Biology subjects

Zhu, Y.

Publications and source records attributed to Zhu, Y..

At least 505 records · Page 28Linked to original sources

bioRxiv2022

Simulations reveal high efficiency and confinement of a population suppression CRISPR toxin-antidote gene drive

Though engineered gene drives hold great promise for spreading through and eventually suppressing populations of disease vectors or invasive species, complications such as resistance alleles and spatial population structure can prevent their success. Additionally, most forms of suppression drives, such as homing drives or driving Y chromosomes, will generally spread uncontrollably between populations with even small levels of migration. The previously proposed CRISPR-based toxin-antidote system called TADE suppression drive could potentially address the issue of confinement and resistance alleles. However, it is a relatively weak form of drive compared to homing drives, which might make it particularly vulnerable to spatial population structure. In this study, we investigate TADE suppression drive using individual-based simulations in continuous space. We find that the drive is actually more confined in continuous space than in panmictic populations, even in its most efficient form with a low cleavage rate in embryos from maternally deposited Cas9. Furthermore, the drive performed well in continuous space scenarios if the initial release requirements were met, suppressing the populations in a timely manner without being severely affected by chasing, a phenomenon in which wild-type individuals avoid the drive by recolonizing empty areas. At higher embryo cut rates, the drive loses its ability to propagate on its own, but a single, widespread release can often still induce rapid population collapse. Thus, if TADE suppression gene drives can be successfully constructed, they may play an important role in control of disease vectors and invasive species when stringent confinement to target populations is desired.

evolutionary biology↗

bioRxiv2022

The Children's Brain Tumor Network (CBTN) - Accelerating Research in Pediatric Central Nervous System Tumors through Collaboration and Open Science

Pediatric brain tumors are the leading cause of cancer-related death in children in the United States and contribute a disproportionate number of potential years of life lost compared to adult cancers. Moreover, survivors frequently suffer long-term side effects, including secondary cancers. The Childrens Brain Tumor Network (CBTN) is a multi-institutional international clinical research consortium created to advance therapeutic development through the collection and rapid distribution of biospecimens and data via open-science research platforms for real-time access and use by the global research community. The CBTNs 32 member institutions utilize a shared regulatory governance architecture at the Childrens Hospital of Philadelphia to accelerate and maximize the use of biospecimens and data. As of August 2022, CBTN has enrolled over 4,700 subjects, over 1,500 parents, and collected over 65,000 biospecimen aliquots for research. Additionally, over 80 preclinical models have been developed from collected tumors. Multi-omic data for over 1,000 tumors and germline material is currently available with data generation for > 5,000 samples underway. To our knowledge, CBTN provides the largest open-access pediatric brain tumor multi-omic dataset annotated with longitudinal clinical and outcome data, imaging, associated biospecimens, child-parent genomic pedigrees, and in vivo and in vitro preclinical models. Empowered by NIH-supported platforms such as the Kids First Data Resource and the Childhood Cancer Data Initiative, the CBTN continues to expand the resources needed for scientists to accelerate translational impact for improved outcomes and quality of life for children with brain and spinal cord tumors.

cancer biology↗

bioRxiv2022

An e-cigarette aerosol generation, animal exposure and toxicants quantification system to characterize in vivo nicotine kinetics in arterial and venous blood

The increasing e-cigarette use worldwide presents an urgent need to characterize their nicotine delivery property, brain stimulation and potential long-term health effects. We constructed an end-to-end system enabling combustible-cigarette (c-cigarette) and e-cigarette aerosol generation, animal exposure, and effect assessment. The system consists of (1) a 10-channel aerosol generator resembling human smoking/vaping scenarios, (2) nose-only and whole-body exposure chambers suitable for long- or short-duration studies, (3) a lab protocol for animal exposure and collecting arterial and venous blood <1 minute after the exposure, and (4) chromatograph and mass spectrometry to quantify nicotine concentrations in aerosol and biospecimens. We applied the system in a proof-of-principle study characterizing in vivo nicotine delivery after e-cigarette aerosol inhalation. Groups of Sprague-Dawley rats were exposed to e-cigarette aerosols for 1, 2 and 4 minutes, respectively. Arterial and venous blood samples were collected immediately after the exposure. We also directly compared nose-only and whole-body exposure approaches. After nose-only e-cigarette aerosol exposure, the nicotine concentration in arterial blood was substantially higher (11.32 ng/mL in average) than in veins. Similar arterio-venous concentration difference was observed in whole-body exposure experiments. In summary, we described a complete system ideal for e- and c-cigarette in vivo nicotine kinetics and long-term health research. Our findings highlight arterial blood as the suitable bio-specimen for e-cigarette nicotine delivery studies. HighlightO_LIWe constructed a combustible- and e-cigarette aerosol generation - exposure - effect assessment system resembling real world human smoking/vaping scenarios. C_LIO_LIProof-of-principle study characterized in vivo nicotine delivery from e-cigarette aerosol to arterial and venous blood at high temporal resolution. C_LIO_LIAfter exposure, the nicotine concentration was substantially higher (11.32 ng/mL) in arterial blood than in veins. C_LIO_LIOur results suggest arterial blood as the suitable bio-specimen to study nicotine delivery and brain stimulation. C_LI

pharmacology and toxicology↗

bioRxiv2022

Smaller total and subregional cerebellar volumes in posttraumatic stress disorder: a mega-analysis by the ENIGMA-PGC PTSD workgroup

BackgroundThe cerebellum critically contributes to higher-order cognitive and emotional functions such fear learning and memory. Prior research on cerebellar volume in PTSD is scant and has neglected neuroanatomical subdivisions of the cerebellum that differentially map on to motor, cognitive, and affective functions. MethodsWe quantified cerebellar lobule volumes using structural magnetic resonance imaging in 4,215 adults (PTSD n= 1640; Control n=2575) across 40 sites from the from the ENIGMA-PGC PTSD working group. Using a new state-of-the-art deep-learning based approach for automatic cerebellar parcellation, we obtained volumetric estimates for the total cerebellum and 28 subregions. Linear mixed effects models controlling for age, gender, intracranial volume, and site were used to compare cerebellum total and subregional volume in PTSD compared to healthy controls. The Benjamini-Hochberg procedure was used to control the false discovery rate (p-FDR < .05). ResultsPTSD was associated with significant grey and white matter reductions of the cerebellum. Compared to controls, people with PTSD demonstrated smaller total cerebellum volume. In addition, people with PTSD showed reduced volume in subregions primarily within the posterior lobe (lobule VIIB, crus II), but also the vermis (VI, VIII), flocculonodular lobe (lobule X), and cerebellar white matter (all p-FDR < 0.05). Effects of PTSD on volume were consistent, and generally more robust, when examining symptom severity rather than diagnostic status. ConclusionsThese findings implicate regionally specific cerebellar volumetric differences in the pathophysiology of PTSD. The cerebellum appears to play an important role in high-order cognitive and emotional processes, far beyond its historical association with vestibulomotor function. Further examination of the cerebellum in trauma-related psychopathology will help to clarify how cerebellar structure and function may disrupt cognitive and affective processes at the center of translational models for PTSD.

neuroscience↗

bioRxiv2022

Multiomics reveal that silk fibroin and sericin differentially potentiate the paracrine functions of mesenchymal stem cells and enhance tissue regeneration

Silk fibroin (SF) and sericin (SS), the two major proteins of silk, are attractive biomaterials that show great potential in regenerative medicine. However, their biochemical interactions with stem cells were not fully understood. Here, we employed multiomics to obtain a global view of the triggered cellular processes and pathways of MSCs by SF and SS. Integrated RNA-seq and proteomics revealed that SF and SS strongly enhanced the paracrine activity of MSCs through differentially activating integrin and glycolytic pathways, rather than directly regulating stem cell fate to initiate multiple but distinct biological processes in MSCs. Those specific paracrine signals of MSCs stimulated by SF and SS effectively promoted skin wound healing by influencing the behaviors of multiple resident cells in skin wound microenvironments. This study provides comprehensive and reliable insights into the cellular interactions with SF and SS, enabling future development of silk-based therapeutics for tissue engineering and stem cell therapy.

bioengineering↗

bioRxiv2022

CorA gene rearrangement triggered the salinity-driven speciation of Poseidoniales

Land-ocean interactions greatly impacted the evolution of coastal life on Earth. However, the geological forces and genetic mechanisms that shaped evolutionary adaptations and allowed microorganisms to inhabit coastal brackish waters remain largely unexplored. Here, we infer the evolutionary trajectory of the ubiquitous heterotrophic archaea Poseidoniales (Marine Group II archaea) across global aquatic habitats. Our results show that their brackish subgroups have evolved through the rearrangement of the magnesium transport gene corA that conferred osmotic-stress tolerance dated to over 600 million years ago. The coastal family of Poseidoniales showed a rapid increase in the evolutionary rate during and in the aftermath of the Cryogenian Snowball Earth (~700 million years ago), possibly in response to the enhanced phosphorus supply and the rise of algae. Our study highlights the close interplay between genetic changes and ecosystem transformations that boosted microbial diversification in the Neoproterozoic continental margins.

evolutionary biology↗

bioRxiv2022

Nhlh1 and Nhlh2, a global transcriptional mechanism regulating commissural axon projection via activating Robo3

SUMMARYCommissural neurons are highly heterogeneous in their developmental origins, neurotransmitter type and function, but all share the common feature of projecting axons across the midline. The floor plate-crossing commissural axons in mammals, from the spinal cord to the midbrain, are guided by a conserved molecular mechanism relying primarily on Netrin-1/DCC/Robo3 signaling. Up to date, we know very little about the upstream transcriptional program that specify commissural axon laterality, neither do we know if a common mechanism operates in all commissural neurons. Here, we identified a pair of highly related helix-loop-helix transcription factors, Nhlh1 and Nhlh2, as a global transcriptional mechanism that controls the laterality of all floor plate-crossing commissural axons. Forced expression of Nhlh1/2 induce ectopic Robo3 expression and contralateral axon projections. And mutant mice deficient in both genes show a remarkable reduction in Robo3 expression and a total lack of ventral commissures from the spinal cord to the midbrain. This global mechanism may interact with neuron type specific mechanism to achieve specific generation of commissural circuits.

neuroscience↗

bioRxiv2022

Transcriptional immunogenomic analysis reveals distinct immunological clusters in pediatric nervous system tumours

To inform immunotherapy approaches in children, we performed an immunogenomic analysis of RNA-seq data from 925 treatment-naive pediatric nervous system tumours (pedNST) spanning 12 cancer types from three public data sets. Within pedNST, we uncovered four broad immune clusters: Pediatric Inflamed (10%), Myeloid Predominant (30%), Immune Neutral (43%) and Immune Excluded (17%). We validated these clusters using immunohistochemistry, methylation immune inference, and segmentation analysis of tissue images. We report shared biology of these immune clusters within and across cancer types, and characterization of specific immune-cell frequencies as well as T- and B-cell repertoires. We found no associations between immune infiltration levels and tumour mutational burden, although molecular cancer entities were enriched within specific immune clusters. Given the heterogeneity within pedNST, our findings suggest personalized immunogenomic profiling is needed to guide selection of immunotherapeutic strategies.

genomics↗

bioRxiv2022

OpenPBTA: An Open Pediatric Brain Tumor Atlas

SummaryPediatric brain and spinal cancer are the leading disease-related cause of death in children, thus we urgently need curative therapeutic strategies for these tumors. To accelerate such discoveries, the Childrens Brain Tumor Network and Pacific Pediatric Neuro-Oncology Consortium created a systematic process for tumor biobanking, model generation, and sequencing with immediate access to harmonized data. We leverage these data to create OpenPBTA, an open collaborative project which establishes over 40 scalable analysis modules to genomically characterize 1,074 pediatric brain tumors. Transcriptomic classification reveals that TP53 loss is a significant marker for poor overall survival in ependymomas and H3 K28-altered diffuse midline gliomas and further identifies universal TP53 dysregulation in mismatch repair-deficient hypermutant high-grade gliomas. OpenPBTA is a foundational analysis platform actively being applied to other pediatric cancers and inform molecular tumor board decision-making, making it an invaluable resource to the pediatric oncology community. In BriefThe OpenPBTA is a global, collaborative open-science initiative which brought together researchers and clinicians to genomically characterize 1,074 pediatric brain tumors and 22 patient-derived cell lines. Shapiro, et. al create over 40 open-source, scalable modules to perform cancer genomics analyses and provide a richly-annotated somatic dataset across 58 brain tumor histologies. The OpenPBTA framework can be used as a model for large-scale data integration to inform basic research, therapeutic target identification, and clinical translation. HighlightsOpenPBTA collaborative analyses establish resource for 1,074 pediatric brain tumors NGS-based WHO-aligned integrated diagnoses generated for 641 of 1,074 tumors RNA-Seq analysis infers medulloblastoma subtypes, TP53 status, and telomerase activity OpenPBTA will accelerate therapeutic translation of genomic insights

genomics↗

bioRxiv2022

Droplet microfluidics forward for tracing target cells at single-cell transcriptome resolution

The rapid promotion of single-cell omics in various fields has begun to help solve many problems encountered in research including precision medicine, prenatal diagnosis, and embryo development. Meanwhile, single-cell techniques are also constantly updated with increasing demand. For some specific target cells, the workflow from droplet screening to single-cell sequencing is a preferred option, which also should reduce the impact of operation steps such as demulsification and cell recovery. We developed an all-in-droplet method integrating cell encapsulation, target sorting, droplet picoinjection, and single-cell transcriptome profiling on chips to achieve a labor-saving monitor of TCR-T cells. As a proof of concept, in this research, TCR-T cells were encapsulated, sorted, and performed single-cell transcriptome sequencing (scRNA-seq) by injecting reagents into droplet. It avoided the tedious operation of droplet breakage and re-encapsulation between droplet sorting and scRNA-seq. Moreover, convenient device operation will accelerate the progress of chip marketization. The strategy achieved an excellent recovery performance of single cell transcriptome with a median gene number over 4000 and a cross-contamination rate of 8.2 {+/-} 2%. Furthermore, this strategy allows us to develop a device with high integrability to monitor infused TCR-T cells, which will promote the development of adoptive T cell immunotherapy and their clinical application.

cell biology↗

bioRxiv2022

Tilt In Place Microscopy (TIPM): a simple, low-cost solution to image neural responses to body rotations

Animals use information about gravity and other destabilizing forces to balance and navigate through their environment. Measuring how brains respond to these forces requires considerable technical knowledge and/or financial resources. We present a simple alternative: Tilt In Place Microscopy (TIPM). TIPM is a low-cost and non-invasive way to measure neural activity following rapid changes in body orientation. Here we used TIPM to study vestibulospinal neurons in larval zebrafish during and immediately after roll tilts. Vestibulospinal neurons responded with reliable increases in activity that varied as a function of ipsilateral tilt amplitude. TIPM differentiated tonic (i.e. sustained tilt) from phasic responses, revealing coarse topography of stimulus sensitivity in the lateral vestibular nucleus. Neuronal variability across repeated sessions was minor relative to trial-to-trial variability, allowing us to use TIPM for longitudinal studies of the same neurons across two developmental timepoints. There, we observed global increases in response strength, and systematic changes in the neural representation of stimulus direction. Our data extend classical characterization of the body tilt representation by vestibulospinal neurons and establish TIPMs utility to study the neural basis of balance, especially in developing animals. Significance StatementVestibular sensation influences everything from navigation to interoception. Here we detail a straight-forward, validated and nearly-universal approach to image how the nervous system senses and responds to body tilts. We use our new method to replicate and expand upon past findings of tilt sensing by a conserved population of spinal-projecting vestibular neurons. The simplicity and broad compatibility of our approach will democratize the study of the brains response to destabilization, particularly across development.

neuroscience↗

bioRxiv2022

SETD2 deficiency promotes renal fibrosis through the TGF-β/Smad signaling pathway in the absence of VHL

Renal fibrosis is the final development pathway and the most common pathological manifestation of chronic kidney disease. An important intrinsic cause of renal fibrosis is epigenetic alterations. SET domain-containing 2 (SETD2) is the sole histone H3K36 trimethyltransferase, catalyzing H3K36 dimethylation to trimethylation. There is evidence that SETD2-mediated epigenetic alterations are implicated in many diseases. However, it is unclear what role SETD2 plays in the development of renal fibrosis. Clinical data indicate that SETD2 is lowly expressed in patients with renal fibrosis. Here, we established genetically engineered mice with SETD2 and VHL deficiency. SETD2 deficiency leads to severe renal fibrosis in VHL-deficient mice. Mechanically, SETD2 maintains the transcriptional level of Smad7, a negative feedback factor of the TGF-{beta}/Smad signaling pathway, thereby preventing the activation of the TGF-{beta}/Smad signaling pathway. Deletion of SETD2 leads to reduced smad7 expression, which results in activation of the TGF-{beta}/Smad signaling pathway and ultimately fibrosis in the absence of VHL. Our findings reveal the role of SETD2-mediated H3K36me3 of Smad7 in regulating the TGF-{beta}/Smad signaling pathway in renal fibrogenesis. Thus, our study provides innovative insights into SETD2 as a potential therapeutic target for the treatment of renal fibrosis.

molecular biology↗

bioRxiv2022

Cryo-EM structure of the type III-E CRISPR-Cas effector gRAMP in complex with TPR-CHAT

The type III-E CRISPR-Cas effector protein, named gRAMP, is the largest single-unit CRISPR-Cas effector. A caspase-like peptidase (TPR-CHAT) gene often co-occurs with gRAMP gene clusters. However, the exact mechanism of the recognition and cleavage of target RNA of Sb-gRAMP, as well as the molecular architecture of the CRISPR-guided caspase complex, remains unclear. Here, we report the cryo-EM structures of the type III-E effector Sb-gRAMP-crRNA in a complex with TPR-CHAT, with and without target ssRNA at 3.0 and 2.9 [A], respectively. The overall structure of the gRAMP-crRNA-ssRNA-TPR-CHAT complex adopts an "L"-shaped conformation, consisting of a copy of gRAMP, a copy of TPR-CHAT, a 37-nt crRNA, and an 18-nt target ssRNA. The data presented in this manuscript reveal the mechanism of recognition of crRNA and target ssRNA by gRAMP, also, this work reports the structure of the CRISPR type III-E effector in complex with the binding partner TPR-CHAT, which provides vital clues for elucidating the functional relation between the CRISPR-Cas system and caspase peptidase.

biochemistry↗

bioRxiv2022

IndepthPathway: an integrated tool for in-depth pathway enrichment analysis based on bulk and single cell sequencing data

Single-cell sequencing (SCS) enables exploring the pathways and processes of cells, and cell populations. However, there is a paucity of pathway enrichment methods designed to tolerate the high noise and low gene coverage of this technology. When gene expression data are noisy and signals are sparse, testing pathway enrichment based on the genes expression may not yield statistically significant results which is particularly problematic when detecting the pathways enriched in less abundant cells that are vulnerable to disturbances. In this project, we developed a Weighted Concept Signature Enrichment Analysis (WCSEA) algorithm specialized for pathway enrichment analysis from single cell transcriptomics (scRNA-seq). WCSEA took a broader approach for assessing the functional relations of pathway gene sets to differentially expressed genes, and leverage the cumulative signature of molecular concepts characteristic of the highly differentially expressed genes, which we termed as the universal concept signature, to tolerate the high noise and low coverage of this technology. We then incorporated WCSEA into a R package called "IndepthPathway" for biologists to broadly leverage this method for pathway analysis based on bulk and single cell sequencing data. Through simulating technical variability and dropouts in gene expression characteristic of scRNA-seq as well as benchmarking on a real dataset of matched single cell and bulk RNAseq data, we demonstrate that IndepthPathway presents outstanding stability and depth in pathway enrichment results under stochasticity of the data, thus will substantially improve the scientific rigor of the pathway analysis for single cell sequencing data. The IndepthPathway package is available through: https://github.com/wangxlab/IndepthPathway.

bioinformatics↗

bioRxiv2022

Global loss of fine-scale chromatin architecture and rebalancing of gene expression during early colorectal cancer development

Although 3D genome architecture can be essential for gene regulation, the biological implications of long-range chromatin interactions in disease remain elusive. In this study, we traced the early evolution and malignant transformation of colorectal cancer by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth from polyps of Familial Adenomatous Polyposis (FAP) patients. Our analysis reveals a substantial progressive loss of genome-wide cis-regulatory connectivity at early stages of malignancy, which correlates with a non-linear effect on gene regulation. Genes with high promoter-enhancer (P-E) connectivity in unaffected mucosa are not correlated with elevated baseline expression, but instead tend to be up-regulated at advanced stages. Inhibition of highly connected promoters preferentially represses gene expression in colorectal cancer cells relative to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P-E connectivity from a redundant state to a rate-limiting one for transcriptional levels. Overall, our study illuminates the intricate interplay between 3D genome architecture and gene regulation during early colorectal cancer progression, and provides valuable insights for potential therapeutic interventions targeting the connectivity of cis-regulatory elements.

genomics↗

bioRxiv2022

An off-the-shelf multi-well scaffold-supported platform for tumour organoid-based tissues

Complex 3D bioengineered tumour models provide the opportunity to better capture the heterogeneity of patient tumours. Patient-derived organoids are emerging as a useful tool to study tumour heterogeneity and variation in patient responses. Organoid cultures typically require a 3D microenvironment that can be manufactured easily to facilitate screening. Here we set out to create a high-throughput, "off-the-shelf" platform which permits the generation of organoid-containing microtissues for standard phenotypic bioassays and image-based readings. To achieve this, we developed the Scaffold-supported Platform for Organoid-based Tissues (SPOT) platform. SPOT is a 3D gel-embedded in vitro platform that can be produced in a 96- or 384-well plate format and enables the generation of flat, thin and dimensionally-defined microgels. SPOT has high potential for adoption due to its reproducible manufacturing methodology, compatibility with existing instrumentation, and reduced within-sample and between-sample variation, which can pose challenges to both data analysis and interpretation. Using SPOT we generate cultures from patient derived pancreatic ductal adenocarcinoma organoids and assess the cellular response to standard-of-care chemotherapeutic compounds, demonstrating our platforms usability for drug screening. We envision 96/384-SPOT will provide a useful tool to assess drug sensitivity of patient-derived organoids and easily integrate into the drug discovery pipeline.

bioengineering↗

bioRxiv2022

ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent in situ cryo-ET structural study. Here, we developed a precise cryogenic fabrication system, the ELI trifocal microscope (ELI-TriScope), by setting up an electron (E) beam, a light (L) beam and an ion (I) beam at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella without sacrificing the throughput. ELI-TriScope was developed starting from a commercial dual-beam scanning electron microscope (SEM) by incorporating a cryo-holder-based transfer system and embedding an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam (FIB) milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome, with an [~]100% success rate, and discovered new in situ structural features of the human centrosome through a subsequent cryo-ET structural study.

biophysics↗

bioRxiv2022

Outer membrane utilisomes mediate oligosaccharide uptake in gut Bacteroidetes

Bacteroidetes are abundant members of the human microbiota, with species occupying the distal gut capable of utilising a myriad of diet- and host-derived glycans. Transport of glycans across the outer membrane (OM) of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, that are thought to operate via a pedal-bin mechanism in which the lids open and close to facilitate substrate binding. However, additional cell surface-exposed lipoproteins, namely surface glycan binding proteins and glycoside hydrolases, play critical roles in the capture and processing of large glycan chains into transport-competent substrates. Despite constituting a crucial mechanism of nutrient acquisition by our colonic microbiota, the interactions between these components in the OM are poorly understood. Here we show that for the levan and dextran utilisation systems of Bacteroides thetaiotaomicron, the additional OM components assemble on the core SusCD transporter, forming stable glycan utilising machines which we term utilisomes. Single particle electron cryogenic electron microscopy (cryo-EM) structures in the absence and presence of substrate reveal concerted conformational changes that rationalise the role of each component for efficient nutrient capture, as well as providing a direct demonstration of the pedal bin mechanism of substrate capture in the intact utilisome.

microbiology↗