Biology subjects

Allison, J.

Publications and source records attributed to Allison, J..

8 recordsLinked to original sources

bioRxiv2025

Bifunctional Architecture Enables Substrate Catalysis and Channeling in Paracoccus TMAO Demethylase

Substrate channeling enhances efficiency and prevents toxicity by directing unstable intermediates between active sites. Trimethylamine N-oxide demethylase (TDM) degrades trimethylamine N-oxide (TMAO) to dimethylamine and formaldehyde (HCHO), but the fate of HCHO has remained unclear. We report cryo-EM structures of TDM in apo, substrate-, and product-bound states that reveal a previously unknown channeling pathway. Combined structural, biochemical, and target molecular dynamics analyses show that HCHO is generated in a catalytic core and guided through a tunnel to a remote tetrahydrofolate (THF)-binding site, where it forms methylene-THF. Thus, TDM emerges as a bifunctional enzyme that unites TMAO demethylation with one-carbon transfer, providing a mechanistic explanation for its role in metabolic efficiency and detoxification.

biophysics↗

bioRxiv2024

Insect abundance and richness response to ecological reclamation on well pads 5-12 years into succession in a semi-arid natural gas field

Natural gas extraction is a critical driver of the economy in western North America. Ecological reclamation is important to ensure surface disturbance impacts associated with natural gas development are not permanent and to assist native biota. Previous studies in semi-arid natural gas fields within Sublette County, Wyoming, USA have shown insects respond favorably to 1-3-year-old well pads undergoing reclamation compared to older successional, reference vegetation communities dominated by Wyoming big sagebrush (Artemisia tridenta spp. Wyomingensis). Here we examined well pads which were initially seeded between 5, 8, 10, 11, and 12 prior to our study. We used a free, image-based software called SamplePoint to quantify vegetation on these well pads and adjacent reference areas from high-resolution cell phone camera pictures. Insects were collected with a sweep net and identified to the family and morphospecies level. Statistical analyses were conducted to compare both vegetation and insect communities between reclamation sites and their paired reference area. We found little statistical difference between vegetation communities across our study, but found significantly more insect abundance in 3 of 5 years and significantly higher family and species richness in 4 of 5 years. Our results suggest reclamation of natural gas well pads within an old successional stand of sagebrush continues to support higher levels of insect biodiversity and abundance for at least 12 years. As insects are the most diverse group of animals on Earth and because they provide a wide array of ecosystem services, our findings suggest ecological reclamation plays an important role in returning biodiversity and ecosystem functionality to a semi-arid and old successional sagebrush-steppe ecosystem.

ecology↗

bioRxiv2023

Tertiary-interaction characters enable fast, model-based structural phylogenetics beyond the twilight zone

Protein structure is more conserved than protein sequence, and therefore may be useful for phylogenetic inference beyond the "twilight zone" where sequence similarity is highly decayed. Until recently, structural phylogenetics was constrained by the lack of solved structures for most proteins, and the reliance on phylogenetic distance methods which made it difficult to treat inference and uncertainty statistically. AlphaFold has mostly overcome the first problem by making structural predictions readily available. We address the second problem by redeploying a structural alphabet recently developed for Foldseek, a highly-efficient deep homology search program. For each residue in a structure, Foldseek identifies a tertiary interaction closest-neighbor residue in the structure, and classifies it into one of twenty "3Di" states. We test the hypothesis that 3Dis can be used as standard phylogenetic characters using a dataset of 53 structures from the ferritin-like superfamily. We performed 60 IQtree Maximum Likelihood runs to compare structure-free, PDB, and AlphaFold analyses, and default versus custom model sets that include a 3DI-specific rate matrix. Analyses that combine amino acids, 3Di characters, partitioning, and custom models produce the closest match to the structural distances tree of Malik et al. (2020), avoiding the long-branch attraction errors of structure-free analyses. Analyses include standard ultrafast bootstrapping confidence measures, and take minutes instead of weeks to run on desktop computers. These results suggest that structural phylogenetics could soon be routine practice in protein phylogenetics, allowing the re-exploration of many fundamental phylogenetic problems.

evolutionary biology↗

bioRxiv2023

Molecular pathways and cellular subsets associated with adverse clinical outcomes in overlapping immune-related myocarditis and myositis

Immune checkpoint therapies (ICTs) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we obtained heart and skeletal muscle biopsies for single-cell RNA sequencing in living patients with cancers treated with ICTs admitted to the hospital with myocarditis and /or myositis (overlapping myocarditis plus myositis, n=10; myocarditis-only, n=1) compared to ICT-exposed patients ruled out for toxicity utilized as controls (n=9) within 96 hours of clinical presentation. Analyses of 58,523 cells revealed clonally expanded CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of tissue-resident myeloid cells expressed Fc{gamma}RIIIa, which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d that were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL-1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results are the first to recognize these myeloid subsets in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/556590v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b5895forg.highwire.dtl.DTLVardef@4dec8corg.highwire.dtl.DTLVardef@1e50cb9org.highwire.dtl.DTLVardef@a653fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-EM structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 [A] resolution (extending to 2.2 [A] at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 [A] resolution) permits accurate assignment of two Na+ sites and the architecture of the substrate binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (KD) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the elevator-with-an-operator mechanism of TRAP transporters.

biochemistry↗

bioRxiv2020

Single-cell Characterization of Acute Myeloid Leukemia and its Microenvironment Following PD-1 Blockade Based Therapy

Acute myeloid leukemia (AML) and effector cells of immune checkpoint blockade (ICB) therapy co-reside in a complex bone marrow (BM) milieu. The interplay of tumor intrinsic and microenvironment (TME) mechanisms that influences the response to ICB-based therapies in AML have not been elucidated. Here we report our analyses of single cell RNA profiling of more than 127,000 BM cells from healthy donors and relapsed/refractory (R/R) AML patients at pre/post treatment with azacitidine/nivolumab, paired with single cell T cell receptor (TCR) repertoire profiles, to uncover factors impacting response and resistance. Loss of chromosome 7/7q conferred an immunosuppressive TME and was associated with resistance to ICB-based therapy in R/R AML. Our trajectory analysis revealed a continuum of CD8+ T cell phenotypes, characterized by differential expression of granzyme B (GZMB) and GZMK. GZMK expression defined a BM residing memory CD8+ T cell subset with stem-like properties likely an intermediary between naive and cytotoxic lymphocytes. Responses to ICB-based therapy were primarily driven by novel and expanded T cell clonotypes. Our findings support an adaptable T cell plasticity in response to PD-1 blockade in AML. Disentangling AML cells from their complex, immune-rich microenvironment revealed characteristics that shaped resistance to ICB-based therapy and could inform strategies to target AML vulnerabilities. SignificanceDetermining the cellular and molecular underpinnings of response and resistance to PD-1 blockade based therapy in AML can guide immune-based therapeutic strategies. Our results reveal AML intrinsic characteristics (chromosome 7/7q status and oxidative stressors) and tumor microenvironment to modulate responses to checkpoint blockers. CD8 cells exist in the bone marrow in a continuum with GZMK expression defining a memory, stem-like T cell population that could play a role in response to therapy.

cancer biology↗

bioRxiv2020

Evaluating aerosol and splatter following dental procedures

BackgroundDental procedures often produce aerosol and splatter which are potentially high risk for spreading pathogens such as SARS-CoV-2. The existing literature is limited. Objective(s)To develop a robust, reliable and valid methodology to evaluate distribution and persistence of dental aerosol and splatter, including the evaluation of clinical procedures. MethodsFluorescein was introduced into the irrigation reservoirs of a high-speed air-turbine, ultrasonic scaler and 3-in-1 spray and procedures performed on a mannequin in triplicate. Filter papers were placed in the immediate environment. The impact of dental suction and assistant presence were also evaluated. Samples were analysed using photographic image analysis, and spectrofluorometric analysis. Descriptive statistics were calculated and Pearsons correlation for comparison of analytic methods. ResultsAll procedures were aerosol and splatter generating. Contamination was highest closest to the source, remaining high to 1-1.5 m. Contamination was detectable at the maximum distance measured (4 m) for high-speed air-turbine with maximum relative fluorescence units (RFU) being: 46,091 at 0.5 m, 3,541 at 1.0 m, and 1,695 at 4 m. There was uneven spatial distribution with highest levels of contamination opposite the operator. Very low levels of contamination ([≤]0.1% of original) were detected at 30 and 60 minutes post procedure. Suction reduced contamination by 67-75% at 0.5-1.5 m. Mannequin and operator were heavily contaminated. The two analytic methods showed good correlation (r=0.930, n=244, p<0.001). ConclusionDental procedures have potential to deposit aerosol and splatter at some distance from the source, being effectively cleared by 30 minutes in our setting.

microbiology↗

bioRxiv2019

Peppy: A Virtual Reality Environment For Exploring The Principles Of Polypeptide Structure

A key learning outcome for undergraduate biochemistry classes is a thorough understanding of the principles of protein structure. Traditional approaches to teaching this material, which include two-dimensional (2D) images on paper, physical molecular modelling kits, and projections of 3D structures into 2D, are unable to fully capture the dynamic, 3D nature of proteins. We have built a virtual reality application, Peppy, aimed at facilitating teaching of the principles of protein secondary structure. Rather than attempt to model molecules with the same fidelity to the underlying physical chemistry as existing, research-oriented molecular modelling approaches, we took the more straightforward approach of harnessing the Unity video game physics engine. Indeed, the simplicity and limitations of our model are a strength in a teaching context, provoking questions and thus deeper understanding. Peppy allows exploration of the relative effects of hydrogen bonding (and electrostatic interactions more generally), backbone {phi}/{psi} angles, basic chemical structure and steric effects on polypeptide structure in an accessible format that is novel, dynamic and fun to use. As well as describing the implementation and use of Peppy, we discuss the outcomes of deploying Peppy in undergraduate biochemistry courses.\n\nSTATEMENTProtein structure is inherently dynamic and three-dimensional, but traditional teaching tools are static and/or two-dimensional. We have developed a virtual reality teaching tool, Peppy, that facilitates undergraduate teaching of the principles of protein structure. We outline how Peppy works in terms of how it is used and what goes on under the hood. We then illustrate its use in undergraduate teaching, where its playful nature stimulated exploration and, thus, deeper understanding.

biochemistry↗