Biology subjects

Brooks, C.

Publications and source records attributed to Brooks, C..

14 recordsLinked to original sources

bioRxiv2026

Mutation choice biases the structure of empirical fitness landscapes

Evolution can be thought of as taking place in a fitness landscape which maps each genotype to a fitness value. This abstraction has motivated significant efforts to quantify the statistical properties of random fitness landscapes with different amounts and types of interactions among loci---broadly referred to as epistasis. New high-throughput experimental techniques measuring the phenotypes of thousands of genotypes promise to bridge this theory with data. Yet, while impressive, thousands is still small relative to the size of genotype space. Moreover, experiments often assay a highly non-random subset of the fitness landscape. For example, a common experimental approach is to generate all possible 2M sequence intermediates between ancestral and evolved sequences differing by M much less than L mutations. Here, we ask to what extent the statistics of an empirical landscape constructed under distinct ascertainment protocols recapitulate the statistics of a random subset of the global landscape in a few well-studied random landscape models. We show that, in most landscapes, selecting mutations which occurred along an adaptive trajectory inflates the variance explained by lower order epistasis, reduces the number of local maxima, and increases the number of accessible paths relative to a random fitness landscape. Selecting the most beneficial or deleterious mutations, on the other hand, results in distortions to these statistics which depend more critically on the amount of epistasis in the global landscape. We then take advantage of several large-scale mutagenesis datasets to simulate the effects of ascertainment in more realistic settings.

genetics↗

bioRxiv2026

Interactions between epistasis and pleiotropy in generic models of complex traits

Evolution in fluctuating environments depends critically on epistatic interactions between mutations and the pleiotropic effects these mutations have on fitness across multiple conditions. However, while these effects of epistasis and pleiotropy have been extensively studied individually, the interactions between them are less well understood. In this study, we analyze how genetic background shapes the effects of mutations across multiple environmental conditions in generic models of complex traits. We show that when epistasis is widespread and random, systematic patterns of cross-environment pleiotropy can emerge naturally. Specifically we outline two regimes, widespread random epistasis (WRE) and structured pleiotropy (SP), in which patterns of global epistasis in one environment can be predicted from those in other conditions. We outline these predictions in several classes of generic null models, and test these predictions in laboratory budding yeast by measuring the fitness effects of a large set of mutations across many genetic backgrounds in several growth environments. We find that only a subset of mutations fall within the WRE and SP regimes, but that in these cases two of our models provide accurate fits to the data. Finally, we use this framework to show how modular, pleiotropic architectures shape long-term evolutionary steady states in fluctuating environments, revealing how tradeoffs between environments can develop from the geometry of shared latent interaction structure.

evolutionary biology↗

bioRxiv2026

Learning protein function through autonomous experimental interaction

Biological AI learns primarily from existing observations, but many questions cannot be answered from available data alone. Here we show that AI can instead acquire knowledge by acting directly on biological systems and learning from the consequences. We developed a closed-loop framework in which autonomous agents design protein variants, construct and characterize them in a robotic laboratory, learn from the resulting experimental feedback, and decide what experiments to perform next. We then allowed the system to operate continuously and without human intervention for approximately one month, during which multiple agents independently explored protein sequence space while learning from shared experimental experience. Applied to glycoside hydrolases, the agents discovered enzymes with substantially altered substrate specificity toward non-native sugars and progressively learned the structure of the underlying sequence-function landscape. The resulting experimental experience also revealed determinants of substrate specificity and protein expression that were not specified as learning objectives. These results demonstrate that AI can autonomously interact with biology over extended periods to acquire knowledge through experience, establishing a framework for biological discovery driven by continuous experimental interaction.

bioengineering↗

bioRxiv2026

Mapping the chaperonin TRiC/CCT interactome in mouse photoreceptors reveals functional significance for energy metabolism

The eukaryotic chaperonin TRiC/CCT is essential for folding a diverse set of proteins, yet its interactome and functional roles in specialized neurons remain incompletely understood. To investigate TRiC-mediated folding in rod photoreceptors, we generated a transgenic mouse line expressing an epitope-tagged Tcp-1 subunit, enabling purification of intact TRiC complexes from retinal tissue. Mass spectrometry identified 226 TRiC-interacting proteins, including known TRiC substrates and co-chaperones as well as numerous novel candidates enriched in RNA processing, cytoskeletal organization, and cell-cycle regulation. Using a TRiC loss-of-function model in which expression of a short splice isoform of phosducin-like protein (PhLPs) competitively inhibits TRiC activity, we observed marked reductions in canonical TRiC substrates, including tubulins, transducin {beta} subunits, and triosephosphate isomerase, as well as secondary alterations in proteins involved in cytoskeletal stability, membrane trafficking, energy metabolism, and phototransduction. Quantitative metabolomic profiling revealed that TRiC deficiency induces a metabolic "energy crisis" characterized by reduced glycolytic- and tricarboxylic acid cycle intermediates, acylcarnitines, ATP, NAD, and NADH, implicating widespread impairment of glucose utilization, mitochondrial bioenergetics, and fatty acid oxidation. Integrative proteomic-metabolomic analysis identified a small subset of proteins, including Rab10 and Anxa1, as potential drivers of these metabolic disruptions, with defective Rab10-dependent GLUT4 trafficking emerging as a plausible mechanism underlying impaired glucose uptake in TRiC-deficient rods. Finally, experiments using a perpetually unfolded G{beta}1 mutant and G{gamma}1-knockout mice demonstrated that substrate overload sequesters TRiC and competitively displaces other clients, exacerbating proteostasis imbalance. Together, our study provides a comprehensive in vivo mapping of the TRiC interactome in mammalian rods, reveals a connection between TRiC-dependent proteostasis and energy metabolism in rods, and indicates a mechanism by which misfolded TRiC substrates exacerbate a proteostasis imbalance that ultimately results in neurodegeneration.

biochemistry↗

bioRxiv2025

The inner membrane complex protein, IMC55, is dispensable for intra-erythrocytic development of Plasmodium falciparum

Plasmodium falciparum, an obligate intracellular Apicomplexan parasite and the causative agent of malaria must reside and replicate within a host cell during the intra-erythrocytic stage. The parasite utilizes a specialized double-membraned organelle, called the inner membrane complex for schizont-stage segmentation and merozoite motility during subsequent re-invasion. In this study, we investigate the function of IMC55, a multi-transmembrane inner membrane complex protein, which we identified as a putative interacting partner of the P. falciparum ER-resident calcium binding protein, PfERC. We have previously shown that PfERC is essential for parasite egress and our data shows that PfERC may also function in parasite invasion into the RBC. The whole genome transposon mutagenesis screen predicts that IMC55 is essential for the intra-erythrocytic asexual cycle. In this work, we show that IMC55 is not required for asexual replication. Following inducible expression of this gene using two conditional systems, we find there is no defect in replicative fitness during the asexual blood stage. Taken together, these data demonstrate that the function of IMC55 is either dispensable or redundant for the Plasmodium falciparum asexual blood stage.

cell biology↗

bioRxiv2024

Syngeneic adipose-derived stromal cells modulate the immune response but have limited persistence within decellularized adipose tissue implants in C57BL/6 mice

The delivery of adipose-derived stromal cells (ASCs) on cell-instructive decellularized adipose tissue (DAT) scaffolds is a promising strategy for stimulating host-derived soft tissue regeneration. However, a better understanding of the mechanisms through which ASCs modulate regeneration in vivo is needed to harness these cells more effectively. In this study, DAT scaffolds, both with and without seeded syngeneic DsRED+ mouse ASCs, were implanted into immunocompetent C57BL/6 mice. Downstream analyses focused on assessing donor ASC persistence and phenotype, as well as the effects of ASC seeding on host macrophage polarization and the perfused host vascular network. Notably, most donor ASCs were cleared from the scaffolds by 2 weeks. Mass spectrometry-based proteomics indicated that the transplanted ASCs maintained their pre-implantation phenotype up to 1 week in vivo, suggesting that the cells were not undergoing programmed cell death. A higher fraction of the infiltrating host macrophages expressed CD68 and Arginase-1 in the ASC-seeded implants up to 1-week post-implantation. Interestingly, a small population of phagocytic macrophages, identified by uptake of DsRED protein, was present in the DAT implants in the first 2 weeks and showed enhanced expression of CD68, Arginase-1, and CD163, along with reduced expression of iNOS. MicroCT angiography revealed a similar perfused vessel network in the seeded and unseeded groups at 4- and 8-weeks post-implantation. Overall, seeding with syngeneic ASCs modulated the host macrophage response to the DAT bioscaffolds at early timepoints, but did not impact long-term regenerative outcomes, potentially due to the rapid clearance of the donor cell population in this model.

cell biology↗

bioRxiv2023

Differential chromatin accessibility and transcriptional dynamics define breast cancer subtypes and their lineages

Breast cancer is a heterogeneous disease, and treatment is guided by biomarker profiles representing distinct molecular subtypes. Breast cancer arises from the breast ductal epithelium, and experimental data suggests breast cancer subtypes have different cells of origin within that lineage. The precise cells of origin for each subtype and the transcriptional networks that characterize these tumor-normal lineages are not established. In this work, we applied bulk, single-cell (sc), and single-nucleus (sn) multi-omic techniques as well as spatial transcriptomics and multiplex imaging on 61 samples from 37 breast cancer patients to show characteristic links in gene expression and chromatin accessibility between breast cancer subtypes and their putative cells of origin. We applied the PAM50 subtyping algorithm in tandem with bulk RNA-seq and snRNA-seq to reliably subtype even low-purity tumor samples and confirm promoter accessibility using snATAC. Trajectory analysis of chromatin accessibility and differentially accessible motifs clearly connected progenitor populations with breast cancer subtypes supporting the cell of origin for basal-like and luminal A and B tumors. Regulatory network analysis of transcription factors underscored the importance of BHLHE40 in luminal breast cancer and luminal mature cells, and KLF5 in basal-like tumors and luminal progenitor cells. Furthermore, we identify key genes defining the basal-like (PRKCA, SOX6, RGS6, KCNQ3) and luminal A/B (FAM155A, LRP1B) lineages, with expression in both precursor and cancer cells and further upregulation in tumors. Exhausted CTLA4-expressing CD8+ T cells were enriched in basal-like breast cancer, suggesting altered means of immune dysfunction among breast cancer subtypes. We used spatial transcriptomics and multiplex imaging to provide spatial detail for key markers of benign and malignant cell types and immune cell colocation. These findings demonstrate analysis of paired transcription and chromatin accessibility at the single cell level is a powerful tool for investigating breast cancer lineage development and highlight transcriptional networks that define basal and luminal breast cancer lineages.

cancer biology↗

bioRxiv2023

Aged male and female Panx3 KO mice develop severe osteoarthritis independent of forced mechanical use.

Osteoarthritis (OA) is a multi-factorial disease associated with aging. As the molecular mechanisms underpinning the pathogenesis of this disease are unclear, there are no disease-modifying drugs to combat OA. Pannexin 3 (PANX3) has been shown to promote cartilage loss during posttraumatic OA. In contrast, the ablation of Panx3 in male mice results in spontaneous full-thickness cartilage lesions at 24 months of age. While protected from traumatic intervertebral disc (IVD) degeneration, Panx3 knockout (KO) mice show signs of IVD disease with altered disc mechanics. Whether the deleterious effects of ablating Panx3 in aging is the result from accumulated mechanical damage is unknown. We used male and female wildtype (WT) and global Panx3 KO C57Bl6 mice aged to 18 months of age. Mice were then randomized to sedentary (SED) or forced treadmill running (FEX) for 6 weeks. Knee joint tissues including patellar tendon, quadriceps and distal patellar enthesis, and synovium were analyzed histologically and through micro-CT, along with lumbar spine IVDs. Half of male and female sedentary Panx3 KO mice developed full-thickness cartilage lesions, severe synovitis, and ectopic fibrocartilage deposition and calcification of the knee joints in comparison to all other conditions. Panx3 KO mice with severe OA show signs of quadriceps and patellar enthesitis, characterized by bone and marrow formation. Forced treadmill running did not seem to exacerbate these phenotypes in male or female Panx3 KO mice; however, it may have contributed to the development of lateral compartment OA. The IVDs of aged Panx3 KO mice displayed no apparent differences to control mice, and forced treadmill running had no further effects in either genotype. We conclude that aged Panx3 KO mice show features of late-stage primary OA including full-thickness cartilage erosion, severe synovitis, and enthesitis. These data suggest that the deletion of Panx3 is deleterious to synovial joint health in aging. LAY SUMMARYOsteoarthritis is a common joint disease, and its incidence can increase with aging. As such, no drugs are available to treat its progression. Recently, we discovered that cellular channels composed of Pannexin 3 (PANX3) proteins may contribute to the health of joint tissues. In this study, we propose a new model for age-related primary OA, using histology, microCT, and pathology assessments under sedentary and forced exercise conditions. We found that aged mice lacking the channel protein PANX3 developed late-stage OA, including severe cartilage loss, joint inflammation, and tendon attachment damage. Half of the sedentary PANX3-deficient mice showed these changes, along with abnormal bone growth. Treadmill running did not make the condition worse but may have caused additional joint damage in some areas. The spine, however, remained unaffected. These findings suggest that deleting Panx3 harms joint health with aging. Importantly, this mouse model mimics human primary OA, even without added factors like diet changes or forced exercise.

physiology↗

bioRxiv2023

Pannexin 3 deletion in mice results in knee osteoarthritis and intervertebral disc degeneration after forced treadmill running.

Pannexin 3 (Panx3) is a glycoprotein that forms mechanosensitive channels expressed in chondrocytes and annulus fibrosus cells of the intervertebral disc (IVD). Evidence suggests Panx3 plays contrasting roles in traumatic versus aging osteoarthritis (OA) and intervertebral disc degeneration (IDD). However, whether its deletion influences the response of joint tissue to mechanical stress is unknown. The purpose of this study was to determine if Panx3 deletion in mice causes increased knee joint OA and IDD after forced treadmill running. Male and female wildtype (WT) and Panx3 knockout (KO) mice were randomized to either a no exercise group (sedentary; SED) or daily forced treadmill running (forced exercise; FEX) from 24 to 30 weeks of age. Knee cartilage, tibial secondary ossification center and IVD histopathology were evaluated by histology. Both male and female Panx3 KO mice developed larger superficial defects of the tibial cartilage after forced treadmill running compared to SED WT mice. Additionally, both male and female Panx3 KO mice developed greater bone area of the tibial secondary ossification center with running. In the lower lumbar spine, both male and female Panx3 KO mice developed histopathological features of IDD after running compared to SED WT mice. These findings suggest that the combination of deleting Panx3 and forced treadmill running induces OA and causes histopathological changes associated with degeneration of the IVDs in mice.

physiology↗

bioRxiv2022

Hierarchical sequence-affinity landscapes shape the evolution of breadth in an anti-influenza receptor binding site antibody

Broadly neutralizing antibodies (bnAbs) that neutralize diverse variants of a particular virus are of considerable therapeutic interest1. Recent advances have enabled us to isolate and engineer these antibodies as therapeutics, but eliciting them through vaccination remains challenging, in part due to our limited understanding of how antibodies evolve breadth2. Here, we analyze the landscape by which an anti-influenza receptor binding site (RBS) bnAb, CH65, evolved broad affinity to diverse H1 influenza strains3, 4. We do this by generating an antibody library of all possible evolutionary intermediates between the unmutated common ancestor (UCA) and the affinity-matured CH65 antibody and measure the affinity of each intermediate to three distinct H1 antigens. We find that affinity to each antigen requires a specific set of mutations - distributed across the variable light and heavy chains - that interact non-additively (i.e., epistatically). These sets of mutations form a hierarchical pattern across the antigens, with increasingly divergent antigens requiring additional epistatic mutations beyond those required to bind less divergent antigens. We investigate the underlying biochemical and structural basis for these hierarchical sets of epistatic mutations and find that epistasis between heavy chain mutations and a mutation in the light chain at the VH-VL interface is essential for binding a divergent H1. Collectively, this work is the first to comprehensively characterize epistasis between heavy and light chain mutations and shows that such interactions are both strong and widespread. Together with our previous study analyzing a different class of anti-influenza antibodies5, our results implicate epistasis as a general feature of antibody sequence-affinity landscapes that can potentiate and constrain the evolution of breadth.

evolutionary biology↗

bioRxiv2022

Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein

Cytoplasmic dynein plays crucial roles in the intracellular transport of organelles and other cargoes. Central to dynein function is the intrinsically disordered N-terminal domain of dynein intermediate chain (IC), which binds the three dimeric dynein light chains at multivalent sites, and dynactin p150Glued and nuclear distribution protein (NudE) at overlapping sites. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography studies of its structure and interactions. Here we use a suite of biophysical methods to reveal how multivalent binding of the three light chains regulate IC interactions with p150Glued and NudE. Using the N-terminal domain or the full-length IC from Chaetomium thermophilum, a tractable species to interrogate IC interactions, we identify a significant reduction in ICs binding affinity for p150Glued and a loss of binding to NudE in contrast to the tight binding observed with small IC constructs. We attribute this difference to autoinhibition caused by strong long-range intramolecular interactions that cover ICs N-terminal single -helix, the site for p150Glued and NudE binding. Reconstitution of IC subcomplexes demonstrate that autoinhibition is differentially regulated by light chains binding underscoring their importance both in assembly and organization of IC, and in selection between multiple binding partners at the same site.

biophysics↗

bioRxiv2022

Computational model of fractal interface formation in bacterial biofilms

Bacteria benefit from cellular heterogeneity: cells differentiate into diverse gene expression states. As colonies grow, cellular phenotypes arrange into spatial patterns. To uncover the functional role of these emergent patterns, we must understand how they arise from cellular growth and mechanical interactions. Here we present a simple, agent-based model to predict patterns of motile and extracellular matrix-producing cells in biofilms of Bacillus subtilis. By incorporating phenotypic inheritance, mechanical interactions, and peripheral motile cell dispersal, our model predicts the emergence of a pattern: matrix cells surround a fractal-like interior motile population. We find that, while some properties of the motile-matrix interface depend on initial conditions, the motile distribution at large radii depends solely on the models growth mechanism. The phenotypic interface exhibits a fractal dimension that increases as biofilms grow but reaches a maximum as the peripheral layer of matrix cells exceeds the capacity of the inner cells to push it out of the way. By varying parameters, we find correlations between the interface fractal dimension and expansion of motile cells. We validate findings using experiments on B. subtilis biofilms in microfluidics. Our model demonstrates the emergence of colony-level phenotypes from single cell-level interactions and cells modifying their own environment.

biophysics↗

bioRxiv2022

Characterization of enterovirus D68 infection in four nonhuman primate species

Human enterovirus D68 (EV-D68) is a globally reemerging respiratory pathogen that is associated with the development of acute flaccid myelitis (AFM) in children. Currently, there are no approved vaccines or treatments for EV-D68 infection, and there is a paucity of data related to the virus and host specific factors that predict disease severity and progression to the neurologic syndrome. Published animal models of EV-D68 infection to date have been limited to mice, cotton rat and ferrets, and investigation of the susceptibility of nonhuman primate (NHP) species to contemporary EV-D68 isolates has not yet been reported. In this study, we challenged juvenile NHPs - cynomolgus macaques, rhesus macaques, pigtailed macaques, and African green monkeys - with one of five different 2014 or 2018 EV-D68 isolates by the respiratory route. Animals were monitored for clinical respiratory and neurologic signs, and serially collected nasal swabs, bronchoalveolar lavage fluid (BALF) and cerebrospinal fluid (CSF) were evaluated for EV-D68 RNA and infectious virus. Infection with 2014 and 2018 EV-D68 isolates resulted in mild respiratory and gastrointestinal disease in some animals, but no evidence of neurological disease. Neither EV-D68 RNA nor infectious virus could be detected from any sample collected from animals challenged with 2014 EV-D68 isolates. Limited viral shedding - based on viral RNA quantified from nasal swabs and BALF - was detected from some animals infected with 2018 EV-D68 isolates. No virus was detectable in CSF. The rate of seroconversion was 100% for cynomolgus macaques infected with the 2018 EV-D68 isolates, but averaged between 0-50% for the 2014 isolates. Based on the results of this study, there is some evidence that infection with 2018 EV-D68 isolates may be more reliable at establishing limited infection than 2014 EV-D68 isolates. Regardless of virus isolate, however, EV-D68 infection of juvenile NHP species resulted in mild and nonspecific clinical disease and limited viral shedding. These data suggest that further refinements to the NHP model system (e.g., immunosuppression and/or direct viral inoculation) may be required to reproduce EV-D68 infection of the central nervous system and the associated AFM phenotype.

microbiology↗

bioRxiv2017

Performance of serological antibody tests for bovine tuberculosis in cattle from infected herds in Northern Ireland

The ability to accurately identify infected hosts is the cornerstone of effective disease control and eradication programs. In the case of bovine tuberculosis, caused by infection with the pathogen Mycobacterium bovis, accurately identifying infected individual animals has been challenging as all available tests exhibit less than 100% discriminatory ability. Here we assess the utility of three serological tests and assess their performance relative to skin test (Single Intradermal Comparative Cervical Tuberculin; SICCT), gamma-interferon (IFN{gamma}) and post-mortem results in a Northern Ireland setting. Furthermore, we describe a case-study where one test was used in conjunction with statutory testing.\n\nSerological tests using samples taken prior to SICCT disclosed low proportions of animals as test positive (mean 3% positive), despite the cohort having high proportions with positive SICCT test under standard interpretation (121/921; 13%) or IFN{gamma} (365/922; 40%) results. Furthermore, for animals with a post-mortem record (n=286), there was a high proportion with TB visible lesions (27%) or with laboratory confirmed infection (25%). As a result, apparent sensitivities within this cohort was very low ([≤]15%), however the tests succeeded in achieving very high specificities (96-100%). During the case-study, 7/670 (1.04%) samples from SICCT negative animals from a large chronically infected herd were serology positive, with a further 10 animals being borderline positive (17/670; 2.54%). 9/17 of these animals were voluntarily removed, none of which were found to be infected (-lesions/-bacteriology) post-mortem; 1 serology test negative animal was subsequently lesion+ and M bovis confirmed at slaughter.\n\nImportanceEradication of bovine tuberculosis (bTB; caused by Mycobacterium bovis) has remained elusive in a number of countries despite long-term coordinated test and cull programs. This can partially be explained by the limitations of available statutory tests; therefore supplementary test platforms that identify additional infected animals would be of significant utility. Overall, during our study three serological tests did not disclose a high proportion of animals as infected in high-risk cattle herds, and exhibited limited ability to disclose animals that were positive to the statutory skin test, the gamma interferon test (IFN{gamma}), or were post-mortem confirmed with M. bovis. These serological tests could be used in a supplementary fashion to the statutory tests in particular circumstances; but may be of limited advantage where parallel use of IFN{gamma} and skin testing is performed, as these tests together tended to disclose the majority of animals with post-mortem evidence of infection in our study cohort.

epidemiology↗