Biology subjects

Barrett, C.

Publications and source records attributed to Barrett, C..

11 recordsLinked to original sources

bioRxiv2026

Unexpected excess heterozygosity in a threatened, mycoheterotrophic, self-pollinating orchid

Self-fertilization results in reduction of heterozygosity over time, accelerated by small population sizes, restricted ranges, and fragmented habitats. Yet some species maintain heterozygosity despite these factors. One is Corallorhiza bentleyi, a threatened, mycoheterotrophic, self-pollinating orchid restricted to Virginia and West Virginia, USA. Reduced representation sequencing was employed across five of the ~12 known localities of C. bentleyi, including representatives of sister species C. involuta from Mexico, to quantify patterns of population structure and genomic diversity. Surveys of C. bentleyi revealed a predominance of the cleistogamous (closed-flowered) growth form. Differentiation was observed between the two species but with weak population structure in C. bentleyi. Surprisingly, excess heterozygosity and negative inbreeding were recovered in both species, counter to expectations. Analyses provided evidence against bioinformatic artifacts (collapsed paralogs), small-sample bias (Robertson effect), and accumulation of mutations along chromosomes due to low recombination associated with selfing (Meselson effect) as explanations for excess heterozygosity. Demographic analyses favored a model of post-glacial divergence among C. bentleyi and C. involuta lacking bottlenecks. Finally, simulations under alternative selection scenarios favored models of overdominance vs. those of negative frequency dependent selection. It was concluded that overdominance, namely selection against selfed homozygotes, is the most likely explanation for elevated heterozygosity in C. bentleyi. Conservation concerns are discussed in light of these findings and the threats of extinction for one of the most vulnerable orchid species in North America.

evolutionary biology↗

bioRxiv2026

Innate Defense Mechanisms Against Nosema ceranae in Hygienic Honey Bee (Apis mellifera) Colonies

The honey bee colony (Apis mellifera) acts as a superorganism, with a dual immune system that operates at the individual and social level. However, the linkages between immune mechanisms across the two levels remain poorly understood, despite the relevance for developing effective breeding strategies to improve honey bee disease resistance. Hygienic behavior involving the removal of unhealthy brood is a key component of honey bee social immunity and is highly effective at limiting parasites and pathogens in the colony. While this form of hygienic behavior can reduce brood diseases, parasites infecting adult bees primarily, such as Nosema ceranae, are not directly impacted by the behavior. However, when using the Unhealthy Brood Odor (UBeeO) assay to quantify hygienic behavior performance, hygienic colonies have been shown to maintain lower Nosema spp. loads over time and overall compared to non-hygienic colonies. To investigate the mechanisms driving reduced Nosema spp. in hygienic colonies, we conducted a series of field and lab experiments to test the innate immune performance of individual bees. We evaluated several factors across hygienic and non-hygienic bees including (1) differences in N. ceranae infection levels, (2) survival probability, (3) Vitellogenin and Hymenoptaecin gene expression, and (4) amount of N. ceranae inoculant consumed. We found that hygienic bees consumed less of the inoculant, exhibited upregulated Vitellogenin gene expression at peak N. ceranae infection, showed a positive relationship between Hymenoptaecin gene expression and N. ceranae infection levels, and had greater survivability when infected with N. ceranae, compared to non-hygienic bees. Here, we present new findings that link colony hygienic behavior performance to individual-level resistance and tolerance mechanisms in response to N. ceranae, suggesting broader implications for the success of selective breeding programs targeting hygienic traits.

immunology↗

bioRxiv2025

Dopamine receptor 1 specific CRISPRa mice exhibit disrupted behaviors and striatal baseline cellular activity

The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion and enhanced acquisition of reward learning in comparison to all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison to their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward-learning. Significance StatementUsing CRISPR-based tools to identify cell-type specific epigenomic and transcriptional mechanisms in disease and behavior has high utility for the neuroscience field. Previous limitations related to implementation of CRISPR-editing systems in mice were thought to be overcome by the generation of transgenic mouse lines, including a novel Cre-dependent dCas9-p300 mouse line. Our data shows however that Drd1-Cre:dCas9-p300 mice, generated from breeding Drd1-Cre mice with the dCas9-p300 mice, have cellular and behavioral disruptions under a nontargeted sgRNA absent state. Overall, these data suggest caution in employing CRISPR-dCas9 systems, particularly transgenic mouse lines, for research investigations.

neuroscience↗

bioRxiv2025

Multiple pathways to the evolution of positive assortment in aggregative multicellularity

The evolutionary transition to multicellularity requires shifting the primary unit of selection from cells to multicellular collectives. How this occurs in aggregative organisms remains poorly understood. Clonal development provides a direct path to multicellular adaptation through genetic identity between cells, but aggregative organisms face a constraint: selection on collective-level traits cannot drive adaptation without positive genetic assortment. We leveraged experimental evolution of flocculating Saccharomyces cerevisiae to examine the evolution and role of genetic assortment in multicellular adaptation. After 840 generations of selection for rapid settling, 13 of 19 lineages evolved increased positive assortment relative to their ancestor. However, assortment provided no competitive advantage during settling selection, suggesting it arose as an indirect effect of selection on cell-level traits rather than through direct selection on collective-level properties. Genetic reconstruction experiments and protein structure modeling revealed two distinct pathways to assortment: kin recognition mediated by mutations in the FLO1 adhesion gene and generally enhanced cellular adhesion that improved flocculation efficiency independent of partner genotype. The evolution of assortment without immediate adaptive benefit suggests that key innovations enabling multicellular adaptation may arise indirectly through cell-level selection. Our results demonstrate fundamental constraints on aggregative multicellularity and help explain why aggregative lineages have remained simple.

evolutionary biology↗

bioRxiv2024

FGFR2 promotes resistance to ALK tyrosine kinase inhibitors and its inhibition acts synergistically with lorlatinib in the treatment of ALK-expressing neuroblastoma

Anaplastic Lymphoma Kinase inhibitors (ALK TKIs) are approved for the treatment of ALK-positive non-small cell lung cancer (NSCLC) and are in clinical trial for ALK-aberrant high-risk neuroblastoma (NB) patients, particularly loratinib. However, resistance to ALK inhibitors can occur in patients, via the activation of bypass-signalling pathways, and there is a need to identify these mechanisms as well as drugs that inhibit them to design therapeutic approaches that prevent resistance, and to treat ALK TKI relapsed/refractory disease. Using genome-wide CRISPR-Cas9 overexpression screens, we identified and validated FGFR2 as a desensitizer to lorlatinib in aberrant ALK-expressing high-risk NB. FGFR2 and FGFR2-associated pathways are up-regulated in lorlatinib-resistant NB cells. Moreover, high-throughput screens using a library of 1,430 FDA approved drugs identified kinase inhibitors including those targeting FGFR2 as efficacious in reducing the survival of lorlatinib resistant NB cells. Hence, the FGFR pathway was investigated as a therapeutic target applying the pan-FGFR inhibitor erdafitinib or the multi-kinase inhibitor ponatinib, resulting in reduced survival of lorlatinib-resitant cells in comparison to their lorlatinib-sensitive counterparts. Moreover, both FGFR inhibitors act synergistically with lorlatinib in vitro and in vivo, using patient-derived xenografts (PDXs) and genetically engineered models (GEMM) of ALK-expressing NB. FGFR2 mRNA expression also correlate with a poorer prognosis for NB patients, regardless of sub-type, suggesting that a broader range of patients may benefit from FGFR inhibitors. Overall, our data suggests that FGFR2 potentially plays roles in lorlatinib resistance in NB and that combined pharmacological inhibition of ALK and FGFR constitutes a therapeutic approach to treat high-risk NB.

cancer biology↗

bioRxiv2024

Placental extracellular vesicles from early-onset but not late-onset preeclampsia induce a pro-vasoconstrictive and anti-vasodilatory state in resistance arteries

BackgroundThe human placenta releases large numbers of extracellular vesicles (EVs) into the maternal circulation throughout pregnancy. In preeclampsia, a hypertensive disorder of pregnancy, the number of EVs increases and the cargo they carry is altered. We investigated whether human placental EVs from pregnancies complicated by preeclampsia directly alter maternal vascular function, a hallmark of the disorder, and if EVs from early-onset or late-onset variants of preeclampsia have different effects on the vasculature. MethodsMacro-EVs, micro-EVs and nano-EVs were isolated from cultured explants of human placentae from women with early-onset or late-onset preeclampsia, or from normotensive women. EVs were injected intravenously into pregnant mice and either at 30 minutes or 24 hours after injection, the mice were euthanized and the function of second order mesenteric arteries were assessed using wire myography. ResultsPlacental EVs from pregnancies with early-onset preeclampsia enhanced vasoconstriction to PE, AngII, and ET-1 whilst impairing vasodilation to ACh and SNP in a time-dependent fashion, most prominently at 24 hours. In contrast, placental EVs from pregnancies with late-onset preeclampsia induced few differences compared to arteries taken from control mice injected with EVs from women with normotensive pregnancies. ConclusionsTo the best of our knowledge, this is the first comparison of vascular function after exposure to the full range of EVs produced by placentae from women with early-onset and late-onset preeclampsia and normotensive women. Placental EVs from early-onset preeclampsia demonstrated the ability to contribute to the development of the high-resistance haemodynamic profiles of women affected by early-onset preeclampsia.

physiology↗

bioRxiv2023

Motifs in SARS-CoV-2 evolution

We present a novel framework enhancing the prediction of whether novel lineage poses the threat of eventually dominating the viral population. The framework is based purely on genomic sequence data, without requiring prior established biological analysis. Its building blocks are sets of co-evolving sites in the alignment (motifs), identified via co-evolutionary signals. The collection of such motifs forms a relational structure over the polymorphic sites. Motifs are constructed using distances quantifying the co-evolutionary coupling of pairs and manifest as co-evolving clusters of sites. We present an approach to genomic surveillance based on this notion of relational structure. Our system will issue an alert regarding a lineage, based on its contribution to drastic changes in the relational structure. We then conduct a comprehensive retrospective analysis of the COVID-19 pandemic based on SARS-CoV-2 genomic sequence data in GISAID from October 2020 to September 2022, across 21 lineages and 27 countries with weekly resolution. We investigate the performance of this surveillance system in terms of its accuracy, timeliness and robustness. Lastly, we study how well each lineage is classified by such a system.

bioinformatics↗

bioRxiv2022

Buying time: detecting VOCs in SARS-CoV-2 via co-evolutionary signals

We present a novel framework facilitating the rapid detection of variants of interest (VOI) and concern (VOC) in a viral multiple sequence alignment (MSA). The framework is purely based on the genomic sequence data, without requiring prior established biological analysis. The frameworks building blocks are sets of co-evolving sites (motifs), identified via co-evolutionary signals within the MSA. Motifs form a weighted simplicial complex, whose vertices are sites that satisfy a certain nucleotide diversity. Higher dimensional simplices are constructed using distances quantifying the co-evolutionary coupling of pairs and in the context of our method maximal motifs manifest as clusters. The framework triggers an alert via a cluster with a significant fraction of newly emerging polymorphic sites. We apply our method to SARS-CoV-2, analyzing all alerts issued from November 2020 through August 2021 with weekly resolution for England, USA, India and South America. Within a week at most a handful of alerts, each of which involving on the order of 10 sites are triggered. Cross referencing alerts with a posteriori knowledge of VOI/VOC-designations and lineages, motif-induced alerts detect VOIs/VOCs rapidly, typically weeks earlier than current methods. We show how motifs provide insight into the organization of the characteristic mutations of a VOI/VOC, organizing them as co-evolving blocks. Finally we study the dependency of the motif reconstruction on metric and clustering method and provide the receiver operating characteristic (ROC) of our alert criterion.

bioinformatics↗

bioRxiv2018

Phylogenomics, biogeography, and evolution in the American palm genus Brahea

Background and AimsSlow rates of molecular evolution at low taxonomic levels hamper studies of relationships among species, and subsequent biogeographic and evolutionary analyses. An example is the genus Brahea, which is among the most poorly understood lineages of American palms and is characterized by a wide variety of growth forms and intermediate morphological features.\n\nMethodsWe generated approximately 400 kb of genome-scale data from all three genomes for the 11 currently described species of Brahea to infer phylogenetic relationships, reconstruct ancestral growth form, estimate ancestral geographic ranges, and test for niche equivalency among closely related species with geographic overlap.\n\nKey ResultsRelationships receive strong support, and conform to previous subgeneric assignments, except for placement of the dwarf species B. moorei within subgenus Erythea. Our robust phylogenetic hypothesis reveals trends in growth form including an overall increase in height in the B. armata clade, and independent evolution of dwarf forms from taller ancestors in the B. pimo and B. dulcis clades. Ancestral range estimation reveals roles of dispersal (e.g. B. edulis on Guadalupe Island) and sympatric speciation in some cases (e.g. in the B. armata clade), but is equivocal in others (e.g. in the B. pimo clade). We find evidence of niche non-equivalency among species within the B. armata clade in northwestern Mexico, and some evidence of niche non-equivalency between B. berlandieri and B. dulcis, the former of which is synonymized under B. dulcis.\n\nConclusionsOur findings have implications for the complex biogeographic history in Central America and Mexico, suggesting that sympatric speciation and dispersal are the predominant processes of species diversification. Future studies should include population-level sampling across the genus, along with morphological and ecological information, to assess distinctness among species and, particularly, levels of gene flow, in an integrative fashion.

evolutionary biology↗

bioRxiv2018

Characterization of Diverse Homoserine Lactone Synthases in Escherichia coli

Quorum sensing networks have been identified in over one hundred bacterial species to date. A subset of these networks regulate group behaviors, such as bioluminescence, virulence, and biofilm formation, by sending and receiving small molecules called homoserine lactones (HSLs). Bioengineers have incorporated quorum sensing pathways into genetic circuits to connect logical operations. However, the development of higher-order genetic circuitry is inhibited by crosstalk, in which one quorum sensing network responds to HSLs produced by a different network. Here, we report the construction and characterization of a library of ten synthases including some that are expected to produce HSLs that are incompatible with the Lux pathway, and therefore show no crosstalk. We demonstrated their function in a common lab chassis, Escherichia coli BL21, and in two contexts, liquid and solid agar cultures, using decoupled Sender and Receiver pathways. We observed weak or strong stimulation of a Lux Receiver by longer-chain or shorter-chain HSL-generating Senders, respectively. We also considered the under-investigated risk of unintentional release of incompletely deactivated HSLs in biological waste. We found that HSL-enriched media treated with bleach is still bioactive, while autoclaving deactivates LuxR induction. This work represents the most extensive comparison of quorum sensing synthases to date and greatly expands the bacterial signaling toolkit while recommending practices for disposal based on empirical, quantitative evidence.

synthetic biology↗

bioRxiv2017

Enhancing Cas9 Activity in Heterochromatin

Recently we demonstrated that closed chromatin composed of Polycomb proteins and histone 3 lysine 27 trimethylation impedes CRISPR-mediated genome editing by blocking the accessibility of chromosomal DNA to spCas9/sgRNA. Editing efficiencies were higher in cells where the same reporter locus had not been repressed, thus we presume that silenced chromatin can be modified to generate a Cas9-accessible state. To test this idea, we exposed the locus to antagonists of Polycomb silencing: Gal4-p65, a targeted transcriptional activator, and UNC1999, a chemical inhibitor of the histone H3K27 methyltransferase EZH2. For both we observed loss of histone trimethylation. Only Gal4-p65 treatment increased target gene expression. Initial Gal4-p65 overexpression impedes Cas9 activity, while a 9-day recovery period leads to enhanced Cas9 efficiency up to 1000 bp from the Gal4 binding site. No enhancement was observed with UNC1999. These results demonstrate the strong influence of transcription-driven chromatin remodeling on CRISPR editing at closed chromatin.

synthetic biology↗