Biology subjects

Walsh, C.

Publications and source records attributed to Walsh, C..

At least 19 recordsLinked to original sources

bioRxiv2026

BigHeart: Mapping connectivity in the adult human heart at the micron scale

The human heart depends on coordinated muscular, electrical, vascular, lymphatic, and neural systems, but their three-dimensional relationships remain unresolved at microscopic resolution across an intact adult organ. Using hierarchical phase-contrast tomography, we generated a continuous three-dimensional structural reference of a whole adult human heart at 8.01 micrometer isotropic voxel size, without sectioning or staining. Organ-wide imaging revealed hierarchical myocardial architecture, and quantitative orientation analysis identified regional differences in myocardial cell aggregate organization. The atrioventricular conduction axis was traced into a distinct subendocardial Purkinje network interfacing with working myocardium. Integrated segmentation showed that coronary vessels, lymphatic collectors, and autonomic nerves occupy shared epicardial corridors. This publicly accessible dataset integrates cardiac systems within a common spatial framework for anatomy, computational modeling, and future molecular atlases.

physiology↗

bioRxiv2026

A Single-Cell Framework for Classifying Human Th17 Pathogenicity Links Acylcarnitine Metabolism to Non-Pathogenic Inflammation in Type 2 Diabetes

Based on in vitro and animal studies, Th17 cells are classified as pathogenic (pTh17) or non-pathogenic (nTh17), but the inability to identify these subsets in primary human samples limits translation. We developed a single-cell ELISA to enrich human Th17s, enabling transcriptomic and flow-cytometric classification. nTh17 cells predominated in Type 2 diabetes and exhibited signatures of acylcarnitine synthesis, while knockdown of CPT1A demonstrated that acylcarnitine metabolism regulates Th17 pathogenicity.

immunology↗

bioRxiv2026

Bridging the microstructural gap in human connectomics using hierarchical phase-contrast tomography as a reference for diffusion MRI in the human brain

Diffusion MRI (dMRI) allows us to image the human connectome non-invasively, yet it provides indirect estimates of axonal orientations based on the diffusion of water molecules in millimeter-scale voxels, hence struggling to resolve complex micrometer-scale fiber geometries. Invasive methods for imaging axonal orientations ex vivo, e.g. histology, are destructive and limited to small volumes, creating a critical need for a non-destructive modality for imaging microscopic fiber orientations in 3D. Here, we use Hierarchical Phase-Contrast Tomography (HiP-CT) to characterize white matter architecture at the microscale. Applying structure-tensor analysis to HiP-CT data, we compute fiber Orientation Distribution Functions and perform tractography analogous to dMRI. Across multiple brain regions, HiP-CT derived fiber architecture shows strong correspondence with that derived from dMRI while revealing substantially greater microstructural complexity. Despite its label-free nature, we demonstrate that vascular structures minimally confound HiP-CT orientation estimates. These results establish HiP-CT as a reference microscopic modality that can complement dMRI in multi-scale studies of white-matter organization.

neuroscience↗

bioRxiv2026

Morphospatial profiling of cancer-associated fibroblasts reveals architectural subtypes of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with an urgent need for biomarkers to predict prognosis and guide treatment. Understanding the complex spatial biology of pancreatic cancer-associated fibroblasts (CAFs) and the broader architecture of the PDAC tumour microenvironment is central to this challenge. Using a multi-omics approach across multiple spatial resolutions in a large human PDAC cohort, we integrate geometry and shape to define discrete morphological CAF subtypes, expanding CAF phenotyping beyond conventional proteomics. We then reveal an architectural and molecular axis of PDAC at tissue level, suggestive of epithelial-stromal co-evolution, with translational implications and prioritisation of stromal targets. Finally, we recapitulate this axis by introducing four unique, internally validated architectural subtypes of PDAC, each characterised by a common microenvironment and CAF enrichment profile. These archetypes outperform conventional pathology in prognostication, and predict response to adjuvant chemotherapy. Collectively, this study establishes a novel morphological paradigm for spatial biology, illuminates the architectural landscape of PDAC, and provides a framework for spatial biomarker discovery to close the translational gap in this devastating disease.

cancer biology↗

bioRxiv2026

Sonogenetic control of cardiomyocytes and cardiac pacing using exogenous Transient Receptor Potential A1 channels

Electronic cardiac pacemakers are the standard of care for treating arrhythmias but, even for wireless pacemakers, they require an intracardiac implant procedure and are invasive. Lead-related complications, including life-threatening infections, and limited suitability in pediatric patients restrict pacemaker use. Biological pacing has emerged as a hardware-free alternative, leveraging cell reprogramming approaches to generate pacemaker-like cardiomyocytes. Despite progress, biological strategies face challenges related to complex signaling pathways, intercellular coupling, durability of effect, efficacy, and safety. In addition, once ion channels or cells are implanted, biological pacemakers offer limited real-time programmability, whereas electronic pacemakers enable continuous adjustment of pacing output based on cardiac performance. Here we propose a hybrid approach that combines genetic sensitization with external control by exploring the feasibility of sonogenetic cardiac pacing. We express the ultrasound-sensitive ion channel hsTRPA1 in cardiomyocytes and use noninvasive ultrasound to modulate channel activity and cardiac function. Using calcium imaging, we show that hsTRPA1 potentiates ultrasound-evoked responses in cardiomyocytes. Furthermore, cardiac expression of hsTRPA1 in mice increases heart rate in response to ultrasound delivered noninvasively through the intact chest, without evidence of a thermal mechanism under the conditions tested. Together, these results establish a proof of concept for sonogenetic cardiac pacing and define an initial acoustic and genetic parameter space compatible with preserving baseline cardiac function. More broadly, this work positions sonogenetics as a minimally invasive, wireless strategy for cardiac rhythm control and motivates future optimization and safety studies toward translation.

bioengineering↗

bioRxiv2026

Hierarchical Phase-Contrast Tomography Imaging: Applicability in biomedical research

ObjectivesHierarchical Phase-Contrast Tomography (HiP-CT) enables non-destructive, multi-scale imaging of whole human organs. We describe how HiP-CT is utilized for biomedical research within the Human Organ Atlas Hub through three case studies: mapping the enteric nervous system (ENS) of the human colon, analysing myocardial and AV conduction architecture in Tetralogy of Fallot (TOF), and characterizing ductal organization in breast carcinoma. The challenges we faced with this novel biomedical data are discussed. MethodsWhole-organ and region-of-interest scans of three types of human organs were acquired at the European Synchrotron Radiation Facility (ESRF) with isotropic voxel sizes ranging from 20 {micro}m to 0.8 {micro}m. For the colon, voxel binning and RootPainter were employed to tackle data size to segment the ENS. For the heart, voxel-wise myocyte orientation mapping was calculated in terabyte-scale datasets with a high-performance computational framework (Cardiotensor). Breast carcinoma samples were correlated with histopathology for structure validation. ResultsHiP-CT revealed the large-scale organization of the ENS in the colon, enabling visualisation of the 3D structures of the ENS across the colon In TOF hearts, analysis uncovered abnormal myocardial structure and heterogeneous conduction system morphology. In breast carcinoma, HiP-CT resolved the full hierarchy of ductal structures and vascular relationships within tumour and peritumoral regions. ConclusionsHiP-CT provides unprecedented, hierarchical insight into intact human organ structure, bridging the gap between histology and radiology. Advances in knowledgeHiP-CT establishes a new ex vivo radiological modality capable of linking microscale pathology to whole-organ context, advancing translational research in neurogastroenterology, cardiology, and oncology

biophysics↗

bioRxiv2026

Evolution of Hierarchical Phase-Contrast Tomography on the European Synchrotron beamlines BM05 and BM18: a whole adult human brain imaging case study

Hierarchical Phase-Contrast Tomography (HiP-CT) was recently developed to enable the ex-vivo imaging of human organs at multiple scales from whole organ down to cellular level. Using whole adult human brain imaging as a case study, this manuscript shows the evolution of this technique from its initial development at the BM05 beamline to its transition and current status at BM18. Thanks to the higher coherence, larger beam size, higher energies and larger propagation distances available at BM18 and due to the European Synchrotrons Extremely Brilliant Source upgrade (ESRF-EBS), this transition resulted in significantly improved data quality, resolution, sensitivity and speed. More recently, the implementation of a new generation of larger sCMOS cameras, helical scanning (including dedicated reconstruction algorithm developments), binning at the chip and projections levels, and the design of high-efficiency optics allowed to progressively improve the trade-off between dose and image quality, while simultaneously reducing scanning times. All these acquisition schemes present the current status of full organ imaging using HiP-CT and represent the constant efforts for the improvement of the technique towards the investigation of human organs in health, disease and aging. SynopsisThis manuscript shows the evolution of Hierarchical Phase-Contrast Tomography (HiP-CT) from its origins at the BM05 beamline to its transition to BM18 (ESRF-EBS). The novel hardware and scanning approach developments resulted in significantly improved data quality, resolution, sensitivity and speed. The case of whole adult brain imaging is presented to demonstrate the current possibilities of full organ imaging with local micron resolution using HiP-CT.

neuroscience↗

bioRxiv2025

A whole-organ multi-scale in silico framework for human kidney haemodynamics informed by hierarchical phase-contrast tomography

Studying human kidney haemodynamics has been limited by the absence of complete vascular maps of the whole organ. Here we utilise previously generated multi-resolution hierarchical phase-contrast tomography (HiP-CT) coupled with a hybrid anatomically-grounded synthetic reconstruction to generate a full arterial-glomerular network of an intact human kidney comprising 1.6 million vessels and over 800,000 glomeruli. Using this anatomically comprehensive structure, we apply physics-based zero-dimensional haemodynamic modelling to quantify blood pressure, flow and simulated filtration rate across the entire organ. We show that the reconstructed human kidney vascular network exhibits order-dependent branching behaviour similar to that of rat kidneys, and that physiologically plausible pressure and flow patterns are recovered only when the full vascular network is represented. We further demonstrate how the kidney responds to macrovascular and microvascular perturbations, including stenosis of the large renal arteries and narrowing or ablation of afferent arterioles. Stenosis and arteriole narrowing exhibit threshold-type behaviour, with kidney perfusion and simulated glomerular filtration rate remaining largely preserved up to [~]50% narrowing, followed by sharp nonlinear declines beyond [~]70%. These predictions emerge in the absence of autoregulatory mechanisms, indicating that vascular geometry and resistance scaling alone contribute to kidney functional deterioration. Together, our framework provides the first organ-wide, data-driven model of human kidney haemodynamics and offers a foundation for future studies of kidney physiology and disease.

bioengineering↗

bioRxiv2025

Resolving near-micron scale features within a whole sheep head using Hierarchical Phase-Contrast Tomography

BackgroundHierarchical Phase-Contrast Tomography (HiP-CT) was developed to image ex vivo intact human soft-tissue organs with local near-micron scale resolution. PurposeWe demonstrate the application of HiP-CT in combination with the recently developed Eikonal Phase Retrieval (EPR) for resolving anatomical features in large hard and soft tissue structures on a sheep head, and show the applicability for zooming to resolve anatomical features with near-micron scale resolution. Materials and MethodsWe imaged an entire skinned sheep head prepared in 70 % ethanol with HiP-CT at an isotropic voxel size of 16.5 {micro}m using the recently developed Eikonal Phase Retrieval (EPR) to reduce artefacts from bones. Local tomography zooms were taken in regions of interest: the eye (4.23 {micro}m voxels) and the coronal suture (2.20 {micro}m voxels). To compare to clinical imaging, we acquired T2-weighted MRI and CT of the sheep head and evaluated the contrast-to-noise ratio for differentiation of white and grey matter in the brain for all modalities. Further evaluations on HiP-CT images include structure tensor analysis for structural orientation in the brain as well as fibre tracking in the coronal suture. ResultsHiP-CT combined with EPR achieved high contrast for both hard and soft tissue. Comparison to MRI showed similar soft-tissue contrast, but much higher spatial resolution. Structure tensor analysis in the brain revealed the orientation of the major white matter bundles. In the eye, near-micron scale features such as retinal layers and the bundles in the optic nerve were visualized. Fiber tracking allowed analysis of the orientation of collagen fiber bundles in the coronal suture. ConclusionThis work highlights the potential of HiP-CT to image a complete sheep head, ex vivo, and hierarchically zoom without sectioning to resolve few-microns features locally, enabling comprehensive three-dimensional visualization of intricate cranial structures and their spatial interrelations. Summary statementTechnical developments in HiP-CT enable ex vivo X-ray imaging of an intact sheep head with local resolution to near-micron scale, demonstrating future viability on a human head. Key resultsO_LIA whole sheep head was imaged with synchrotron-based hierarchical phase-contrast tomography coupled with Eikonal Phase-retrieval with 16.5 {micro}m isotropic voxels. C_LIO_LIThe high contrast for both hard and soft tissue enabled differentiation of brain white and grey matter, the optic nerve and bone features within the skull. C_LIO_LILocal tomography zoom scans in an eye (4.23 {micro}m voxels) and coronal suture (2.20 {micro}m voxels) allowed visualization and analysis of near-micron sized features. C_LI

bioengineering↗

bioRxiv2025

Benchmarking of duplex sequencing approaches to reveal somatic mutation landscapes

Detecting somatic mutations in normal tissues is challenging due to sequencing errors and the low allele fractions of post-zygotic variants. Duplex sequencing greatly reduces errors and can detect mutations at any allele fraction, but systematic, cross-platform comparisons are lacking. We present a comprehensive benchmarking of six duplex sequencing technologies used by the SMaHT Network: CODEC, CompDuplex-seq, HiDEF-seq, NanoSeq, ppmSeq, and VISTA-seq. We evaluated their performance using cord blood DNA, a tumor-normal cell line mixture, and homogenates from six human tissues. Each method shows distinct profiles in genomic footprint, sensitivity, and cost. Despite differences in library construction and sequencing platforms, estimates of mutation rates and mutational signatures are highly concordant. Integration with ultra-deep whole-genome sequencing shows that duplex approaches sensitively capture mutations and signatures beyond embryonic or clonally expanded variants. These results provide a foundation for selecting duplex methods and interpreting their data, enabling scalable single-molecule analyses of somatic mutation landscapes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/692823v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@123b221org.highwire.dtl.DTLVardef@83ba0aorg.highwire.dtl.DTLVardef@2b0ab0org.highwire.dtl.DTLVardef@1cae35e_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2025

The Heterogeneous Nature of Atrioventricular Conduction Tissues in Tetralogy of Fallot demonstrated by Hierarchical Phase-contrast Tomography - redefining the anatomic substrate

ObjectivesPostoperative arrhythmias are frequent after Tetralogy of Fallot (ToF) repair, yet anatomic substrate and preventive strategies remain poorly defined. Using hierarchical phase-contrast tomography (HiP-CT) the atrioventricular conduction system in pediatric ToF specimens was investigated non-destructively and in 3D. MethodsEighteen whole-heart specimens (11 ToF, 7 controls) were imaged at the European Synchrotron, ESRF. Segmentation and 3D renderings demonstrated gross morphology. Morphology, size, depth and course of the non-branching bundle and right bundle branch (RBB) were quantified using custom computational pipelines. Segmentations were visualized in VheaRts, a Unity3D-based XR platform. ResultsThe ToF conduction system was more draped than in controls, bilaterally spanning the septum, resembling early embryonic architecture. The RBB was variable in origin, course and morphologic structure with significantly smaller indexed cross-sectional area in native ToF versus controls (0.05 {+/-} 0.20 vs 0.50 {+/-} 0.20 mm{superscript 2}, p = 0.005). One anomalous fasciculo-ventricular connection and six dead-end tracts were found. Regions at surgical risk included the posterior-inferior margin of the ventricular septal defect (VSD) and the septal crest along its nadir. The superior margin of the VSD at its intersection with the aortic root was free of conduction tissue. The HiP-CT to VR pipeline enabled interactive 3D visualization of conduction pathways relative to key structures. ConclusionsThis first pediatric cardiac HiP-CT series reveals a broader anomalous conduction complex in ToF, including variable RBB origin and hypoplasia, providing insight into preoperative vulnerability, arrhythmia, and surgical risk. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=149 SRC="FIGDIR/small/692356v1_fig7.gif" ALT="Figure 7"> View larger version (50K): org.highwire.dtl.DTLVardef@1b39e89org.highwire.dtl.DTLVardef@16d49c2org.highwire.dtl.DTLVardef@5884borg.highwire.dtl.DTLVardef@136fc24_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7:C_FLOATNO Graphical Abstract C_FIG

developmental biology↗

bioRxiv2025

Integrated multi-omics reveals coordinated Staphylococcus aureus metabolic, iron transport and stress responses to human serum

Bloodstream infections caused by Staphylococcus aureus remain a leading cause of mortality worldwide. Our understanding of S. aureus survival and persistence in human serum, a cell-free fraction of blood hostile for bacteria, is still limited. Here, we applied multivariate data integration methods and network analysis to a multi-omic dataset generated from five clinically prevalent S. aureus genotypes exposed to human serum. We observed and then confirmed using isogenic mutants, the significant roles of gapdhB, sucA, sirA, sstD, and perR in bacterial survival in serum. These data show that metabolic versatility in carbon source usage, iron transport and resistance to oxidative stress are interlinked and central to S. aureus fitness in serum, representing potential S. aureus vulnerabilities that could be exploited therapeutically. IMPORTANCEBloodstream infections caused by Staphylococcus aureus are associated with mortality rates of up to 30%. However, the molecular mechanisms that enable this pathogen to survive in human serum, a nutrient-limited and immunologically hostile environment remain poorly understood. By integrating multi-omic data from five clinically relevant S. aureus genotypes and validating key signatures using mutants, we identified conserved genetic determinants critical for bacterial survival in serum. Our findings highlight the interconnected roles of carbohydrate metabolic flexibility, iron acquisition, and oxidative stress resistance in shaping S. aureus adaptation to serum. This work advances our understanding of microbial strategies to survive in the bloodstream and demonstrates the potential of multi-omic integration to uncover therapeutic vulnerabilities in bacterial pathogens.

microbiology↗

bioRxiv2025

Very low amplitude muscle activity increases probability of motor evoked potentials in healthy individuals and in ALS

IntroductionIn many clinical and research settings, transcranial magnetic stimulation (TMS) intensities are standardised based on resting motor threshold (RMT). It is well-established that contraction of the target muscle increases motor evoked potential (MEP) amplitude and correspondingly decreases RMT. As such, when estimating RMT it is crucial to ensure the target muscle is relaxed. Typically trials in which baseline electromyographic (EMG) amplitude exceeds a specified threshold are rejected. The influence of motor activity below typical rejection thresholds on MEP amplitudes has yet to be established. MethodsWe retrospectively analysed TMS-EMG data collected during RMT measurement in 45 healthy controls (1761 datapoints) and 35 people with amyotrophic lateral sclerosis (ALS, 1238 datapoints). Trials where root mean squared (RMS) baseline EMG amplitude exceeded 10{micro}V were rejected. Generalised linear mixed-effects models were used to assess effects of muscle activity below this rejection threshold on probability of evoking an MEP with peak-to-peak amplitude [≥]50{micro}V. ResultsGreater sub-rejection-threshold activity significantly increases MEP probability in controls (p<0.0004) and people with ALS (p=0.0010). Models predicted a 31-38% increase in MEP probability when baseline RMS-EMG amplitude increased from 1{micro}V to 9{micro}V. Sub-rejection- threshold baseline activity was significantly greater in ALS than controls (p=0.0055). DiscussionWe have shown for the first time that higher EMG amplitudes below a typical rejection threshold markedly increase probability of evoking an MEP with peak-to-peak amplitude [≥]50{micro}V. Researchers should take measures to account for effects of sub-rejection-threshold activity on RMT, particularly in populations where baseline activity may be elevated, such as in ALS.

neuroscience↗

bioRxiv2025

Multi-omic dereplication of antibiotic production by diffusion chamber isolated bacteria from Australian soils

Soil bacteria are a major source of clinically useful antibiotics, yet the majority of soil-dwelling microorganisms remain uncultivable by standard laboratory methods. To access this untapped microbial diversity, we employed microbial diffusion chambers to isolate bacteria from ten Australian soil samples. A total of 1,218 bacterial isolates were recovered, representing a diverse collection spanning 61 genera from 32 families, covering the major known phyla of soil bacteria. Antibiotic activity screening revealed that 16% of isolates inhibited the growth of at least one of E. coli or S. aureus, with 120 isolates displaying activity against multidrug-resistant pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE). Mass spectrometry-based dereplication using GNPS identified known antibiotics in 33% of bioactive strains, including actinomycin D, nonactins, and valinomycin. Genomic analysis confirmed the presence of corresponding biosynthetic gene clusters (BGCs), while targeted analysis of selected strains uncovered production of additional antibiotics such as nigericin and streptothricin that were not initially detected by mass spectrometry. Our results demonstrate that diffusion chambers enhance bacterial recovery from soil and show the benefits of a combined pipeline including bioactivity screening, mass spectrometry, and genomics for effective antibiotic dereplication and discovery. Impact StatementThis study delivers a significant advance in natural product discovery by demonstrating that microbial diffusion chambers can dramatically improve the recovery of diverse soil bacteria with antibiotic-producing potential from Australian soil samples. By integrating in situ cultivation with high-throughput screening, mass spectrometry-based dereplication and genome mining, this study yielded more than 1,200 bacterial isolates--including many with activity against multidrug-resistant pathogens--and confirms the production of both known and previously undetected antibiotics. The discovery of streptothricin production via genomics, despite its absence from mass spectrometry data, underscores the power of a multi-layered dereplication strategy. This work not only validates the utility of diffusion chambers for unlocking the "rare biosphere" of uncultivable microbes but also highlights critical methodological refinements to diversify antibiotic-producing strains beyond Streptomyces. The findings will inform and accelerate future efforts to discover urgently needed antibiotics from environmental microbiomes. Data SummaryThe whole-genome sequences were deposited in the National Center for Biotechnology Information National Library of Medicine under BioProject accession number PRJNA1272227.

microbiology↗

bioRxiv2025

Tau-Associated Neuronal Loss in the Intermediate Nucleus of the Human Hypothalamus (VLPO Analog): Unveiling the Basis of NREM Sleep Dysfunction in PSP and Alzheimer's Disease

Sleep disturbances are prevalent in Alzheimers disease (AD) and Progressive Supranuclear Palsy (PSP), often exacerbating disease progression. Understanding the neuropathological basis of these disturbances is essential for identifying potential therapeutic targets. This study investigates the intermediate nucleus (IntN) of the human hypothalamus--a key sleep-regulating region analogous to the rodent ventrolateral preoptic area (VLPO)--to assess neuronal loss and tau pathology in AD and PSP. Using postmortem brain tissue, we applied unbiased stereology to quantify galanin-expressing neurons and phosphorylated tau (p-tau) accumulation. Among 26 cases analyzed, both AD and PSP exhibited significant neuronal loss in the IntN, with PSP showing the most pronounced reduction (84.9% fewer neurons than healthy controls [HC]). In AD, neuronal loss correlated with Braak staging, with late-stage AD cases (Braak 5-6) demonstrating a 76.9% reduction in galanin-expressing neurons compared to HC, while non-galanin neurons exhibited a more moderate decline (45.7%). In PSP, extensive neuronal loss precluded a clear assessment of p-tau burden. These findings suggest a differential neuronal vulnerability to tau pathology across diseases, aligning with distinct sleep disturbances observed in each condition. PSP, characterized by severe insomnia despite preserved wake-promoting neurons, may be explained by the near-total loss of NREM sleep-regulating neurons. In contrast, AD exhibits a progressive decline in both wake- and sleep-promoting neurons, contributing to excessive daytime sleepiness and sleep fragmentation. This study provides critical insights into the selective neuronal vulnerabilities underlying sleep dysfunction in tauopathies, emphasizing the need for targeted interventions to mitigate sleep disturbances in these disorders.

neuroscience↗

bioRxiv2024

The complete genome sequence of the crayfish pathogen Candidatus Paracoxiella cheracis n.g. n.sp. provides insight into pathogenesis and the phylogeny of the Coxiellaceae family

The Coxiellaceae bacterial family, within the order Legionellales, is defined by a collection of poorly characterized obligate intracellular bacteria. The zoonotic pathogen and causative agent of human Q fever, Coxiella burnetii, represents the best characterized member of this family. Coxiellaceae establish replicative niches within diverse host cells and rely on their host for survival, making them challenging to isolate and cultivate within a laboratory setting. Here we describe a new genus within the Coxiellaceae family that has been previously shown to infect economically significant freshwater crayfish. Using culture-independent long-read metagenomics, we reconstructed the complete genome of this novel organism and demonstrate that the previously referred to as Candidatus Coxiella cheraxi represents a novel genus within this family, herein denoted Candidatus Paracoxiella cheracis. Interestingly, we demonstrate that Candidatus P. cheracis encodes for a complete, putatively functional Dot/Icm type 4 secretion system that likely mediates the intracellular success of this pathogen. In silico analysis defined a unique repertoire of Dot/Icm effector proteins and highlighted homologues of several important C. burnetii effectors including a homologue of CpeB that was demonstrated to be a Dot/Icm substrate in C. burnetii. IMPORTANCEUsing long-read sequencing technology we have uncovered the full genome sequence of Candidatus Paracoxiella cheracis, a pathogen of economic importance in aquaculture. Analysis of this sequence has revealed new insight into this novel member of the Coxiellaceae family, demonstrating that it represents a new genus within this poorly characterized family of intracellular organisms. Importantly, the genome sequence reveals invaluable information that will support diagnostics and potentially both preventative and treatment strategies within crayfish breeding facilities. Candidatus P. cheracis also represents a new member of Dot/Icm pathogens that rely on this system to establish an intracellular niche. Candidatus P. cheracis possesses a unique cohort of putative Dot/Icm substrates that constitute a collection of new eukaryotic cell biology manipulating effector proteins.

microbiology↗

bioRxiv2024

Efficient secretion of a plastic degrading enzyme from the green algae Chlamydomonas reinhardtii

Plastic pollution has become a global crisis, with microplastics contaminating every environment on the planet, including our food, water, and even our bodies. In response, there is a growing interest in developing plastics that biodegrade naturally, thus avoiding the creation of persistent microplastics. As a mechanism to increase the rate of polyester plastic degradation, we examined the potential of using the green microalga Chlamydomonas reinhardtii for the expression and secretion of PHL7, an enzyme that breaks down post-consumer polyethylene terephthalate (PET) plastics. We engineered C. reinhardtii to secrete active PHL7 enzyme and selected strains showing robust expression, by using agar plates containing a polyester polyurethane (PU) dispersion as an efficient screening tool. This method demonstrated the enzymes efficacy in degrading ester bond-containing plastics, such as PET and bio-based polyurethanes, and highlights the potential for microalgae to be implemented in environmental biotechnology. The effectiveness of algal-expressed PHL7 in degrading plastics was shown by incubating PET with the supernatant from engineered strains, resulting in substantial plastic degradation, confirmed by mass spectrometry analysis of terephthalic acid (TPA) formation from PET. Our findings demonstrate the feasibility of polyester plastic recycling using microalgae to produce plastic-degrading enzymes. This eco-friendly approach can support global efforts toward eliminating plastic in our environment, and aligns with the pursuit of low-carbon materials, as these engineered algae can also produce plastic monomer precursors. Finally, this data demonstrates C. reinhardtii capabilities for recombinant enzyme production and secretion, offering a "green" alternative to traditional industrial enzyme production methods. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/619606v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@108e29dorg.highwire.dtl.DTLVardef@55ceaborg.highwire.dtl.DTLVardef@173793eorg.highwire.dtl.DTLVardef@1d14c3_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Micro to macro scale anatomical analysis of the human hippocampal arteries with synchrotron Hierarchical Phase-Contrast Tomography

PurposeTo date, no non-invasive imaging modality has been employed to profile the structural intricacies of the hippocampal arterial microvasculature in humans. We hypothesised that synchrotron-based imaging of the human hippocampus would enable precise characterisation of the arterial microvasculature. MethodsTwo preserved human brains from, a 69-year-old female and a 63-year-old male body donors were imaged using hierarchical phase-contrast tomography (HiP-CT) with synchrotron radiation at multiple voxel resolutions from 25.08 m down to 2.45 m. Subsequent manual and semi-automatic artery segmentation were performed followed by morphometric analyses. These data were compared to published data from alternative methodologies. ResultsHiP-CT made it possible to segment in context the arterial architecture of the human hippocampus. Our analysis identified anterior, medial and posterior hippocampal arteries arising from the P2 segment of the posterior cerebral artery on the image slices. We mapped arterial branches with external diameters greater than 50 m in the hippocampal region. We visualised vascular asymmetry and quantified arterial structures with diameters as small as 7 m. ConclusionsThrough the application of HiP-CT, we have provided the first imaging visualisation and quantification of the arterial system of the human hippocampus at high resolution in the context of whole brain imaging. Our results bridge the gap between anatomical and histological scales.

neuroscience↗