Biology subjects

Bolton, M.

Publications and source records attributed to Bolton, M..

6 recordsLinked to original sources

bioRxiv2026

Student-Driven Microbiome Exploration: A Low-Cost 16S rRNA Sequencing Curriculum for Undergraduate Biology Education

Microbiome science is increasingly important in modern biology education because microbial communities influence human health, ecosystems, and environmental processes. However, undergraduate microbiome instruction is often limited by the high cost and technical complexity of sequencing-based workflows, restricting opportunities for authentic student-driven research. To address this challenge, we developed a low-cost, inquiry-based curriculum that enables undergraduate students to conduct complete microbiome studies using 16S rRNA gene sequencing. The module integrates project design, environmental sample collection, microbial cell processing, PCR amplification, sequencing, and bioinformatic analysis using open-source tools such as QIIME 2. Cost-reduction strategies included centrifugation-based cell collection and a surfactant-assisted direct PCR workflow that eliminated the need for commercial DNA extraction kits. Students designed independent research projects investigating microbial communities in local environments, including campus water sources and gym equipment surfaces. Assessment data from post-course surveys, knowledge checks, and student research products demonstrated strong learning gains in microbiome concepts, molecular biology techniques, scientific communication, and computational analysis. Students reported high confidence in PCR, experimental design, and microbiome interpretation, while also identifying bioinformatics as the most challenging yet rewarding component of the curriculum. All participants expressed increased interest in future research in microbiology or bioinformatics. Overall, this curriculum provides an accessible, scalable framework for integrating next-generation sequencing into undergraduate education while promoting inquiry-driven learning, student ownership, and engagement in authentic scientific research.

microbiology↗

bioRxiv2024

INGEsT: An Open-Source Behavioral Setup for Studying Self-motivated Ingestive Behavior and Learned Operant Behavior

Ingestive behavior is driven by negative internal hunger and thirst states, as well as by positive expected rewards. Although the neural substrates underlying feeding and drinking behaviors have been widely investigated, they have primarily been studied in isolation, even though eating can also trigger thirst, and vice versa. Thus, it is still unclear how the brain encodes body states, recalls the memory of food and water reward outcomes, generates feeding/drinking motivation, and triggers ingestive behavior. Here, we developed an INstrument for Gauging Eating and Thirst (INGEsT), a custom-made behavioral chamber which allows for precise measurement of both feeding and drinking by combining a FED3 food dispenser, lickometers for dispensing liquid, a camera for behavioral tracking, LED light for optogenetics, and calcium imaging miniscope. In addition, in vivo calcium imaging, optogenetics, and video recordings are well synchronized with animal behaviors, e.g., nose pokes, pellet retrieval, and water licking, by using a Bpod microprocessor and timestamping behavioral and imaging data. The INGEsT behavioral chamber enables many types of experiments, including free feeding/drinking, operant behavior to obtain food or water, and food/water choice behavior. Here, we tracked activity of insular cortex and mPFC Htr3a neurons using miniscopes and demonstrate that these neurons encode many aspects of ingestive behavior during operant learning and food/water choice and that their activity can be tuned by internal state. Overall, we have built a platform, consisting of both hardware and software, to precisely monitor innate ingestive, and learned operant, behaviors and to investigate the neural correlates of self-motivated and learned feeding/drinking behaviors.

neuroscience↗

bioRxiv2023

Ptth regulates lifespan through innate immunity pathway in Drosophila

The prothoracicotropic hormone (PTTH) is a well-known neuropeptide that regulates insect metamorphosis (the juvenile-to-adult transition) by inducing the biosynthesis of steroid hormones. However, the role of PTTH in adult physiology and longevity is largely unexplored. Here, we show that Ptth loss-of-function mutants are long-lived and exhibit increased resistance to oxidative stress in Drosophila. Intriguingly, we find that loss of Ptth blunt age-dependent upregulation of NF-{kappa}B signaling specifically in fly hepatocytes (oenocytes). We further show that oenocyte-specific overexpression of Relish/NF-{kappa}B blocks the lifespan extension of Ptth mutants, suggesting that PTTH regulates lifespan through oenocyte-specific NF-{kappa}B signaling. Surprisingly, adult-specific knockdown of Ptth did not prolong lifespan, indicating that PTTH controls longevity through developmental programs. Indeed, knockdown of PTTH receptor Torso in prothoracic gland (PG) during fly development prolongs lifespan. To uncover the developmental processes underlying PTTH-regulated lifespan, we perform a developmental transcriptomic analysis and identify an unexpected activation of NF-{kappa}B signaling in developing oenocytes during fly metamorphosis, which is blocked in Ptth mutants. Importantly, knockdown of Relish/NF-{kappa}B specifically in oenocytes during early pupal stages significantly prolongs the lifespan of adult flies. Thus, our findings uncover an unexpected role of PTTH in controlling adult lifespan through temporal and spatial activation of NF-{kappa}B signaling in developing hepatocytes and highlight the vital role of developmental NF-{kappa}B signaling in shaping adult physiology. Significance StatementDespite the strong link between animal development and adult lifespan, we know little about how developmental programs impact adult longevity, and when and where such programs are activated during development. Here, we demonstrate that loss of insect hormone PTTH prolongs lifespan and healthspan by repressing chronic inflammation in Drosophila. Intriguingly, we demonstrate that PTTH regulates adult lifespan through temporal and spatial activation of NF-{kappa}B signaling in developing hepatocytes during insect metamorphosis. These findings provide novel insights into the developmental programs that impact adult longevity.

physiology↗

bioRxiv2022

IgG3 subclass antibodies recognize antigenically drifted influenza viruses and SARS-CoV-2 variants through efficient bivalent binding

The constant domains of antibodies are important for effector functions, but less is known about how they can affect binding and neutralization of viruses. Here we evaluated a panel of human influenza virus monoclonal antibodies (mAbs) expressed as IgG1, IgG2 or IgG3. We found that many influenza virus-specific mAbs have altered binding and neutralization capacity depending on the IgG subclass encoded, and that these differences result from unique bivalency capacities of the subclasses. Importantly, subclass differences in antibody binding and neutralization were greatest when the affinity for the target antigen was reduced through antigenic mismatch. We found that antibodies expressed as IgG3 bound and neutralized antigenically drifted influenza viruses more effectively. We obtained similar results using a panel of SARS-CoV-2-specific mAbs and the antigenically advanced B.1.351 strain of SARS-CoV-2. We found that a licensed therapeutic mAb retained neutralization breadth against SARS-CoV-2 variants when expressed as IgG3, but not IgG1. These data highlight that IgG subclasses are not only important for fine-tuning effector functionality, but also for binding and neutralization of antigenically drifted viruses. SignificanceInfluenza viruses and coronaviruses undergo continuous change, successfully evading human antibodies elicited from prior infections or vaccinations. It is important to identify features that allow antibodies to bind with increased breadth. Here we examined the effect that different IgG subclasses have on monoclonal antibody binding and neutralization. We show that IgG subclass is a determinant of antibody breadth, with IgG3 affording increased neutralization of antigenically drifted variants of influenza virus and SARS-CoV-2. Future studies should evaluate IgG3 therapeutic antibodies and vaccination strategies or adjuvants that may skew antibody responses toward broadly reactive isotypes.

microbiology↗

bioRxiv2018

Response to a Synthetic Pheromone Source by OX4319L, a Self-Limiting Diamondback Moth (Lepidoptera: Plutellidae) Strain, and Field Dispersal Characteristics of Its Progenitor Strain

The Diamondback Moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a global pest that infests vegetable and field crops within the Brassica family. A genetically engineered strain of P. xylostella, OX4319L, carrying a self-limiting gene, has shown potential for managing P. xylostella populations, using sustained releases of OX4319L male moths. In order for such a strain to provide control, the transgenic individuals must exhibit attraction to female P. xylostella sex pheromone and adequate dispersal in the field. In this study, we tested these key traits. First, we compared the responses of the OX4319L male moths to a synthetic female sex pheromone source in wind tunnel trials to those of males from three other strains. We found that OX4319L males responded comparably to strains of non-engineered males, with all males flying upwind towards the pheromone source. Second, we used mark-release-recapture studies of a wildtype P. xylostella strain, from which the OX4319L strain was originally developed, to assess dispersal under field conditions. Released males were recaptured using both pheromone-baited and passive traps within a 2.83 ha circular cabbage field, with a recapture rate of 7.93%. Males were recaptured up to the boundary of the field at 95 m from the central release point. The median dispersal of males was 14 m. These results showed the progenitor strain of OX4319L retained its ability to disperse within a host field. The results of these experiments are discussed in relation to the potential for the effective use of engineered male-selecting P. xylostella strains under field conditions.

zoology↗

bioRxiv2018

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsino[e] fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii, and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.\n\nOriginality and significance statementGenes involved in secondary metabolite (SM) production are often clustered together to form biosynthetic pathways. These pathways frequently have highly conserved keystone enzymes which can complicate allocation of a biosynthetic gene cluster (BGC) to the cognate SM. In our study, we utilized a combination of comparative genomics, phylogenetic analyses and biochemical approaches to reliably identify BGCs for perylenequinone toxins and DHN-melanin in multiple plant pathogenic fungi. Furthermore, we show that earlier studies that aimed to identify these perylenequinone pathways were misdirected and actually reported DHN-melanin biosynthetic pathways. Our study outlines a reliable approach to successfully identify fungal SM pathways.

microbiology↗