain unrecorded, potentially distorting our understanding of network structure and dynamics. We test the utility of four structural models and a new coverage-deficit model for predicting missing links in both simulated and empirical bipartite networks. We find they can perform well, but that the predictive power of structural models varies with the underlying network structure. Predictions can be improved by ensembling multiple models. Sample-coverage estimators of the number of missed interactions are highly correlated with the number of missed interactions, but strongly biased towards underestimating the true number of missing links. Augmenting observed networks with most-likely missing links improves estimates of qualitative network metrics. Tools to identify likely missing links can be simple to implement, allowing the prioritisation of research effort and more robust assessment of network properties.

ecology↗

bioRxiv2017

Designing oil palm landscapes to retain biodiversity using insights from a key ecological indicator group

Oil palm expansion threatens biodiverse ecosystems across the tropics. However, palm oil is a widely used and profitable crop, so identifying strategies that mitigate the impact of oil palm expansion on biodiversity is important. Riparian reserves (strips of forest along rivers) are protected in many countries for hydrological reasons and also support species that would not otherwise persist in oil palm. However, management guidelines for riparian zones have been informed by relatively few ecological studies. We assessed how the structural features and landscape context of riparian reserves in Sabah, Malaysia affected dung beetle communities. We also tested the use of flight intercept traps to study movement of dung beetles along linear forest corridors. Overall, dung beetle abundance in riparian reserves was 54% lower than in logged forest areas, but all species observed in the logged forest were found in at least one riparian reserve site and both species richness and diversity increased with reserve width. Distance from a large block of continuous forest affected dung beetle community composition but not species richness, abundance, or functional diversity. The amount of forest cover in the surrounding landscape improved the retention of species within riparian reserves, and increases in vegetation complexity corresponded with higher functional richness and functional dispersion. The flight intercept traps did not indicate that there is net movement of individuals out of logged forest areas into the riparian reserves. The species richness of 30 m reserves (the suggested requirement of reserves in Sabah) was only 10% lower than in logged forest, but our data indicate that riparian reserves of at least 50 - 80 m are needed for species richness and diversity to equal that in nearby logged forest. These findings, particularly if they apply more widely to forest-dependent taxa, should be taken into account when setting policy and sustainability guidelines for oil palm plantations, both in areas undergoing conversion from forest and in existing oil palm plantations where forest restoration is required.

ecology↗

bioRxiv2017

Riparian reserves in oil palm plantations may provide movement corridors for invertebrates

Tropical forests are becoming increasingly fragmented through conversion to agriculture, with negative consequences for biodiversity. Movement of individuals among the remaining fragments is critical to allow populations of forest-dependent taxa to persist. In SE Asia, conversion of forests to oil palm plantations is a particular threat. Many oil palm dominated landscapes retain forested riparian reserves along streams and rivers, but the extent to which these riparian reserves are used for movement, relative to the oil palm matrix, is poorly understood. We used mark-release-recapture methods to investigate the movement of five moth taxa in riparian reserves connected to a continuous forest area within oil palm matrix in Sabah, Malaysian Borneo. Moths were recaptured on average 68m from the release point, and the mean time to recapture was between 2 and 3 days. Moths showed a significant tendency to move within forested riparian reserves than into adjacent oil palm. When moving within riparian reserves moths also showed a tendency to orient their movement away from the continuous forest into the riparian reserve rather than towards continuous forest or into oil palm. Overall, our results show a role for riparian reserves as movement corridors through oil palm plantations for some invertebrate species, strengthening the case for their retention and re-establishment.

ecology↗