Biology subjects

Allen, A.

Publications and source records attributed to Allen, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Scale- and genotype-dependent spatial concordance of Mycobacterium bovis in cattle and badgers

Mycobacterium bovis causes tuberculosis (TB) in cattle, badgers (Meles meles), other wildlife, and humans, posing major animal and public health challenges in Britain and Ireland. Shared genotypes between cattle and badgers have been reported at regional scales, but whether this concordance holds at finer spatial scales, where control interventions operate, and whether it varies by genotype combination, remains unknown. Resolving this is critical for interpreting cross-species transmission risk and designing targeted control measures. We analyzed 1,248 M. bovis isolates from cattle and badgers (Northern Ireland, 1986-2018), including samples from a small, densely sampled intervention research area, using Multi-Locus Variable Number of Tandem Repeats Analysis (MLVA) and kernel discriminant analysis (KDA) across multiple spatial scales (25-95% density contours). We identified 36 MLVA types with four shared between species and one (MLV06) endemic to the area. Spatial concordance varied by type combination and scale: MLVA004-MLVA297 showed strong, bidirectional, scale-robust concordance; MLVA006-MLVA297 was concordant only at fine spatial scales; MLVA004-MLVA006 showed weak, asymmetric concordance; and no concordance was detected when all three types were analysed together. Our findings reveal spatial concordance between shared M. bovis genotypes varied by genotype combination and scale, indicating genotype-specific dynamics within this multi-host system.

ecology↗

bioRxiv2026

TIMS-Bench: Towards community standards for benchmarking untargeted trapped ion mobility metabolomics tools and datasets

Untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics is an important technology for unbiased discovery of small molecules in biomedical (e.g., drug discovery to diagnostics), animal, plant, environmental, and microbial research. Over the past decade, ion mobility has added an additional dimension to the triplet of MS1, MS2, and retention time, helping resolve co-eluting or isomeric features in an LC-MS/MS that aid in compound identification. Here, we focused on evaluating the current trapped ion mobility spectrometry (TIMS)-amenable feature-finding tools (MZmine 4.9, MS-DIAL 5.5, and MetaboScape 2025 14.0.3) for pre-processing of metabolomics data generated using a popular ion mobility mass spectrometry (IM-MS) technique, TIMS. We leveraged ten public and three benchmark TIMS datasets to evaluate these tools for their strengths and weaknesses. Our results show that MZmine consistently identified the highest number of features and confidently annotated features; however, this performance was accompanied by an increased number of false positives, due to peak splitting, as well as reduced accuracy in collision cross section (CCS) measurements. In contrast, MetaboScape achieved the highest fraction of high-quality MS2 spectra, reflecting a more conservative feature detection strategy. MS-DIAL demonstrated balanced performance, identifying features that other tools missed. Finally, we publicly release the ground-truth datasets and code to support future developments in improving IMS data analysis.

bioinformatics↗

bioRxiv2026

PDE4 Inhibitor Apremilast Rebalances Inflammatory Responses to Pseudomonas aeruginosa Infection in CF Rats

CFTR modulator therapies have transformed CF care, yet chronic airway inflammation persists in many people with cystic fibrosis (pwCF) even after long-term highly effective modulator therapy (HEMT). Because of the adverse side effects or the incompatibility with CFTR modulators, the use of traditional anti-inflammatory therapies is very limited in CF. Hence, new therapeutic strategies that rebalance inflammation without worsening infection with immunosuppression are needed. We evaluated the selective phosphodiesterase 4 (PDE4) inhibitor apremilast (Apr) for its ability to modulate dysregulated inflammation in humanized CF (G551D) rats acutely challenged with Pseudomonas aeruginosa. Apr is an approved anti-inflammatory therapeutic strategy for several chronic inflammatory conditions, but it has not been well studied in CF. In the humanized CF (G551D) rats, a short prophylactic Apr regimen significantly preserved lung function and reduced lung injury, accompanied by broad modulation of inflammatory responses, notably within Th1 and Th17 axes. Importantly, Apr did not cause a significant increase in bacterial burden. Just as importantly, Apr did not reduce CFTR mRNA or protein in vivo, and it increased G551D-CFTR phosphorylation critical for channel gating in vitro, supporting mechanistic compatibility with HEMT. These findings suggest Apr as a potential adjunct to CFTR modulators to rebalance airway inflammation while preserving host defense.

immunology↗

bioRxiv2025

Highly Efficient Multiplexed Genome Engineering and Clone Selection to Enable Next Generation Induced Pluripotent Stem Cell (iPSC)-based Cell Therapies

Human induced pluripotent stem cells (iPSCs) have revolutionized regenerative medicine and cellular therapies. To improve the functionality and safety of iPSC-based therapies, genome engineering has been employed to disrupt gene expression and introduce therapeutic transgenes. However, current genome editing methods face significant challenges, including labor-intensive procedures, low efficiency, and safety concerns. Here, we report a novel and efficient approach for multiplex genome engineering in iPSCs using Integrase-mediated Programmable Genomic Integration (I-PGI) technology. I-PGI combines CRISPR-mediated genome editing and site-specific integrases, enabling precise gene insertion without DNA double-strand breaks. By optimizing I-PGI components and protocols, we achieved multiple gene knockouts and knock-ins of up to five transgenes in a single process. Additionally, we developed a streamlined workflow for enrichment, deposition, and screening of single-cell clones with the desired genome edits. This approach significantly accelerates and improves the precision of multiplex iPSC engineering, paving the way for next-generation iPSC-based therapies.

genomics↗

bioRxiv2025

Loss of tight junction protein claudin 18 uncovers alveolar epithelial stem cell plasticity and emergence of non-fibrogenic transitional progenitors

Persistence of senescent alveolar transitional progenitors following lung injury is implicated in the pathogenesis of fibrosis. We identified transitional cells in uninjured Cldn18 knockout (KO) mouse lungs distinct from previously reported damage-associated transitional progenitors (DATPs) with a less fibrogenic transcriptomic profile. Cldn18 KO mice are protected from bleomycin-induced fibrosis, with early restoration of cellular homeostasis. Lineage tracing implicates accelerated differentiation as a mechanism for protection from fibrosis, leading us to name these cells regeneration-associated transitional progenitors (RATPs). Multiome confirms that RATPs and DATPs are epigenetically distinct, with RATPs comprised of RATP2s and RATP1s based on epigenomic proximity to AT2s and AT1s, respectively, and suggests dynamic regulatory remodeling during AT2-to-AT1 differentiation, with NKX2.1 and AP-1 active in early transitions and TEAD factors in later stages. These results reveal an unexpected role for Cldn18 in regulation of AEC plasticity, while identification of RATPs challenges the notion that persistence of transitional alveolar cells is invariably pathologic.

cell biology↗

bioRxiv2025

Hierarchical microstructural tissue growth of the gray and white matter of human visual cortex during the first year of life

Development of gray and white matter tissue microstructure is critical for the emergence of sensory and cognitive functions. However, it is unknown how microstructural tissue properties of the human visual system develop in the first year of human life. Here, we use tissue relaxation rate (R1) obtained using quantitative MRI to measure the longitudinal development of gray and white matter in brain areas spanning three visual processing streams: dorsal, lateral, and ventral, during the first year of life. R1 in gray and white matter of all visual regions in the three processing streams increases postnatally, indicating microstructural tissue growth. R1 increases faster between 0-6 months than 6-12 months, and faster in white matter than gray matter, with white matter R1 surpassing that of gray matter after two months of age. Strikingly, this microstructural growth is hierarchical: across all streams, early visual areas are more mature at birth than higher-level areas but develop more slowly postnatally than higher-level areas. The exception is TO1 (MT) which is similar to V1: it is microstructurally more mature at birth and develops slower than neighboring areas. Overall, our findings provide the first comprehensive measurement of microstructural tissue growth in infancy across three visual processing streams and propose a new hypothesis that functional development of the visual cortex may be coupled with microstructural development and follows a similar hierarchical trajectory.

neuroscience↗

bioRxiv2025

Oncogene SETDB1s Dual Role in Endometrial Cancer: Driving Tumor Progression and Immune Escape

Oncogene SETDB1, an H3K9 methyltransferase, drives tumorigenesis in various cancers. Using endometrial cancer (EC) as a model, we discovered SETDB1s dual mechanisms in driving EC tumorigenesis and mediating immune evasion. SETDB1 knockout (SETDB1-/-) tumor-bearing mice exhibited prolonged survival of up to 100 days. Transcriptomic profiling of SETDB1-/- EC cells revealed decreased expression of oncogenes (POLR2A, MSH6, FLNA) and increased expression of tumor suppressor genes (PGR, RERG, ZNF582), which indicates that SETDB1 intrinsically promotes EC tumor proliferation by regulating these downstream genes. SETDB1 repressed repeat elements and the interferon pathway, mediating immune evasion extrinsically by inhibiting anti-tumor macrophage infiltration. ChIP-seq analysis showed SETDB1 binding at pericentromeric regions on many chromosomes and numerous ZNFs. Loss of SETDB1 resulted in abnormal cell division. SETDB1-/- tumors displayed reduced proliferation markers (Ki67, pHH3) and increased macrophage infiltration. Mechanistically, SETDB1 promotes CD47 (a dont-eat-me signal) and represses CCL5 and CXCL9 (macrophage and T-cell recruiting chemokines), contributing to immune evasion. M1-like macrophages killed more SETDB1-/- cells in co-culture. Additionally, SETDB1 knockout in mouse EC cells reduced tumor growth in C57BL/6 mice, with increased macrophage and CD4+ T-cell infiltration. Our results indicates that elevated SETDB1 and its target genes can predict higher tumor grade and worse survival, suggesting that targeting SETDB1 could be a promising therapeutic strategy for EC.

cancer biology↗

bioRxiv2025

How infant brains fold: Sulcal deepening is linked to development of sulcal span, thickness, curvature, and microstructure

Cortical folding begins in utero as sulci emerge and continues postnatally as sulci deepen. However, the timeline and mechanisms underlying postnatal sulcal development remain unknown. Using structural and quantitative magnetic resonance imaging in infants from birth to one year of age, we longitudinally measured macroanatomical and microstructural development in major sulci that emerge in utero between the 16th and 31st gestational weeks. We find that sulci that emerge earlier in utero are deeper at birth and deepen at a slower rate postnatally than later emerging sulci. Sulci also become wider, thicker, and microstructurally denser, while their curvature decreases. Notably, mean sulcal depth is predicted by a weighted sum of sulcal span, thickness, curvature, and tissue microstructure, with differential weights across sulci. Analysis of local depth along the sulcus also reveals that deeper portions of sulci (fundi) have higher curvature and higher microstructural density than the surrounding sulcal walls. These data reveal that postnatal sulcal deepening is nonuniform and depends on the time of emergence in utero, tissue microstructure, and multiple macroanatomical factors. Together, these findings have important ramifications for theories of cortical folding and elucidating neurodevelopmental disorders and delays.

neuroscience↗

bioRxiv2025

Design of solubly expressed miniaturized SMART MHCs

The precise recognition of specific peptide-MHC (pMHC) complexes by T-cell receptors (TCRs) plays a key role in infectious disease, cancer and autoimmunity. A critical step in many immunobiological studies is the identification of T-cells expressing TCRs specific to a given pMHC antigen. However, the intrinsic instability of empty class-I MHCs limits their soluble expression in Escherichia coli (E. coli) and makes it very difficult to characterize even a small fraction of possible pMHC/TCR interactions. To overcome this limitation, we designed small proteins which buttress the peptide binding groove of class I MHCs, replacing {beta}2-microglobulin ({beta}2m) and the heavy chain 3 domain, and enable soluble expression of both H-2Db and A*02:01 in E. coli. We demonstrate that these soluble, monomeric, antigen-receptive, truncated (SMART) MHCs retain both peptide- and TCR-binding specificity, and that peptide-bound structures of both allomorphs are similar to their full-length, native counterparts. With extension to the majority of HLA alleles, SMART MHCs should be broadly useful for probing the T-cell repertoire in approaches ranging from yeast display to T-cell staining. SignificanceDespite the critical role that TCR/pMHC interactions play in human health, it has remained difficult to produce reagents necessary to study them. Requirements for refolding or sequence optimization limit immunologists and biochemists ability to characterize diverse pMHC/TCR interactions. Here, we develop a de-novo designed protein domain that stabilizes the H-2Db and A*02:01 class I MHC allomorphs, allowing soluble expression in E. coli without the need for a stabilizing peptide, and improving display on the yeast surface, while maintaining peptide and TCR binding interactions. These features facilitate a wide range of experiments to more fully understand the nature of pMHC/TCR interactions, and pave the way for the development of stabilizing domains for all MHC allomorphs.

biochemistry↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2025

Defining the limits of plant chemical space: challenges and estimations

The plant kingdom, encompassing nearly 400,000 known species, produces an immense diversity of metabolites, including primary compounds essential for survival and secondary metabolites specialized for ecological interactions. These metabolites constitute a vast and complex phytochemical space with significant potential applications in medicine, agriculture, and biotechnology. However, much of this chemical diversity remains unexplored, as only a fraction of plant species have been studied comprehensively. In this work, we estimate the size of the plant chemical space by leveraging large-scale metabolomics and literature datasets. We begin by examining the known chemical space, which, while containing at most several hundred thousand unique compounds, remains sparsely covered. Using data from over 1,000 plant species, we apply various mass spectrometry-based approaches--a formula prediction model, a de novo prediction model, a combination of library search and de novo prediction, and MS2 clustering--to estimate the number of unique structures. Our methods suggest that the number of unique compounds in the metabolomics dataset alone may already surpass existing estimates of plant chemical diversity. Finally, we project these findings across the entire plant kingdom, conservatively estimating that the total plant chemical space likely spans millions, if not more, with the vast majority still unexplored.

bioinformatics↗

bioRxiv2024

Evolution of thermal tolerance in marine diatoms: Metabolic strategies under heat stress.

In the last decade, numerous laboratory experiments have demonstrated that when marine phytoplankton are exposed to thermal stress, they can evolve high temperature tolerance in a short time (weeks to months). This evolutionary potential may ensure the persistence of marine phytoplankton species under current and future global warming. However, the effect of such adaptation on the phytoplankton interaction with the environment and other organisms depends on how cellular metabolism shifts during the evolutionary process. In order to elucidate which cellular strategies allow the emergence of thermo-tolerant populations, we analyzed the proteomics response of a marine diatom (Chaetoceros simplex) to both thermal acclimation and evolutionary adaptation. We found that high temperature-tolerant populations exhibit a conservative cellular strategy when acclimated to high, above-optimal temperature, where recycling and reallocation is favored at the expense of new structures biosynthesis. While this strategy gives the populations that evolved high temperature tolerance an advantage under thermal stress, the shift to resource reallocation may explain the absence of high-temperature adaptation when cells are exposed to low nitrate availability.

ecology↗

bioRxiv2024

Design of facilitated dissociation enables control over cytokine signaling duration

Protein design has focused primarily on the design of ground states, ensuring they are sufficiently low energy to be highly populated1. Designing the kinetics and dynamics of a system requires, in addition, the design of excited states that are traversed in transitions from one low-lying state to another2,3. This is a challenging task as such states must be sufficiently strained to be poorly populated, but not so strained that they are not populated at all, and because protein design methods have generally focused on creating near-ideal structures4-7. Here we describe a general approach for designing systems which use an induced-fit power stroke8 to generate a structurally frustrated9 and strained excited state, allosterically driving protein complex dissociation. X-ray crystallography, double electron-electron resonance spectroscopy, and kinetic binding measurements demonstrate that incorporating excited states enables design of effector-induced increases in dissociation rates as high as 6000-fold. We highlight the power of this approach by designing cytokine mimics which can be dissociated within seconds from their receptors.

biochemistry↗

bioRxiv2024

Hormonal contraceptive intake during adolescence and cortical brain measures in the ABCD Study

Adolescence is a critical period in human development, marked by rapid changes in brain structure and function, influenced, in part, by sex steroids. During adolescence, females may initiate the use of hormonal contraception, which suppresses ovarian production of estradiol and progesterone. Here, we examined the impact of hormonal contraceptive use on adolescent brain structures using magnetic resonance imaging data from 1,234 individuals from the ABCD Study (average age = 14 years). We investigated differences in cortical thickness, surface area, and volume in hormonal contraceptive-users compared to non-users. Statistically significant differences were observed in several regions, and differences in cortical thickness of the paracentral gyrus survived familywise error correction. Estradiol, testosterone and DHEA levels negatively correlated with cortical thickness, surface area, and volume across both hormonal contraceptive-users and non-users only at an uncorrected threshold. These findings highlight the need to further study longitudinal effects of hormonal contraceptives and endogenous hormone changes on brain development in the ABCD Study and related datasets as these data become available.

neuroscience↗

bioRxiv2024

First record of the seagrass Halophila stipulacea (Forskkal) Ascherson in the waters of the continental United States (Key Biscayne, Florida)

The first record of Halophila stipulacea is reported for the continental waters of the United States. In August 2024, a small meadow was identified inside Crandon Marina on Key Biscayne, Florida, USA. Following surveys have revealed that H. stipulacea has spread to adjacent areas immediately outside of the marina, often growing either in close proximity to, or interspersed with, the native seagrasses Thalassia testudinum, Syringodium filiforme, and Halodule wrightii. This serves as an initial report and extends the geographic scope of this introduced species in the Western Atlantic basin.

ecology↗

bioRxiv2024

Controlling semiconductor growth with structured de novo protein interfaces

Protein design now enables the precise arrangement of atoms on the nanometer length scales of inorganic crystal nuclei, opening up the possibility of templating the growth of metal oxides including semiconductors. We designed proteins presenting regularly repeating interfaces containing functional groups that organize ions and water molecules, and characterized their ability to bind to and template metal oxides. Two interfaces promoted the growth of hematite under conditions that otherwise resulted in the formation of magnetite. Three interfaces promoted ZnO nucleation under conditions where traditional ZnO-binding peptides and control proteins were ineffective. Designed cyclic assemblies with these ZnO nucleating interfaces lining interior cavities promoted ZnO growth within the cavity. CryoEM analysis of a designed octahedral nanocage revealed atomic density likely corresponding to the growing ZnO directly adjacent to the designed nucleation promoting interfaces. These findings demonstrate that designed proteins can direct the formation of metal oxides not observed in biological systems, opening the door to protein-semiconductor hybrid materials. One Sentence SummaryWe describe the design of structured protein interfaces that bind to, promote, and localize the growth of zinc oxide and hematite, inorganic materials which are not found in biological systems.

bioengineering↗

bioRxiv2024

Ankyrin-B is required for the establishment and maintenance of lens cytoarchitecture, mechanics, and clarity

Summary StatementThis study illustrates a vital role for ankyrin-B in lens architecture, growth and function through its involvement in membrane protein and spectrin-actin cytoskeletal organization and stability The transparent ocular lens is essential for vision by focusing light onto the retina. Despite recognizing the importance of its unique cellular architecture and mechanical properties, the molecular mechanisms governing these attributes remain elusive. This study aims to elucidate the role of ankyrin-B (AnkB), a membrane scaffolding protein, in lens cytoarchitecture, growth and function using a conditional knockout (cKO) mouse model. AnkB cKO mouse has no defects in lens morphogenesis, but exhibited changes that supported a global role for AnkB in maintenance of lens clarity, size, cytoarchitecture, and stiffness. Notably, absence of AnkB led to nuclear cataract formation, evident from P16. AnkB cKO lens fibers exhibit progressive disruption in membrane organization of the spectrin-actin cytoskeleton, channel proteins, cell-cell adhesion, shape change, loss and degradation of several membrane proteins (e.g., NrCAM. N-cadherin and aquaporin-0) along with a disorganized plasma membrane and impaired ball-and-socket membrane interdigitations. Furthermore, absence of AnkB led to decreased lens stiffness. Collectively, these results illustrate the essential role for AnkB in lens architecture, growth and function through its involvement in membrane protein and cytoskeletal organization.

cell biology↗

bioRxiv2024

Design of High Affinity Binders to Convex Protein Target Sites

While there has been progress in the de novo design of small globular miniproteins (50-65 residues) to bind to primarily concave regions of a target protein surface, computational design of minibinders to convex binding sites remains an outstanding challenge due to low level of overall shape complementarity. Here, we describe a general approach to generate computationally designed proteins which bind to convex target sites that employ geometrically matching concave scaffolds. We used this approach to design proteins binding to TGF{beta}RII, CTLA-4 and PD-L1 which following experimental optimization have low nanomolar to picomolar affinities and potent biological activity. Co-crystal structures of the TGF{beta}RII and CTLA-4 binders in complex with the receptors are in close agreement with the design models. Our approach provides a general route to generating very high affinity binders to convex protein target sites.

biochemistry↗