Biology subjects

Allen, J. L.

Publications and source records attributed to Allen, J. L..

14 recordsLinked to original sources

bioRxiv2026

Multimodal Imaging of the Cellular and Extracellular Microenvironment on the Same Formalin-Fixed Paraffin-Embedded Tissue Section

Same-tissue section multimodal imaging is a powerful strategy that spatially profiles tissue histology, cell populations and molecular composition while maximizing tissue economy, preserving spatial molecular relationships, and increasing co-registration capacity. However, performing multiple modalities on the same tissue section can destroy or chemically alter the tissue, compromising downstream data. Here, we systematically assess integration of picrosirius red staining, hematoxylin and eosin staining, and multiplexed immunofluorescence into N-glycan and extracellular matrix peptide matrix-assisted laser/desorption ionization imaging mass spectrometry (IMS) workflows. We evaluate alterations in tissue morphology, stain efficiency, IMS feature intensity as well as IMS feature localization after upstream modality integration. We propose an optimized multimodal sequence that maximizes data quality and follows a very specific order of: autofluorescence microscopy, multiplexed immunofluorescence, picrosirius red staining, N-glycan IMS, hematoxylin and eosin staining, and extracellular matrix peptide IMS. Overall, this work develops an optimized multimodal workflow that comprehensively images tissue morphology, collagen fibers, and cell populations at single-cell resolution as well as multiplexed N-glycan composition and multiplexed extracellular matrix peptides with post-translational modification status from a single tissue section.

biochemistry↗

bioRxiv2026

Endosome motility controls light-responsive reproductive development and secondary metabolite production in Aspergillus

Filamentous fungi, such as Aspergillus species, use microtubule transport to move early endosomes. Other cargos, such as peroxisomes and mRNAs, "hitchhike" on early endosomes to move throughout the long hyphae of these organisms. In Aspergillus nidulans, peroxisomes hitchhike on early endosomes using the endosomal protein PxdA and the peroxisomal protein AcbdA. The HookA adaptor protein links endosomes to microtubule motors. Here, we set out to explore the physiological functions of peroxisome hitchhiking and endosome motility. Aspergillus nidulans has a complex life cycle that includes asexual and sexual reproduction. A. nidulans and other fungi within the Pezizomycotina subphylum are also notable for the vast number of secondary metabolites they produce. Light and other environmental conditions influence developmental decisions and secondary metabolite production. Here, we found that sexual reproduction is favored in the absence of endosome motility, even in the light, which normally promotes asexual reproduction. RNA sequencing of strains lacking PxdA-marked motile early endosomes showed altered expression of genes involved in development. Unexpectedly, we also observed altered expression of genes involved in secondary metabolism in strains lacking endosome motility and peroxisome hitchhiking. Using mass spectrometry, we found that the loss of endosome motility affected the biosynthesis of secondary metabolites, including sterigmatocystin, a carcinogenic mycotoxin that is a food contaminant. Finally, in a pathogenic species, Aspergillus fumigatus, we found that deletion of its pxdA homolog also significantly altered secondary metabolite production. Our work uncovers an unexpected link between organelle motility, developmental decisions in response to light, and secondary metabolite production in filamentous fungi.

cell biology↗

bioRxiv2025

Rarity and reproductive biology drive population genomics of obligate symbioses

Modern perspectives on obligate symbioses increasingly recognize their dynamism, complexity, and global importance. Population genomics has been key to this advancement, yet the extension of existing frameworks to reproducibly integrate across multiple genotype-phenotype-environment relationships remains elusive. We generated 962 metagenomes for six lichens, obligate symbioses between fungi and algae, with contrasting traits sampled at a landscape scale. Core neutral population metrics were primarily driven by rarity and reproductive biology, while genes underlying expression regulation were under selection in response to land use and climate across all species. Examined within a comparative framework, these results illustrate that symbiont interactions are a major factor influencing microevolutionary trajectories, especially neutral processes. We present a conceptual framework for population-scale evolution in obligate symbioses that centers on biotic interactions.

evolutionary biology↗

bioRxiv2025

Helitrons are enriched in lichenized fungi with long generation lengths and small distribution sizes

Mobile genetic elements (MGEs) have the potential to drive genome evolution by introducing mutations and causing structural instability and chromosomal rearrangements, particularly under conditions like environmental or genetic stress. In this study, we generated 18 new long-read based reference genomes for lichenized fungi, which form obligate mutualistic symbioses with algae or cyanobacteria. We used the new genomes and 10 publically available genomes to investigate the relationships between species traits (i.e., dominant reproductive mode, distribution size, and generation length) and the abundance and spatial distribution of MGEs using a phylogenetic comparative framework. We found that species with smaller distribution sizes and longer generation lengths had a higher genomic DNA transposon load. Specifically, their genomes were enriched with Rolling Circle transposons, which contradicts previous research that has identified high proportions of retrotransposons in rare species. Disproportionate distributions of MGEs in rare and range-restricted species may disrupt genomic stability, decrease fitness, and be reflective of species experiencing a greater degree of stress. Conversely, greater MGE activity may be an important source of novel genetic diversity in isolated populations with limited gene flow. Further research is needed to understand the potential mechanisms driving MGE proliferation in rare species genomes, and if MGE content is predictive of increased extinction risk.

genomics↗

bioRxiv2025

A Multimodal Imaging Pipeline for the Discovery of Molecular Markers of Cellular Neighborhoods

Insights into tissue microenvironments are constrained by profiling cellular and molecular components independently. To address this, we developed an integrated, multimodal workflow combining imaging mass spectrometry and highly multiplexed immunofluorescence on the same sample. Custom computational pipelines enable precise spatial co-registration, facilitating direct integration of cellular and molecular profiles. CODEX-derived cellular and neighborhood masks enable mining of IMS-derived lipid data to identify molecular markers of cell types and spatial neighborhoods in human kidney tissue. We discovered distinct carnitine distributions in cortical and medullary regions, indicating metabolic heterogeneity driven by microenvironment-specific oxygen levels and energy demands. Glomerular cell-type-specific analysis found ether-linked phosphatidylcholine and sphingomyelin, species associated with slit diaphragm integrity, localized to podocytes. Immune cell neighborhoods exhibited molecular signatures consistent with cell signaling and activation pathways in damaged tissue regions. This multi-omic framework links cellular organization with molecular signatures to unveil cellular and molecular relationships underlying tissue function and pathology.

molecular biology↗

bioRxiv2024

MOTOR MODULES ARE LARGELY UNAFFECTED BY PATHOLOGICAL WALKING BIOMECHANICS: A SIMULATION STUDY

BackgroundMotor module (a.k.a. muscle synergy) analysis has frequently been used to provide insight into changes in muscle coordination associated with declines in walking performance, to evaluate the effect of different rehabilitation intervention, and more recently, to control exoskeletons and prosthetic devices. However, it remains unclear whether changes in muscle coordination revealed via motor module analysis stem from pathological walking biomechanics or pathological neural control. This distinction has important implications for the use of motor module analysis for rehabilitation interventions and device design. Thus, this study aims to elucidate the extent to which motor modules emerge from pathological walking biomechanics. MethodsWe conducted a series of computer simulations using OpenSim Moco to simulate abnormal biomechanics by manipulating speed, asymmetry, and step width in a three-dimensional musculoskeletal model. We extracted motor modules using nonnegative matrix factorization from the muscle activation from each simulation. We then examined how alterations in walking biomechanics influenced the number and structure of extracted motor modules and compared the findings to previous experimental studies. ResultsThe motor modules identified from our simulations were similar to those identified from previously published experiments of non-pathological walking. Moreover, our findings indicate that the same motor modules can be used to generate a range of pathological-like waking biomechanics by modulating their recruit timing over the gait cycle. These results contrast with experimental studies in which pathological-like walking biomechanics are accompanied by a reduction in motor module number and alterations in their structure. ConclusionsThis study highlights that pathological walking biomechanics do not necessarily require pathological motor modules. In other words, changes in number and structure of motor modules can be a valuable indicator of alterations in neuromuscular control and may therefore be useful for guiding rehabilitation interventions and controlling exoskeletons and prosthetic devices in individuals with pathological walking function.

bioengineering↗

bioRxiv2023

An end-to-end pipeline for succinic acid production at an industrially relevant scale using Issatchenkia orientalis

As one of the top value-added chemicals, succinic acid has been the focus of numerous metabolic engineering campaigns since the 1990s. However, microbial production of succinic acid at an industrially relevant scale has been hindered by high downstream processing costs arising from neutral pH fermentation. Here we describe the metabolic engineering of Issatchenkia orientalis, a non-conventional yeast with superior tolerance to highly acidic conditions, for cost-effective succinic acid production. Through deletion of byproduct pathways, transport engineering, and expanding the substrate scope, the resulting strains could produce succinic acid at the highest titers in sugar-based media at low pH (pH 3) in fed-batch fermentations using bench-top reactors, i.e. 109.5 g/L in minimal medium and 104.6 g/L in sugarcane juice medium. We further performed batch fermentation in a pilot-scale fermenter with a scaling factor of 300x, achieving 63.1 g/L of succinic acid using sugarcane juice medium. A downstream processing comprising of two-stage vacuum distillation and crystallization enabled direct recovery of succinic acid, without further acidification of fermentation broth, with an overall yield of 64.0%. Finally, we simulated an end-to-end low-pH succinic acid production pipeline, and techno-economic analysis and life cycle assessment indicate our process is financially viable and can reduce life cycle greenhouse gas emissions by 34-90% relative to fossil-based production processes. We expect I. orientalis can serve as a general industrial platform for the production of a wide variety of organic acids.

synthetic biology↗

bioRxiv2022

A multiscale atlas of the molecular and cellular architecture of the human kidney

Tissue atlases provide foundational knowledge on the cellular organization and molecular distributions across molecular classes and spatial scales. Here, we construct a comprehensive spatio-molecular lipid atlas of the human kidney from 29 donor tissues using integrated multimodal molecular imaging. Our approach leverages high spatial resolution matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) for untargeted lipid mapping, stained microscopy for histopathological assessment, and tissue segmentation using autofluorescence microscopy. With a combination of unsupervised, supervised, and interpretive machine learning, the atlas provides multivariate lipid profiles of specific multicellular functional tissue units (FTUs) of the nephron, including the glomerulus, proximal tubules, thick ascending limb, distal tubules, and collecting ducts. In total, the atlas consists of tens of thousands of FTUs and millions of mass spectrometry measurements. Detailed patient, clinical, and histopathologic information allowed molecular data to be mined based on these features. As examples, we highlight the discovery of how lipid profiles are altered with sex and differences in body mass index.

biochemistry↗

bioRxiv2021

Direct Imaging and Identification of Proteoforms up to 70 kDa from Human Tissue

Imaging of proteoforms in human tissues is hindered by low molecular specificity and limited proteome coverage. Here, we introduce proteoform imaging mass spectrometry (PiMS), which increases the size limit for proteoform detection and identification by 4-fold compared to reported methods, and reveals tissue localization of proteoforms at <80 m spatial resolution. PiMS advances proteoform imaging by combining ambient nanospray desorption electrospray ionization (nano-DESI) with ion detection using individual ion mass spectrometry (I2MS). We demonstrate the first proteoform imaging of human kidney, identifying 169 of 400 proteoforms <70 kDa using top-down mass spectrometry and database lookup from the human proteoform atlas, including dozens of key enzymes in primary metabolism. PiMS images reveal distinct spatial localizations of proteoforms to both anatomical structures and cellular neighborhoods in the vasculature, medulla, and cortex regions of the human kidney. The benefits of PiMS are poised to increase proteome coverage for label-free protein imaging of tissues. TeaserNano-DESI combined with individual ion mass spectrometry generates images of proteoforms up to 70 kDa.

biochemistry↗

bioRxiv2021

Autofluorescence microscopy as a label-free tool for renal histology and glomerular segmentation

Automated spatial segmentation models can enrich spatio-molecular omics analyses by providing a link to relevant biological structures. We developed segmentation models that use label-free autofluorescence (AF) microscopy to recognize multicellular functional tissue units (FTUs) (glomerulus, proximal tubule, descending thin limb, ascending thick limb, distal tubule, and collecting duct) and gross morphological structures (cortex, outer medulla, and inner medulla) in the human kidney. Annotations were curated using highly specific multiplex immunofluorescence and transferred to co-registered AF for model training. All FTUs (except the descending thin limb) and gross kidney morphology were segmented with high accuracy: >0.85 F1-score, and Dice-Sorensen coefficients >0.80, respectively. This workflow allowed lipids, profiled by imaging mass spectrometry, to be quantitatively associated with segmented FTUs. The segmentation masks were also used to acquire spatial transcriptomics data from collecting ducts. Consistent with previous literature, we demonstrated differing transcript expression of collecting ducts in the inner and outer medulla.

bioinformatics↗

bioRxiv2021

Assessing the role of cold-shock protein C: A novel regulator of Acinetobacter baumannii biofilm formation and virulence

Acinetobacter baumannii is a formidable opportunistic pathogen that is notoriously difficult to eradicate from hospital settings and can spread quickly via healthcare personnel despite preventive measures. This resilience is often attributed to a proclivity for biofilm formation, which grants A. baumannii a higher tolerance towards external stress, desiccation, and antimicrobials. Despite this, little is known regarding the mechanisms orchestrating A. baumannii biofilm formation. Herein, we performed RNA-seq on biofilm and planktonic populations for the multidrug resistant isolate, AB5075, and identified 438 genes with altered expression. To assess the potential role of genes upregulated within biofilms, we tested the biofilm forming capacity of their respective mutants from an A. baumannii transposon library. In so doing, we uncovered 24 genes whose disruption led to reduced biofilm formation. One such element, cold shock protein C (cspC), produced a mucoidal, non-mucoviscous colony phenotype. RNA-sequencing of this mutant revealed the down regulation of pili and fimbriae in the cspC mutant, which would explain the decreased biofilm observed. Using MIC assays, we note that the mutant displayed increased antibiotic tolerance that we hypothesize is mediated by overexpression of multi-drug efflux pumps and altered mRNA stability of their corresponding transcriptional repressor. Finally, we show that CspC is required for survival during oxidative stress and challenge by the human immune system, and plays a pivotal role during systemic infection. Collectively, our work identifies a cadre of new biofilm associated genes within A. baumannii and provides insight into the global regulatory network of this emerging human pathogen.

microbiology↗

bioRxiv2021

Young Adults Recruit Similar Motor Modules Across Walking, Turning, and Chair Transfers

Moving about in the world during daily life requires executing and successfully shifting between a variety of functional tasks, such as arising from a chair or bed, walking, turning, and navigating stairs, etc. Moreover, moving about during daily life requires not only navigating between different functional tasks but also performing these tasks in the presence of mental distractions. However, little is known about underlying neuromuscular control for executing and shifting between these different tasks. In this study, we investigated muscle coordination across walking, turning, and chair transfers by applying motor module (aka muscle synergy) analysis to the Timed-Up-and-Go (TUG) test with and without a secondary cognitive dual task. We found that healthy young adults recruit a small set of common motor modules across the subtasks of the TUG test and that their composition is robust to cognitive distraction. Instead, cognitive distraction impacted motor module activation timings such that they became more consistent. This work is the first to demonstrate motor module generalization across multiple tasks that are both functionally different and crucial for healthy mobility. Overall, our results suggest that the central nervous system may draw from a "library" of modular control strategies to navigate the variety of movements and cognitive demands required of daily life. New & NoteworthyWe demonstrated that healthy young adults recruit a small set of motor modules across subtasks of the Timed-Up-and-Go test (i.e., walking, turning, and chair transfers). Moreover, we showed that motor module composition, but not activation timing, is robust to cognitive distractions. These results support the hypothesis that healthy young adults recruit from a "library" of motor modules and modulate their activation timing to meet the different mechanical and cognitive demands required to navigate daily life.

neuroscience↗

bioRxiv2019

Spatial distribution of Salmonella enterica in poultry shed environments observed by intensive longitudinal environmental sampling

During a series of epidemiological studies to investigate the transmission of salmonellae within poultry environments, intensive longitudinal sampling within caged sheds revealed that both the number and location of sample collection within this environment were important to ensure the best chance of detecting Salmonella spp. Multiple serovars of Salmonella enterica subspecies enterica were detected in each shed; 5% of all samples contained more than one serovar. Samples collected on the north side of the shed (OR = 1.77, 95% CI [1.17, 2.68]), on the sheltered side of the shed (OR = 1.90, 95% CI [1.26, 2.89]) and during winter (OR = 48.41, 95%CI [23.56, 104.19]) were more likely to be positive for salmonellae. The distribution of salmonellae within a shed was not homogenous, as differences were identified in the within-shed distribution of Salmonella enterica subspecies enterica serovar Typhimurium ({chi}2 (27, 1,538) = 54.4, P < 0.001), and Salmonella enterica subspecies enterica serovar Infantis ({chi}2 (27, 1,538) = 79.8, P < 0.0001). The difference in sample prevalence by serovar and spatial location within a shed indicates that there are important shed micro-environmental factors that influence survival and/or distribution of salmonellae. These factors should be taken into consideration when undertaking environmental surveillance for salmonellae in flocks housed in caged sheds. IMPORTANCERoutine epidemiological surveillance for salmonellae in poultry relies initially on environmental sampling. Our study confirmed that the sampling methodology conducted within a poultry environment is a non-trivial part of sampling design. The spatial locations of sample collection within a shed revealed differences in both the sample prevalence and serovar of salmonellae detected.

epidemiology↗

bioRxiv2018

Motor module generalization across balance and walking is reduced after stroke

Here, we examined features of muscle coordination associated with reduced walking performance in chronic stroke survivors. Using motor module (a.k.a. muscle synergy) analysis, we identified differences in the modular control of overground walking and standing reactive balance in stroke survivors compared to age-similar neurotypical controls. In contrast to previous studies that demonstrated reduced motor module number post-stroke, our cohort of stroke survivors did not exhibit a reduction in motor module number compared to controls during either walking or reactive balance. Instead, the pool of motor modules common to walking and reactive balance was smaller, suggesting a reduction in generalizability of motor module function across behaviors. The motor modules common to walking and reactive balance tended to be less variable and more distinct, suggesting more reliable output compared to motor modules specific to one behavior. Indeed, higher levels of motor module generalization was associated with faster walking speeds in stroke survivors. Further, recruitment of a common independent plantarflexor module across both behaviors was associated with faster walking speeds. Our work is the first to show that motor module generalization across walking and balance may help to distinguish important and clinically-relevant differences in walking performance across stroke survivors that would have been overlooked by examining only a single behavior. Finally, as similar relationships between motor module generalization and walking performance have been demonstrated in healthy young adults and individuals with Parkinsons disease, our work suggests that motor module generalization across walking and balance may be important for well-coordinated walking.\n\nNew and NoteworthyOur study is the first to simultaneously examine neuromuscular control of walking and standing reactive balance in stroke survivors. We show that motor module generalization across these behaviors (i.e., recruiting common motor modules) is reduced compared to neurotypical controls, which is associated with slower walking speeds. This is true despite no difference in motor module number between groups within each behavior, suggesting that motor module generalization across walking and balance is important for well-coordinated walking.

bioengineering↗